Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions

PII: DOI: Reference:

S0045-2068(16)30186-9 http://dx.doi.org/10.1016/j.bioorg.2016.08.007 YBIOO 1941

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

1 August 2016 8 August 2016 10 August 2016

Please cite this article as: A. Chadha, S. Venkataraman, R. Preetha, S.K. Padhi, Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/ j.bioorg.2016.08.007

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions

Anju Chadha a,b *, Sowmyalakshmi Venkataramana, Radhakrishnan Preethaa,c and Santosh Kumar Padhid a

Laboratory of Bioorganic Chemistry, Department of Biotechnology, bNational Center for

Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, Tamil Nadu, India *

Corresponding Author: Tel: 91-44-2257 4106 (O); Fax: 044-2257 4102; Email: [email protected]

c

Present address: Department of Food and Process Engineering, School of Bioengineering, SRM

University, Kattankulathur-603203, Tamil Nadu, India d

Biocatalysis and Enzyme Engineering Laboratory, Department of Biochemistry, School of Life

Sciences, University of Hyderabad, Hyderabad – 500 046, India

1

Abstract

This review highlights the importance of the biocatalyst, Candida parapsilosis for oxidation and reduction reactions of organic compounds and establishes its versatility to generate a variety of chiral synthons. Appropriately designed reactions using C. parapsilosis effect efficient catalysis of organic transformations such as deracemization, enantioselective reduction of prochiral ketones, imines, and kinetic resolution of racemic alcohols via selective oxidation. This review includes the details of these biotransformations, catalyzed by whole cells (wild type and recombinant strains), purified enzymes (oxidoreductases) and immobilized whole cells of C. parapsilosis. The review presents a bioorganic perspective as it discusses the chemo, regio and stereoselectivity of the biocatalyst along with the structure of the substrates and optical purity of the products. Fermentation scale biocatalysis using whole cells of C. parapsilosis for several biotransformations to synthesize important chiral synthons/industrial chemicals is included. A comparison of C. parapsilosis with other whole cell biocatalysts for biocatalytic deracemization and asymmetric reduction of carbonyl and imine groups in the synthesis of a variety of enantiopure products is presented which will provide a basis for the choice of a biocatalyst for a desired organic transformation. Thus, a wholesome perspective on the present status of C. parapsilosis mediated organic transformations and design of new reactions which can be considered for large scale operations is provided. Taken together, C. parapsilosis can now be considered a ‘reagent’ for the organic transformations discussed here.

Keywords

Candida

parapsilosis,

Deracemization,

Kinetic

resolution,

Asymmetric

reduction,

Oxidoreductases, Protein engineered carbonyl reductases, Immobilized C. parapsilosis, Fermentation scale biotransformation

2

Contents

1. Introduction

2. Reactions catalyzed by whole cells of Candida parapsilosis 2.1. Deracemization 2.1.1 Aliphatic and aromatic diols 2.1.2. Aryl ethanols and allylic alcohols 2.1.3. Aryl α-hydroxy esters 2.1.4. β-Hydroxy acid esters 2.1.5. Aliphatic β-hydroxy esters

2.2. Asymmetric reduction 2.2.1 Aromatic α-oxoaldehydes 2.2.2 Aromatic α-ketoesters 2.2.3 Aromatic ketones & diketones 2.2.4 Aliphatic ketones, keto esters & nitro ketones 2.2.5 Aryl keto amides 2.2.6 Aryl imines

2.3. Kinetic resolution 2.3.1 Aliphatic diols 2.3.2 Allylic secondary alcohols

3. Immobilized whole cells of Candida parapsilosis as biocatalyst

4. Optimization and scale up of biocatalysis using whole cells of Candida parapsilosis

5. Candida parapsilosis vs other whole cell biocatalysts for oxidation-reduction reactions 5.1. Deracemization 5.2. Asymmetric reductions 3

6. Oxidoreductases of Candida parapsilosis as biocatalyst

7. Genetically engineered carbonyl reductases from Candida parapsilosis as biocatalyst

8. Conclusion

9. References

1. Introduction

Biocatalytic methods are increasingly being employed for the preparation of a variety of chiral precursors which have wide biological applications [1, 2]. The yeast, Candida sp. is widely reported to mediate the syntheses of a broad range of chiral compounds [3] - both, as whole cells and isolated enzymes. In addition to being a common source of lipases [4, 5]. Candida sp. is also an important source of oxidoreductases. The oxidoreductases mediated reactions reported from Candida sp. provide important insights into their catalytic mechanisms during biotransformations. This opens up the possibilities of developing novel biocatalytic applications using the purified enzymes or whole cells. For instance, the whole cells of Candida viswanathii are reported for the reduction of hetero aryl methyl ketones to the (S)- alcohols with high enantiomeric excess (ee) [6]; Candida chilensis are used to produce enantiomerically pure (R)-allylic alcohols from the respective α,β-unsaturated ketones [7]. The carbonyl reductase enzymes from Candida viswanathii [8-10] and Candida boidinii [11-14] have also been reported in a number of biotransformations. Other species of Candida, such as C. macedoniensis [15, 16], C. utilis [17, 18], C. tropicalis [19, 20], C. floricola [21, 22] and C. magnolia [23, 24] are also known to biocatalyze redox reactions. Xylose reductase from C. tenuis shows a broad substrate acceptance and in addition to xylitol, it reduces a number of aromatic carbonyl substrates [2528]. Xylose reductases are also reported from C. intermedia [29], C. guilliermondii [30], C. boidinii [31] and C. tropicalis [32, 33]. In addition, another important oxidoreductase of the Candida sp. is the formate dehydrogenase (FDH) from C. boidinii which is commonly used as an enzyme in cofactor regeneration (enzyme-coupled approach) [34-36]. 4

Isolated enzymes/whole cells from Candida sp. are also reported for the large scale production of pharmaceuticals and fine chemicals. Candida sorbophila MY 1833 was used for asymmetric reduction to produce (R)-N-(2-hydroxy-2-pyridin-3-yl-ethyl)-2-(4-nitrophenyl)acetamide), an intermediate of the drug with -3-agonist activity used in the treatment of hypertension and coronary disease [37, 38]. FDH from Candida boidinii was combined with phenylalanine dehydrogenase (PDH) in recombinant E. coli to produce (S)-2-amino-5-(1,3dioxolan-2-yl)-pentanoic acid, a chiral precursor used in the synthesis of the antihypertensive drug, omapatrilat, which inhibits the angiotensin-converting enzyme (ACE) [39, 40].

The availability of an enzyme in adequate amounts is a challenge when an isolated enzyme is used for biotransformation instead of whole cells. A practical solution to this problem is to clone and overexpress the desired enzyme. Oxidoreductases, particularly carbonyl reductases from different Candida sp. have been cloned and overexpressed [15, 41-45]. Although genetic engineering techniques i.e. cloning and overexpression of enzymes have opened new avenues in biocatalysis, it is a challenge to develop newer improved and sustainable green technology to obtain the desired enzymes in large quantities, which can be used to prepare enantiomerically pure compounds in bulk quantities.

Among the known Candida sp., Candida parapsilosis is now established as a whole cell biocatalyst for organic oxidation-reduction reactions. The products are enantiomerically pure molecules which are important chiral synthons and have industrial applications [46, 47]. By careful choice of reaction conditions, C. parapsilosis has been shown to catalyze deracemization, enantioselective reduction of prochiral ketones, kinetic resolution of racemic alcohols via selective oxidation to produce a host of chiral compounds e.g. aliphatic and aromatic diols, α-and β-hydroxy esters, allylic alcohols, aryl ethanols and amines. Biocatalytic reduction of imine bond to the respective enantiomerically pure amines is an important transformation. C. parapsilosis (ATCC 7330) can asymmetrically reduce aryl imines to give optically pure amines with good yields (55-80%) and excellent optical purity (95->99%) [48]. In addition, the chemical analogy between the reduction of a carbonyl group (C=O) and an imine (C=N) extends the scope of the biocatalyst, C. parapsilosis for biotransformations. More recently, the biocatalyst C. parapsilosis 5

(ATCC 7330) has been utilized for oxidation reactions. Sivakumari et al. reported the biooxidation of aromatic (activated) primary alcohols to aldehydes in high yields (up to 86%) in mild reaction conditions using hexane: water (48:2) biphasic system [49]. Using the same biocatalyst, the enantioselective oxidation of secondary alcohols were reported to produce the corresponding ketone and the enantiomerically enriched alcohols [50, 51] which are discussed in the later part of this review.

The review also presents important biocatalytic reactions mediated by C. parapsilosis such as deracemization, asymmetric reduction of prochiral ketones, imines, resolution of racemic alcohols through selective oxidation. The understanding of the oxidoreductases from C. parapsilosis at the molecular level are also discussed. The review presents a consolidated view of the different reactions catalyzed by C. parapsilosis with a perspective

to design new

biocatalytic reactions for large scale reactions and also at the lab level.

2. Reactions catalyzed by whole cells of Candida parapsilosis

2.1.Deracemization

2.1.1 Aliphatic and aromatic diols

C. parapsilosis mediated deracemization produces a variety of chiral secondary alcohols (Table 1). The pioneering work on the preparation of optically pure alcohols using C. parapsilosis was reported in 1990 [52]. It was found that C. parapsilosis IFO 0708 selectively transformed (R)-1,2-pentanediol, to the (S)-enantiomer. Thus (S)-1,2-pentane diol was produced from its racemate in 24 h with 100% ee, 93% molar yield. In another study, deracemization of racemic (RS)-1-(2’,3’-dihydrobenzo[b]furan-4’-yl)-ethane-1,2-diol using C. parapsilosis ATCC 52820 produced the corresponding chiral (R)-diol, in 80% ee [53]. Nie et al. screened bacteria, yeasts and molds for the conversion of racemic 1-phenyl-1,2-ethanediol (PED) to (S)-PED, an important chiral building block for liquid crystals and pharmaceuticals. Among the microbes tested, C. parapsilosis CCTCC M203011 showed high activity and produced (S)-PED in 92% yield and 98% ee [54] (Scheme 1). The intermediate α-hydroxyacetophenone was obtained as a 6

result of the stereoselective oxidation of the (R)-enantiomer thus deracemization mechanism was identified as stereoinversion. The intermediate undergoes subsequent addition to produce the (S)enantiomer (Scheme 1). A novel method for NADPH regeneration in cells was proposed using pentose metabolism in microorganisms. In this study xylose addition in reaction batches regenerates more NADPH as compared with those that do not contain xylose. It was observed that the catalytic efficiency and sustainability of the resting cells of C. parapsilosis improved due to the metabolism of xylose during deracemization [55]. Later, the same group of researchers developed a highly competent resin-based in situ substrate feeding and product removal (ISSFPR) methodology for Candida parapsilosis-catalyzed deracemization of racemic 1-phenyl1,2-ethanediol (PED) to produce (S)-PED with 97% enantiomeric excess (ee) and 85% yield [46].

Scheme 1. Deracemization of racemic PED by C. parapsilosis CCTCC M203011

Emphasizing the need for optimizing parameters in biotransformations, it was reported that enhancement of oxygen supply (at 300 rpm) on the deracemization of (RS)-1-phenyl-1,2ethane diol using C. parapsilosis resulted in (S)-alcohol with higher ee (98%) and yield (83%), as opposed to low aeration (200 rpm), which gave a moderate ee (82%) and yield (78%) [56]. Also, the deracemization of (RS)-1-phenyl-1,2-ethane diol catalyzed by C. parapsilosis CCTCC M203011 when performed in aqueous-two phase system showed an improved substrate loading from 15 g/L to 30 g/L and produced (S)-1-phenyl-1,2-ethane diol in higher ee (>99%) and yield (96%) [57].

2.1.2. Arylethanols and allylic alcohols

7

Amrutkar et al. reported the deracemization of racemic 1-(1-naphthyl) ethanol to produce (R)-1-(1-naphthyl) ethanol via stereoinversion using C. parapsilosis MTCC 1965. Under optimized conditions (R)-1-(1-naphthyl)ethanol was obtained in excellent ee (~100%)ee and yield (88%) [58] (Scheme 2).

Scheme 2. Deracemization of racemic (RS)-1-(1-naphthyl)ethanol by C. parapsilosis MTCC 1965

C. parapsilosis ATCC 7330 catalyzed the deracemization of racemic 1-arylethanols and 4-phenyl-2-butanol to the (R)-enantiomer (ee up to >99%) (Table 1). The deracemization of racemic 1-aryl ethanol occurs via dynamic kinetic resolution. The enantioselective oxidation of the (S)-enantiomer and reduction of the subsequent ketone intermediate gives the racemic alcohol which gets kinetically resolved and enriches the (R)-enantiomer [59]. Chiral allylic alcohols are important due to their stereodirecting propensity in diastereoselective reactions [60]. They are chiral precursors for the preparation of verapamil and baclofen and hence considered as important substrates for both synthetic and mechanistic studies. A simple biocatalyst mediated one-pot reaction using whole cells of C. parapsilosis ATCC 7330 was developed that involves the deracemization of racemic allylic alcohols by dynamic kinetic resolution to produce the (R)allylic alcohols in good yields (68–79%) and high ee (76–99%) in less than 3 h under mild reaction conditions [61] (Scheme 3).

8

Scheme 3. Deracemization of racemic allylic alcohols by C. parapsilosis ATCC 7330

Different aryl and alkyl substituted enantiomerically pure propargyl alcohols were also prepared with excellent ee (up to >99%) and isolated yields (up to 87%) by deracemization using whole cells of Candida parapsilosis ATCC 7330. The biocatalyst displayed high substrate specificity towards alkyl substituted propargyl alcohols with different chain lengths and induced a shift in the stereoselectivity from ‘R’ to ‘S’ with a subsequent increase in the chain length of the alkyl group. The enantiopure (R)-4-(3-hydroxybut-1-ynyl)benzonitrile, (R)-4-(biphenyl-4yl)but-3-yn-2-ol, (S)-ethyl3-hydroxy-5-phenylpent-4-ynoate and (S)-4-phenylbut-3-yne-1,2-diol were obtained using this strategy was reported for the first time [62]. 2.1.3. Aryl α-hydroxy esters

C. parapsilosis ATCC 7330 is also reported as an efficient biocatalyst for the deracemization of aryl α-hydroxy esters (Table 2). These optically pure aryl α-hydroxy esters are valuable chiral precursors for ACE inhibitors and antihypertensive drugs [63]. These included an array of aryl substituted α-hydroxy esters which exhibited highlighting steric and electronic effects on biocatalytic deracemization. The substrates can be used as probes to understand the active site of the enzymes involved. A detailed study carried out with ethyl mandelate involved dual enzymatic reactions viz. oxidation of one of the enantiomer i.e. ‘R’ to the keto intermediate followed by its subsequent reduction by a (S)-specific reducing enzyme which predominantly produced the ‘S’ enantiomer [64] (Scheme 4).

9

Scheme 4. Mechanism of C. parapsilosis ATCC 7330 catalyzed deracemization of ethyl mandelate

Deracemization of racemic methyl and ethyl mandelates using the whole cells of C. parapsilosis ATCC 7330 produced their (S)-enantiomers in 70-74% isolated yield and 99% ee in 1 h. The same biocatalyst also catalyzed the deracemization of racemic 2-hydroxy-3arylpropanoic esters (Scheme 5). These substrates are structurally homomandelates. The biotransformation concluded with the formation of (S)-2-hydroxy-3-propanoic esters in 62-70% chemical yield and 45-60% ee in 4 h [64].

Scheme 5. Deracemization of racemic 2-hydroxy-3-arylpropanoic esters by C. parapsilosis ATCC 7330

Although C. parapsilosis ATCC 7330 catalyzed deracemization of homomandelate esters resulted in moderate ee of the products, the same biocatalyst on deracemization of racemic methyl and ethyl esters of 2-hydroxy-4-arylbutanoic acid produced the corresponding (S)enantiomers in 91-99% ee and 85–90% yield (Scheme 6). In addition, the harvested resting cells of C. parapsilosis ATCC 7330 catalyzed the deracemization of six different esters of 2-hydroxy-

10

4-arylbutanoic acid e.g. methyl and ethyl esters of unsubstituted, p-Me, p-Cl and o-hydroxy-4phenylbutanoic acid in 1 to 1.5 h [63, 64].

Scheme 6. Deracemization of racemic 2-hydroxy acid esters using whole cells of C. parapsilosis ATCC 7330

Biocatalytic deracemization of racemic methyl and ethyl 2-hydroxy-4-arylbut-3-enoates using C. parapsilosis ATCC 7330 produced the respective (S)-enantiomers in 90-99% ee and 6575% yield in 1.5 to 2 h (Scheme 7). Irrespective of the electronic property of the substituent on the aromatic ring of alkyl 2-hydroxy-4-arylbut-3-enoates, the deracemized products were formed in high enantiopurity and yields. However, substrates with di-substitutions on the aromatic ring e.g. 2,4-dichloro and 2,5-dimethoxy compounds (R'= Me, Et, Scheme 7), on deracemization gave products (absolute configuration not determined) with 52-72% chemical yield and 50-65% ee in 4 h. The poor ee could be due to the steric effects of these substrates. Substrates like benzyl ester of 2-hydroxy-4-(p-chlorophenyl) but-3-enoic acid and methyl ester of 2-hydroxy-4-(1naphthyl) but-3-enoic acid could not be deracemized using the resting cells of C. parapsilosis as the products were obtained in 10 and 3% ee respectively possibly due to lack of selectivity in the oxidation step [64]. C. parapsilosis ATCC 7330 catalyzed deracemization of methyl and ethyl 2hydroxy-4-arylbut-3-enoates to give their corresponding (S)-enantiomeric products due to the transfer of the pro-R hydride from the cofactor to the Re-face of the carbonyl group (Prelog's rule) and vice-versa [65]. The (R)-enantiomeric products of the deracemization of allyl alcohols (Scheme 3) are due to the variations in the priority of the functional groups in assigning the (R)and (S)-configurations where the stereopreference of the enzymes involved still transfers the proR hydride from the cofactor to the Re-face of the carbonyl group of the prochiral ketone [64].

11

Scheme 7. Deracemization of racemic alkyl 2-hydroxy-4-arylbut-3-enoates using whole cells of C. parapsilosis ATCC 7330

Whole resting cells of C. parapsilosis ATCC 7330 mediated the deracemization of aryl unsubstituted and substituted (3E,5E)-alkyl-2-hydroxy-6-aryl-hexa-3,5-dienoates to produce the respective enantiomerically pure (S)-dienoates (ee: 42 to >99%; yields: 51–80%) (Scheme 8). The compounds with β,γ,ω,δ-unsaturated α-hydroxy groups exhibit antibacterial and cytotoxic properties, for example avalleneol and lachnellins B & C [66].

Scheme 8. C. parapsilosis ATCC 7330 catalyzed deracemization of racemic (3E,5E)-alkyl-2hydroxy-6-arylhexa-3,5-dienoates

In the case of C. parapsilosis ATCC 7330 mediated deracemization of aryl substituted (3E,5E)-alkyl-6-aryl-2-hydroxyhexa-3,5-dienoates, it was shown that ortho/para substitutents [electron withdrawing or donating] in the phenyl ring gave decreased chemical yields (51–74%) and ee (42–99%) as compared to aryl unsubstituted dienoates (yields: 73–80% and ee: 93–99%) [66]. 12

The alkyl-2-hydroxy-4-arylbut-3-ynoates (Scheme 9) were deracemized using whole resting cells of C. parapsilosis ATCC 7330 with excellent chemo- and enantioselectivity to produce their (S)-enantiomers [ee:up to >99%); yields: (up to 81%)]. The enantiomerically pure propargylic alcohols are important chiral precursors for immense biologically important compounds like mifepristone, petrosynol, efavirenz etc. [67]. These bioreactions were carried out in water medium under mild conditions for 1–4 h.

Scheme 9. C. parapsilosis ATCC 7330 catalyzed deracemization of racemic alkyl-2-hydroxy-4arylbut-3-ynoates

Deracemization of racemic (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates using C. parapsilosis ATCC 7330 produced one antipode i.e. (S)-enantiomer in high ee (up to >99%) and isolated yields (up to 79%) [68] (Scheme 10).

Scheme 10. C. parapsilosis ATCC 7330 catalyzed deracemization of racemic (3E)-alkyl-4(hetero-2-yl)-2-hydroxybut-3-enoates

Substrates shown in Scheme 10 also contain an allyl alcohol moiety and just like the alcohols shown in Scheme 7, their deracemization products were also found to be (S) 13

enantiomers. Analysis of various aromatic 2-hydroxy esters deracemized by C. parapsilosis ATCC 7330 discussed above showed that structures with larger carbon frameworks between the hydroxy and the aromatic ring take more time for deracemization compared to the simple structures. For example, the reaction time for the deracemization of mandelates is 1 h, followed by alkyl 2-hydroxy-4-phenylbutanoates (1 to 1.5 h), alkyl 2-hydroxy-4-arylbut-3-enoates (1.5 to 2 h), (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates (3 h), alkyl-2-hydroxy-4-arylbut-3ynoates (1 to 4 h) and (3E,5E)-alkyl-2-hydroxy-6-arylhexa-3,5-dienoates (3 h) among others. An exception here is alkyl 2-hydroxy-3-arylpropanoates which took 4 h. Further analysis of the enantioselectivity of the C. parapsilosis ATCC 7330 catalyzed deracemization of different aromatic 2-hydroxy esters indicated that structures with even (2n, n=0,1,2; n= number of carbon atoms between the hydroxyl and aromatic ring) carbon framework, were preferred substrates for this multi-enzyme based whole cell deracemization process that yielded up to 99% ee of the products while structures with odd (2n+1, n=0) carbon framework i.e. alkyl 2-hydroxy-3arylpropanoic esters resulted in 34-60% ee and 62-70% yield. This also probably explains the longer reaction time which these substrates required. Alkyl 2-hydroxy-4-arylbut-3-enoates and (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates despite having two possible geometric isomers have the same number of carbons between the hydroxyl and the aromatic ring and showed comparable enantioselectivity (90-99% ee) in the C. parapsilosis ATCC 7330 catalyzed deracemization. These ee and isolated yields are comparable to the ee and yields of C. parapsilosis ATCC 7330 mediated deracemization of other substrates carrying four carbons between the hydroxyl (-OH) and aromatic ring as mentioned above. 2.1.4. β-Hydroxy esters Whole cells of C. parapsilosis ATCC 7330 catalyzed the deracemization of several βhydroxy acid esters (Scheme 11), (Table 2). The resting cells of this yeast catalyze the deracemization of racemic alkyl 3-hydroxy 3-aryl propionates and form their (S)-enantiomers with 96-99% ee and 62-75% isolated yields. However, deracemization of ethyl 3-hydroxy 3-(2methyl phenyl) propionate resulted in poor ee (15%) and 75% yield [69].

14

Scheme 11. C. parapsilosis ATCC 7330 catalyzed deracemization of racemic β-hydroxy acid esters

Optically pure alkyl 3-(hetero-2-yl)-3-hydroxypropanoates are industrially important precursors for

the preparation of important

pharmaceuticals

like duloxetine

[70],

tetrahydropyrans [71], methyl 2-(hetero-2-yl(hydroxy)methyl)acrylate, methyl 2-(hetero-2-yl)-3hydroxy-phenylpropanoate and heteroaryl aminoalkanols [72]. A biocatalytic protocol was reported

for the deracemization of racemic alkyl 3-(hetero-2-yl)-3-hydroxypropanoates to

produce the respective enantiomerically pure (S)-enantiomers which gave high ee (89 to >99%) and yields (58–75%) using the resting cells of C. parapsilosis ATCC 7330 [72] (Scheme 12). Electron donating groups present in the hetero aryl -hydroxy ester did not influence the yield and ee, while, the electron withdrawing, –NO2 group resulted in moderate ee (89%) and lower yield (58%). The molecules of Scheme 12 are -hydroxy acid esters similar to those described in Scheme 11, except for the presence of heterocyclic ring instead of a phenyl ring. The reaction conditions for deracemization of these sets of molecules were identical.

Scheme 12. Deracemization of racemic alkyl 3-(hetero-2-yl)-3-hydroxypropanoates using whole cells of C. parapsilosis ATCC 7330

15

In addition to stereochemical preference in a racemate, C. parapsilosis ATCC 7330 exhibits this preference even towards geometrical isomers. Deracemization of a number of cisand trans-aryl secondary alcohols were studied and it was observed that C. parapsilosis ATCC 7330 showed preference towards the E-isomers over Z-isomers. The biocatalytic reduction of E4-phenylbut-3-en-2-one to (R, E)-4-phenylbut-3-en-2-ol in 98% ee and 96% conversion by whole cells of C. parapsilosis ATCC 7330 while the Z-isomer of the ketone remained unreacted bears testimony for this stereochemical preference. Furthermore, the cell free extract of this biocatalyst confirmed that the geometrical preference of the biocatalyst is due to the enzymes and not due to factors like restricted entry into the cell [73]. 2.1.5. Aliphatic β-hydroxy esters

C.

parapsilosis

ATCC

7330-catalyzed

deracemization

of

various

alkyl-3-

hydroxybutanoates gave anti-Prelog products; (R)-enantiomers were obtained with yields of up to 71 % and >99 % ee. Unlike aryl α/β, the deracemization of aliphatic β-hydroxy esters did not occur in water with ethanol as cosolvent [65]. Therefore, the reaction conditions were reoptimized in Tris-HCl buffer medium (pH 8.5) using acetone as cosubstrate for efficient cofactor regeneration with dimethyl formamide/dimethyl sulphoxide as cosolvent. Using optimized conditions, the racemic methyl, ethyl, propyl, butyl, t-butyl and allyl-3-hydroxybutanoates were deracemized to produce anti Prelog (R)-enantiomers except for pentyl, iso-propyl and iso-amyl3-hydroxybutanoates which were obtained as (S)-enantiomers (Prelog products). In addition, a notable decrease in the optical purity of substrates with branched chain on the ester side (64–81 %) was observed compared to their linear counterparts (79 to >99 %) [74] (Scheme 13).

Scheme 13. Deracemization of racemic alkyl 3-hydroxybutanoates using whole cells of C. parapsilosis ATCC 7330 16

The deracemization of ethyl 3-hydroxy 4,4,4-trifluorobutanoate was reported for the first time using C. parapsilosis ATCC 7330. The reaction was carried out at pH 8.5 (Tris-HCl buffer) using acetone as cosubstrate and ethanol as cosolvent to produce (S)-ethyl 3-hydroxy 4,4,4trifluorobutanoate [ee (96%); yield: (65%)] [75] (Scheme 14).

Scheme 14. Deracemization of racemic ethyl 3-hydroxy 4,4,4-trifluorobutanoate using whole cells of C. parapsilosis ATCC 7330

2.2. Asymmetric reduction

Enantioselective biocatalytic reduction of ketones is a powerful tool for the production of enantiopure compounds (Tables 3, 4), which are high value chiral precursors and are used in various industrial applications. Many biocatalysts are known to catalyze this reaction [65, 76, 77]. An account of C. parapsilosis mediated asymmetric reduction reactions are as follows. 2.2.1 Aromatic α-oxoaldehydes

Mahajabeen and Chadha reported a one-pot, synthesis of enantiomerically pure 1-phenyl 1,2-ethanediols (PEDs) through asymmetric reduction of several substituted aromatic αoxoaldehydes using C. parapsilosis ATCC 7330 in good yields (up to 70%) and with high ee (>99%) [78]. The position of the substituent on the phenyl ring of the substrates has a significant influence on the optical purity and the absolute configuration of the product. Presence of a substituent at meta- and para-positions of the aromatic ring of α-oxoaldehydes gave the corresponding (S)-PEDs, while chloro group at the ortho-position gave the (R)-1-(2chlorophenyl) ethane-1,2-diol (Scheme 15).

17

Scheme 15. Asymmetric reduction of aromatic -oxoaldehydes using the whole cells of C. parapsilosis ATCC 7330 2.2.2 Aromatic α-ketoesters

Asymmetric reduction of alkyl 2-oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates catalyzed by C. parapsilosis ATCC 7330 produced the corresponding (S)-2-hydroxy esters in high ee (93–99%) and good yields (58–71%) [79] (Scheme 16).

Scheme 16. C. parapsilosis ATCC 7330 catalyzed asymmetric reduction of (A) alkyl 2-oxo-4arylbutanoates; and (B) alkyl 2-oxo-4-arylbut-3-enoates

2.2.3 Aromatic ketones

18

Patil et al. studied the bioreduction of 1-benzosuberone using various Candida sp. like C. parapsilosis, C. viswanathii and C. melibiosa in phosphate buffer (pH 6.5) containing 5% v/v 2propanol in 120 h. All the biocatalysts screened gave (S)-1-benzosuberol with >99% ee and low yield (up to 22%) [10] (Scheme 17).

Scheme 17. Asymmetric reduction of 1-benzosuberone using different Candida sp.

In the biotransformation of (R,S)-PED to its optically active (S)-enantiomer using C. parapsilosis CCTCC M203011, presence of xylose (8 g/L) increased the optical purity and the yield of the (S)-enantiomer by 14% and 10%, respectively. Furthermore, the reusability of the whole cells up to 4 cycles without any loss in the ee of the (S)-enantiomer (98%) was observed. It was shown that the addition of xylose as cosubstrate increased the productivity of the reaction and also increased the sustainability of the cells for repeated use without much loss in activity [80]. Mahapatra et al. studied the asymmetric reduction of an enantiopure tricyclic scaffold i.e. (1S,9R)-9-hydroxymethyl-11-oxatricyclo- dodeca-2,4,6-trien-8-one using the growing culture of C. parapsilosis NBRC 1396. The corresponding alcohol i.e Prelog product in 97% de and 68% yield was obtained [81]. Bhuniya et al. used a growing culture of C. parapsilosis NBRC 1396 for the asymmetric reduction of 1-tetralone, 1-indanone and 4-chromanone based scaffolds. The keto functionality in all the thirteen substrates were reduced in a Prelog fashion to their corresponding alcohols in high ee (97-99%) and 67-82% yields [82], (Table 4). Recently, the regio- and enantioselective bioreduction of aryl diketones was reported using the resting cells of C. parapsilosis ATCC 7330 to produce enantiomerically enriched hydroxy ketones with high ee(up to 98%) and yields (up to 75%). %). In this study, α-diketones took lesser time (up to 8 h) to give Prelog products [(S)-hydroxy ketones] while β-diketones produced anti-Prelog products [(R)hydroxy ketones] in 30 h [83].

19

2.2.4 Aliphatic ketones, keto esters & nitro ketones

Different biocatalysts were employed for the preparation of optically pure 1,3-butane diols, industrially important chiral synthon using asymmetric reduction. Among them, Kluyveromyces lactis IFO 1267 produced the (R)-1,3-butanediol in high ee (93%), while C. parapsilosis IFO 1396 gave the (S)-product in high ee (98%) [84]. Changes in cultivation conditions and addition of cosubstrates have marked effect on the enantioselectivity of biocatalytic reductions. In the bioreduction of methyl acetoacetate by C. parapsilosis DSM 70125 cells grown in medium containing dodecanoic acid, showed maximum specific activity and a marked change in enantioselectivity to form (R)-alcohol with 40% ee was seen. The cells grown in medium containing glucose or glycerol with ethyl-3-oxohexanoate as the inducer gave the (S)-product alcohol in up to 29% ee [85]. C. parapsilosis ATCC 7330 when grown in glycerol containing medium, reduced ethyl-4 chloro-3-oxobutanoate (COBE) to (R)-ethyl-4chloro-3-hydroxybutanote (CHBE) (ee >99%) while glucose and sucrose grown cells produced (S)-CHBE (>99%) [86, 87]. It was shown that, the (S)-specific bioreduction of prochiral COBE was carried out by an NADH dependent alcohol dehydrogenase, while glycerol induced an NADH dependent aldehyde reductase which catalyzed the (R)-specific asymmetric reduction of COBE. This was confirmed by the effect of inhibitors on COBE reductase from C. parapsilosis ATCC 7330. The benefit of the developed methodology is that by simply altering the carbon source in the growth medium, both the optical antipodes of CHBE could be produced in high optical purity using the same biocatalyst (Scheme 18).

Scheme 18. Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using C. parapsilosis ATCC 7330 grown using different carbon sources 20

Whole cells of C. parapsilosis ATCC 7330 when used for bioreduction of alkyl-3oxobutanoates gave their corresponding enantiomerically pure alkyl-3-hydroxybutanoates in good yields (up to 72%) and excellent ee (up to >99%). Most of the ketoesters gave the (S)-alkyl hydroxyesters by this method except the methyl ester which resulted in its (R)-enantiomer (Scheme 19). Using this method, the enantiomerically enriched (S)-isoamyl-3-hydroxybutanoate (ee: 79%) was produced from isoamyl-3-oxobutanoate and was reported for the first time [88].

Scheme 19. Asymmetric reduction of alkyl 3-oxobutanoates using C. parapsilosis ATCC 7330 Another efficient for the biocatalytic reduction of ethyl 4,4,4-trifluoro-3-oxobutanoate in water was also reported using C. parapsilosis ATCC 7330 to give (S)-ethyl 3-hydroxy 4,4,4trifluorobutanoate with good yield (68%) and ee (66%); the ee was further increased to 84% ee (yield 60%) upon treatment with allyl alcohol as inhibitor [75] (Scheme 20).

Scheme 20. Asymmetric reduction of ethyl 4,4,4-trifluoro-3-oxobutanoate using C. parapsilosis ATCC 7330 with allyl alcohol as inhibitor

The biocatalytic reduction of aliphatic nitro ketones was carried out in aqueous medium using the resting cells of Candida parapsilosis ATCC 7330 in much lesser time (4 h) and the biocatalyst showed excellent chemoselectivity. It reduces the carbonyl group in preference to the nitro group (Scheme 21). The biocatalytic reduction of 1-nitro-butan-2-one, 1-nitro-pentan-2one, 3-methyl-1-nitro-butan-2-one and 1-cyclohexyl-2-nitroethanone to produce (R)-alcohols [ee 21

up to 79%, yield up to 74%] and 1-nitro-hexan-2-one and 1-nitro-heptan-2-one to produce (S)alcohols [ee up to 81%, yield up to 76%] were reported for the first time [89].

Scheme 21. Asymmetric reduction of nitro ketones using C. parapsilosis ATCC 7330

2.2.5 Aryl keto amides Optically pure aromatic α-hydroxy amides are chiral synthons for the preparation of bradykinin 1 selective antagonists or inverse-agonists. Whole resting cells of Candida parapsilosis ATCC 7330 were utilized for the bioreduction of various aryl α-keto primary and secondary amides to produce optically enriched (R)-α-hydroxy amides (Scheme 22). Among the substrates studied, primary (R)-α-hydroxy amides were produced with good ee (12-94%) and yields (74-97%) compared to the secondary (R)-α-hydroxy amides (ee: 16-75%; yield: up to 86%) [90].

Scheme 22. Asymmetric reduction of aryl α-keto primary and secondary amides using C. parapsilosis ATCC 7330

2.2.6 Aryl imines 22

Biocatalytic reduction of C=N bond is an efficient method to obtain enantiomerically enriched amines as these molecules are important precursors of compounds with broad industrial applications. A highly enantioselective one pot, novel biocatalytic method for the asymmetric reduction of aryl imines was reported (Scheme 23). The C. parapsilosis ATCC 7330 catalyzed the bioreduction of aromatic imines in water to produce optically enriched (R)-secondary amines in good yields (55–80%) and excellent ee (95–>99%) [48] thus establishing a biocatalytic method for the preparation of chiral secondary amines using C. parapsilosis ATCC 7330.

. Scheme 23. Asymmetric reduction of aromatic imines using the whole cells of C. parapsilosis ATCC 7330

2.3. Kinetic resolution

Enzymatic kinetic resolution (KR) is an important strategy where the fast reacting enantiomer of the racemic reactant is converted into product while the slow reacting enantiomer remains unreacted. This reactivity mainly depends on the selectivity of the enzyme towards the two opposite stereoisomers. Hydrolases are widely used in the KR of a variety of substrates mainly by hydrolysis or esterification reactions. Several C. parapsilosis strains also carry out KR, however the mechanism involves either (a) hydrolysis/esterification or (b) enantioselective oxidation. It is known that the hydrolysis of a racemic ester using an esterase, results in an enantiopure ester and an alcohol; while in enantioselective oxidation, one of the enantiomer of the racemic alcohol gets selectively oxidized to the prochiral ketone leaving the other enantiopure alcohol unreacted. This shows the versatility of C. parapsilosis towards different biotransformations and selectivity of the corresponding enzymes in this multi-enzymatic 23

biosystem. In contrast to deracemization, the theoretical chemical yield of enantiopure alcohols obtained by KR of racemic alcohols cannot exceed 50% but it is an important biocatalytic strategy to synthesize enantiomerically pure compounds from their corresponding racemates, to produce both enantiomers.

2.3.1 Aliphatic diols

Matsuyama et al. studied enantio-selective oxidation of racemic 1,3-butanediol (BDO) where the (S)-1,3-butanediol (BDO) in the racemate gets selectively oxidized leaving the (R)diol unreacted. In this study, several microbes were screened for the biocatalytic resolution of racemic 1,3-BDO. C. parapsilosis IFO 1396, was found to have varying rates of oxidation for the two antipodes of racemic 1,3-BDO [84]. This was the most practical process to produce (R)1,3-BDO, a chiral precursor for the preparation of azetidinone derivatives with high ee (95%) and 50% yield (Scheme 24).

Scheme 24. Kinetic resolution of racemic 1,3-BDO using whole cells of C. parapsilosis IFO 1396

Yamamoto et al. cloned a gene that encodes a secondary alcohol dehydrogenase from C. parapsilosis (CpSADH) which catalyzes the enantioselective oxidation of (S)-1,3-BDO into 4hydroxy-2-butanone. The whole cells of recombinant E. coli JM109 carrying cloned CpSADH in an expression plasmid was used to convert racemic 1,3-BDO (555 mM) into (R)-1,3-BDO (272 mM) in 94% ee and 49% yield [91].

2.3.2 Allylic secondary alcohols

24

Racemic allylic alcohols and 4-phenylbutan-2-ols were enantioselectively oxidized by the resting cells of C. parapsilosis ATCC 7330 produced their corresponding optically pure (R)alcohols. This biocatalytic reaction performed under mild reaction conditions selectively oxidized the (S)-enantiomer of the racemate to the keto compound (yield: 37–46%) and the (R)alcohol remains unreacted (yield: 37–52% and ee: 46 to >99%) [50]. The biocatalyst also exhibits excellent chemoselectivity between primary and secondary alcohols. For instance, in the oxidation of racemic aryl 1,2-diols the secondary alcohol was preferentially oxidized to the primary alcoholic group; the R-enantiomer undergoes selective oxidation to the corresponding keto alcohol (yield: 18–54%) leaving the S-diol unreacted (yield: 31–69% and ee from 14% to >99%) in isooctane–water (48 : 2 v/v) medium [51].

There

exists

several

examples

of

KR

by

enantioselective

acylation

/esterification/hydrolysis/transesterification of appropriate substrates catalyzed by different strains of C. parapsilosis, which are not part of oxidation-reduction system. The selective oxidation of a single enantiomer of the racemic substrate during deracemization by stereoinversion is also an example of KR which is considered an integral part of the deracemization process.

3. Immobilized whole cells of Candida parapsilosis

Immobilization of microbial cells provides multiple benefits over free cells such as reusability, reaction control i.e. ability to stop the reaction by removing the biocatalyst, stability of the enzyme/s, high cell densities and cell loads, reduced risk for microbial contamination and simplified work up system [92]. Immobilization of biocatalyst can be done by using several methods namely- adsorption, entrapment, crosslinking, covalent bonding and encapsulation [93]. Immobilized whole cells are utilized in the industrial preparation of (S)-naproxen [94], 1,5dimethyl-2-piperidone (1,5-DMPD, Xolvone), acrylamide from acrylonitrile [95] etc. and also used for various environmental applications [96].

25

The use of immobilized C. parapsilosis is reported for different biocatalytic reactions e.g. deracemization of many different racemic secondary alcohols and enantioselective bioreduction of prochiral ketones. Deracemization of ethyl 3-hydroxy-3-phenyl propanoate, and aryl substituted β-hydroxy esters by alginated whole cells of C. parapsilosis ATCC 7330 produced the corresponding (S)enantiomers similar to Scheme 11. In this study, the cells were entraped in sodium alginate [97]. The method is most commonly used for its robustness, cost effectiveness and importantly, the matrix support protects the biocatalyst from microbial contamination. Ethyl 3-hydroxy-3-aryl propanoates with electron withdrawing (m-Br, and p-NO2) and electron donating groups (p-Me, p-Et, o-OMe, p-OMe and p-Cl) attached to the phenyl ring were deracemized to give the (S)alcohols in 72–99% ee and 41–75% isolated yields. However ethyl 3-NO2-3-hydroxy propanoate on deracemization resulted in only 26% ee and 66% yield. Deracemization of ethyl 3-hydroxy-5phenyl-pent-4-enoate and ethyl 3-hydroxy-5-phenyl pentanoate resulted in >99% ee, 28% yield and 87% ee, 10% yield respectively of their (S)-enantiomers. Ethyl 3-(2,4-dichlorophenyl)-3hydroxy propanoate on deracemization resulted in (S)-enantiomer (82% ee and 53% yield), indicating that the o,p-diCl substitution does not adversely affect the deracemization process [98]. Ethyl 3-hydroxy-3-(2-methylphenyl) propanoate, ethyl 3-hydroxy-3-(3-nitrophenyl) propanoate and ethyl 3-hydroxy-4-phenyl butanoate gave poor optical purity of the products (9, 26 and 13% ee respectively) on deracemization. Not only ethyl, but also racemic methyl, npropyl and n-butyl esters of 3-hydroxy-3-phenyl propanoates produced the (S)-alcohols (ee: 6799%; yield: 47-71%) by deracemization. Ethyl 3-hydroxy-3-napthalen-1-yl propanoate, ethyl 3anthracen-9-yl-3-hydroxy propanoate were not deracemized possibly due to steric reasons. Reusability of immobilized whole cells of C. parapsilosis ATCC 7330 was also well studied using ethyl-3-hydroxy-3-phenyl propanoate for the deracemization reaction [98]. A notable decrease in activity was observed with the increase in number of cycles of re-use. The activity of the biocatalyst was monitored by using the deracemization of ethyl 3-hydroxy-3-phenyl propanoate to produce the (S)-alcohol as the model reaction. The immobilized cells could be reused for up to three cycles with a gradual loss of activity (5%) and the sixth to eighth cycles showed a loss of 12-15% of activity. Mechanistic investigation of the deracemization of aryl 3hydroxy esters using a deuterated substrate (ethyl 3-deutero-3-hydroxy-3-phenyl propanoate) 26

revealed that the (R)-enantiomer undergoes enantioselective oxidation to its respective ketoester, which undergoes enantiocomplementary reduction to give the (S)-enantiomer (yield 75%); ee >99%) [97]. Zhang et al. described an efficient biocatalytic method for the synthesis of optically pure (S)-4-(trimethylsilyl)-3-butyn-2-ol, a chiral precursor in the synthesis of 5-lipoxygenase inhibitors. Calcium alginate encapsulated whole cells of C. parapsilosis CCTCC M203011 under optimum conditions catalyzed the biocatalytic reduction of 4-(trimethylsilyl)-3-butyn-2-one (TMSB) into enantiopure (S)-4-(trimethylsilyl)-3-butyn-2-ol ((S)-TMSBOL), in >99% ee and 81% yield [99]. Although the substrate TMSB was unstable under neutral or alkaline conditions, the acidic range of pH between 3.0 and 6.0 stabilized the substrates in the efficient synthesis of enantiopure (S)-TMSBOL.

In addition to immobilization, role of ionic liquids has been studied on several C. parapsilosis mediated biotransformations. Lou et al. designed a new, two-phase reaction systems using biocompatible water-immiscible ionic liquids (ILs)

as an alternative to conventional

organic solvents. The biocatalytic reduction of TMSB to (S)-TMSBOL was reported using calcium alginate encapsulated cells of C. parapsilosis CCTCC M203011 in various ILs [100]. Among them, 1-butyl-3-methylimidazolium hexafluorophosphate (C4MIM·PF6), was found to be the most effective IL for this bioreduction and under optimized conditions the product was obtained in 98% yield and >99% ee. Cells retained greater relative activity in the [C4MIM]PF6 IL system than in water system, even after repeated use up to twelve cycles (83% vs 34%). Zhang et al. reported the enantioselective bioreduction of acetyltrimethylsilane (ATMS) to antiPrelog (R)-1-trimethylsilylethanol [(R)-1-TMSE], using calcium alginate encapsulated C. parapsilosis CCTCC M203011 cells in water-immiscible ILs (Scheme 25). Under optimized reaction conditions, (R)-1-TMSE was produced in maximum yields (99%) and ee (>99%). The immobilized C. parapsilosis CCTCC M203011 cells could be used repeatedly for eight batches in the C4MIM.PF6/buffer biphasic system as they still maintained ~87% of their initial activity [101]. Thus the operational stability of encapsulated C. parapsilosis when used in IL seems higher than when used in the buffer.

27

Scheme 25. Immobilized C. parapsilosis CCTCC M203011 catalyzed asymmetric reduction of ATMS to (R)-1-TMSE in water immiscible IL-buffer biphasic system

Biocatalytic reduction of TMSB to (S)-TMSBOL, by calcium alginate encapsulated C. parapsilosis CCTCC M203011 cells was examined using hydrophilic dialkylimidazolium-based ILs to enhance the asymmetric reduction process. Among fourteen different hydrophilic ILs, 1(2-hydroxyl)ethyl-3-methylimidazolium nitrate (C2OHMIM.NO3) showed the highest initial reaction rate with best product yield and ee. The initial reaction rate, yield and ee of the (S)TMSBOL under the optimal conditions, were 17.3 µmol/h gram of cells, 95% and >99%, respectively. In the above study it was found that the immobilized cells could be recycled for ten cycles in C2OHMIM.NO3 containing a cosolvent system and still retaining >83% of their initial activity [102].

4. Optimization and scale up of biotransformations using the whole cells of C. parapsilosis

Matsuyama et al. developed an industrial scale biocatalytic method for the production of (R)-1,3-BDO by stereospecific microbial redox reactions [84]. The experiments were done on a large scale (2000 L) using C. parapsilosis IFO 1396 and enantioselective oxidation of (S)-1,3BDO was carried out using bulk amount of the harvested biomass with racemic 1,3-BDO. However, the yield of (R)-1,3-BDO was only 16% with chemical purity 99% and 94% ee. Improvements on the fermentation process of C. parapsilosis CCTCC M203011 for the production of (S)-PED by stereoinversion reaction were also reported [103]. In this work, 28

stereoinversion capabilities of numerous strains of C. parapsilosis were studied and C. parapsilosis CCTCC M203011 is reported as the best biocatalyst for the production of (S)-PED. The important medium (MgSO4, ZnSO4, and KH2PO4) constituents were identified using Plackett–Burman

method. The reaction conditions for the preparation of (S)-PED were

optimized using response surface methodology and it was observed that the yield of the product increased from 75 to 96% and the ee from 84 to 97 %. Nie et al. studied the dissolved oxygen concentration during cell growth, which was found to enhance the efficiency of C. parapsilosis mediated (R,S)-1-phenyl-1,2-ethanediol (PED) deracemization. The increase in the speed of agitation from 200 to 300 rpm at 1.5 vvm aeration rate, a marked improvement in the cell growth as well as the activity of the key enzymes involved in the process was observed which in turn enhanced dissolved oxygen concentration and (S)-PED was obtained with higher ee (98%) and yield (83%) [56].

Kaliaperumal et al. have reported the optimization of different culture parameters namely- size of the inoculum, culture age of the biocatalyst etc. and reaction parameters like the addition of co-substrate, pH of the reaction medium and substrate concentrations for the biocatalytic reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-ethyl 4-chloro-3hydroxybutanoate (CHBE) in water using the resting cells of C. parapsilosis ATCC 7330 [87] (Scheme 26). Under optimized reaction conditions, the final concentration of (S)-CHBE, its optical purity, isolated yield and space time yield were found to be1.38 M (230 g/L), >99, 95% and 115 mmol/h respectively.

Scheme 26. Asymmetric reduction of COBE by C. parapsilosis ATCC 7330

Reportedly, optimization of reaction parameters play a crucial role in the high ee of the products formed in various C. parapsilosis ATCC 7330 mediated biotransformations. Effect of cosolvents, pH, effect of different carbon sources and use of specific enzyme inhibitors on optimization improved ee and yields [74, 75, 88, 89]. 29

5. C. parapsilosis vs other whole cell biocatalysts for oxidation-reduction reactions

C. parapsilosis is an established biocatalyst for deracemization of racemic diols (aryl and aliphatic), α-hydroxy esters, β-hydroxy esters (aliphatic and aromatic), α -hydroxy propargylic esters, allylic alcohols and aryl ethanols; asymmetric reduction of aliphatic ketones and esters, lactones, diketones, arylketones, nitro ketones, aryl α-keto amides, aromatic esters (saturated and unsaturated), oxo aldehydes and imines. Numerous other microbes have also been used for oxidation-reduction reactions. The following section presents a comparative account of C. parapsilosis with other whole cell systems for oxidation-reduction reactions (Tables 5 & 6).

5.1. Deracemization

Deracemization of aliphatic diols such as (RS)-pentane-1, 2-diol and (RS)-butane-1, 3diol (1-3-BDO) was reported using C. parapsilosis IFO 0708 and C. parapsilosis IFO 1396 respectively (Table 5). Deracemization using C. parapsilosis IFO 0708 produced ~100% ee for the (S)-pentane-1,2-diol within 24 hours while C. maltosa produced 95% ee for the same product after 48 hours [52]. Screening of different microbes, for the biocatalytic kinetic resolution of racemic 1,3-BDO was reported. However, the reaction catalyzed by C. parapsilosis IFO 1396 was found to be the best practical process to produce (R)-1,3-BDO with high % of ee and yield [84] (Table 5). Similarly, in another screening study for deracemization of (RS)-1-phenyl-1,2ethanediol, C. parapsilosis CCTCC M203011 was established as the most productive biocatalyst to produce (S)-PED with ee (98%) and yield (92%) [54]. Deracemization of the phenylethan-1,2diol using G. candidum IFO 5767 as biocatalyst with ~100% yield (69% isolated yield) and ~100% ee for its (S)- enantiomer production is also reported [104].

Efficient deracemization reactions are also reported from our research group for a wide array of α-hydroxy esters such as (RS)-2-hydroxy n- aryl alkyl esters, (RS)-2-hydroxy-4arylbutanoic acid ester, (RS)-2-hydroxy-4-arylbut-3-enoic acid ester, (RS)-(3E)-alkyl-4-(hetero2-yl)-2-hydroxybut-3-enoate, (RS)- (3E,5E)-ethyl-2-hydroxy-6-arylhexa-3,5-dienoate, and (RS)alkyl-2-hydroxy-4-arylbut-3-ynoate using C. parapsilosis ATCC 7330 [63, 64, 66-68] (Table30

2). The above mentioned compounds are not yet reported in the literature using any other whole cell biocatalysts with comparable yields and ee as with C. parapsilosis ATCC 7330. However, (S)-mandelic acid and its derivatives were prepared by enantioselective deacylation of 2-acetoxy2-phenylacetic acid and its derivatives using Pseudomonas sp. ECU1011 [105]. Deracemization of secondary aliphatic alcohols, benzylic alcohols and propargylic alcohols [106, 107] is well studied using other whole cell biocatalysts however, deracemization of α-hydroxy esters is well documented with C. parapsilosis only.

Apart from the above mentioned substrates C. parapsilosis ATCC 7330 deracemized a variety of β-hydroxyesters such as (RS)- ethyl 3-hydroxy 3-phenyl propionate, (RS)- methyl 3hydroxy 3-phenyl propionate and (RS)-alkyl 3-(hetero-2-yl)-3-hydroxypropanoates (Table 5). In this context, the remarkably short reaction time (3 hours) was reported for deracemization of βhydroxyesters. Notably, C. parapsilosis ATCC 7330 mediated deracemization of racemic allylic alcohols produced the (R)-enantiomers in high ee (up to >99%) and isolated yields (up to 79%) within 3 hours [61]. Voss et al. also described deracemization of allylic alcohol using a combination of whole cells and isolated enzyme 1) Alcaligenes fecalis DSM 13975 for (R)specific oxidation and alcohol dehydrogenase from Rhodococcus ruber 44541 for (S)-specific reduction and 2) Rhodococcus erythropolis DSM 43066 for (S)-specific oxidation and alcohol dehydrogenase from Lactobacillus kefir 44541 for (R)-specific reduction. The above mentioned catalytic (asymmetric) tandem reactions took 16 h for biocatalytic oxidation -reduction in the presence of NAD+ in a one-pot transformation and produced the (S)-allylic alcohol with 94% ee [108].

C. parapsilosis ATCC 7330 efficiently deracemized racemic 1-arylethanols and 4phenyl-2-butanol (Table 5). The reaction occurs via dynamic kinetic resolution in 3 h [59] while stereoinversion of aryl alcohols by G. candidum IFO 5767 was also achieved under aerobic conditions in 24 h [104]. Aliphatic β-hydroxyesters undergoes deracemization using C. parapsilosis ATCC 7330 to produce predominantly the (R)-enantiomers with high ee (up to >99%) in 18-24 h via a stereoinversion mechanism [74]. Nakamura et al. described the deracemization of methyl 3-hydroxybutanoate and methyl 3-hydroxypentanoate employing Geotrichum candidum IFO 5767 in 24 h [104] (Table 5). In another study, Voss et al. reported 31

the deracemization of ethyl 3-hydroxybutanoate using multiple purified enzymes with the external addition of expensive cofactors in 16 h [108, 109]. The deracemization of different aliphatic β-hydroxyesters were scarcely reported and the biotransformation of these βhydroxyesters were performed efficiently using C. parapsilosis ATCC 7330 [74]. The biocatalytic deracemization of ethyl 3-hydroxy-4,4,4-trifluorobutanoate is not reported using any other biocatalyst except C. parapsilosis ATCC 7330 [75].

5.2. Asymmetric reductions

The reduction of 4-hydroxy-2-butanone by C. parapsilopsis IFO 1396 produced (S)- 1,3butanediol (ee: 97%) [84] (Table 6). Among different biocatalysts screened, C. parapsilopsis was found to be the best candidate for producing high yield (97%) of (S)- enantiomer by biocatalytic reduction of 4-(trimethyl)but-3-yn-2-one [100], although other biocatalysts namely, Rhodotorula sp. (14%) and Geotrichum candidum (5%) produced the maximum ee (>99%) [99]. In such reactions, along with ee, it is important to consider yields especially if the asymmetric reduction is being considered for industrial (scale-up) applications. Interestingly, asymmetric reduction of another aliphatic compound, COBE to (S)-CHBE in water using resting cells of C. parapsilosis ATCC 7330 was reported with 99% ee and 96% yield within 10 minutes reaction time [87] (Table 6). On the other hand, the biocatalytic reduction of COBE using different microorganisms such as Lactobacillus kefir [110] and Aureobasidium pullalans reportedly needed longer reaction times (14 to 36 h). Zhou et al. reported the reduction of COBE to produce (S)-CHBE in 55% ee [111]. Dahl et al. studied the effect of sugar, heat treatment and allyl alcohol on the stereoselectivity of COBE reduction using baker's yeast which resulted in the formation of (S)-CHBE (conversion: 100%; ee 90%) in 19 h [112]. Stereoselective reduction of COBE was also catalyzed by G. candidum SC 5469 cells grown in glucose as carbon source to produce (S)-CHBE in 83% yield and 96% ee [113]. Whole cells of Lactobacillus kefir catalyzed the reduction of COBE to (S)-CHBE in 97% yield and >99% ee, however the biotransformation took 14 h [110]. Daucus carota roots cells/tissue were reported to carry out enantioselective reduction of COBE in 60 h to produce (S)-CHBE in 50% yield and 90% ee [114]. Asymmetric reduction of COBE was also experimented with different plant cells/tissues and the biotransformations were carried out for 100 h [115]. Fresh apple (M. pumila) carrot (D. carota), 32

cucumber (C. sativus), potato (S. tuberosum), radish (R. sativus) and sweet potato (I. batatas) produced (S)-CHBE (ee: up to 93%; yield: up to 49%) while onion (A. cepa) produced (R)CHBE (ee: 80%; yield: 59%). Aureobasidium pullulans CGMCC1244 efficiently catalyzed the asymmetric reduction of COBE into (S)-CHBE in 95% molar conversion and 98% ee, however the bioconversion was performed for 24 h [116]. From the above analysis of various whole cell biocatalyst catalyzed reductions of COBE, it is clear that C. parapsilosis ATCC 7330 seems to be the most efficient biocatalyst for this asymmetric reduction.

Asymmetric reduction of alkyl 2-oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates using C. parapsilosis ATCC 7330 gave the corresponding (S)-2-hydroxy compounds in high ee (93–99%) and yields (58–71%) within 4 h [79]. Later, ethyl (R)-2-hydroxy-4-phenylbutyrate (R)HPBE), a chiral precursor of angiotensin converting enzyme (ACE) inhibitors, was prepared using the biocatalytic reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) [117]. In the above study, Candida krusei SW2026, was suspended in aqueous/[Bmim]PF6 biphasic system with an optimum reaction time of 10 h for the production of (R)-enantiomer >99% ee and 95% yield was reported for the first time. Baskar et al. studied the asymmetric reduction of 2-oxo-4-arylbut-3enoates using cell cultures of Daucus carota to produce the (R)-enantiomer predominantly (ee up to >99%) and yield (up to 73%) [118]. However, biocatalytic reduction of 2-oxo-4-arylbut-3enoates has not yet been reported with any other microorganisms other than C. parapsilosis ATCC 7330 [79]. At the same time, bioreduction of aliphatic and aromatic α–keto esters and β– keto esters is well documented using different microorganisms [119-124].

Biocatalytic asymmetric reduction for synthesis of diols is also well documented in literature. Optically pure (S)-1-phenyl-1, 2-ethanediols are prepared in good yields (up to 70%) and high ee (>99%) via asymmetric reduction of different substituted aryl -oxoaldehydes using C. parapsilosis ATCC 7330 [78]. In this study, the reaction occurs in a single-pot involving the formation of 2-hydroxy-1-phenylethanone (intermediate) in 3 h as compared to the multienzymatic synthesis of (S)-1-phenyl-1,2-ethanediol in 22 h [125]. Biocatalytic asymmetric reduction of different acyclic, cyclic and polycyclic ketones was studied using different microorganisms and Diplogelasinospora grovessii IMI 171018 was found to be the best

33

biocatalyst which even reduced the aldehyde group of cyclic ketones but not the conjugated C=C bond with a longer reaction time (72 h) [126].

Biocatalytic reduction of various alkyl-3-oxobutanoates was reported to produce (S)hydroxy esters (ee: up to >99%) in much lesser time (2 h 15 min) compared to the methods reported using other microorganisms [127-129]. This study emphasized the fact the enantioselectivity of the biocatalyst can be improved by optimization of reaction parameters. Several other biocatalytic methods were also reported for the asymmetric reduction of the fluorinated β–keto ester (Table 6). Among different whole cell biocatalysts, C. parapsilosis ATCC 7330 showed best results in terms of high ee and lesser reaction time. [75]. Biocatalytic reduction of different aliphatic α, β and γ- nitro ketones was performed using C. parapsilosis ATCC 7330 resulted in the formation of respective enantiomerically enriched nitro alcohols in 4 h. Using Comamonas testosteroni as biocatalyst the asymmetric reduction of different aryl and aliphatic nitro ketones was reported. Among the substrates, the bioreduction of 1-nitro-octan-2one resulted in the (S)-alcohol with good ee (>99%) and yield (47%) in 48 h [130]. The asymmetric reduction of 4-nitro-butan-2-one and 5-nitro-pentan-one was performed using C. parapsilosis ATCC 7330 to produce the (S)-nitro alcohols. The (S)-4-nitro-butan-2-ol is a chiral precursor of (+)-brefeldin A, a natural product with wide biological applications and in the preparation of (S)-sulcatol [89]. The optically pure (S)-5-nitro-pentan-2-ol is reported to be a chiral precursor for various pheromone preparations. Nakamura et al. studied the biocatalytic reduction of these nitro ketones using baker's yeast to produce the (S)-nitro alcohols with high ee (up to 99%) and yields (up to 66%) [131, 132]. Numerous reports on the biocatalytic reduction of 4-nitro-2-butanone by baker's yeast to produce the (S)-alcohol with high ee (up to 99%) and yields (up to 74%) was also available [133, 134]. Fantin et al. reported the microbial reduction of β- and γ-nitro ketones using different strains of Yarrowia lipolytica, Saccharomyces cerevisiae and Rhizopus sp. among others. Among them, Yarrowia lipolytica selectively produced the (S)enantiomer of 5-nitro-pentan-2-ol (ee: 60-90%; yields: up to ~100%) while different strains of Saccharomyces cerevisiae produced both enantiomers 5-nitro-pentan-2-ol (ee: up to >99%; yields: up to 97%) [135]. The asymmetric reduction of ketopantoyl lactone to pantoyl lactone was studied in a variety of microorganisms. Among them, strains of C. parapsilosis IFO 0708 &

34

0585 and R. minuta IFO 0920 were found to convert ketopantoyl lactone to D-pantoyl lactone with high ee (up to 94%) [136, 137] (Scheme 27).

Scheme 27. Asymmetric reduction of ketopantoyl lactone using the whole cells of C. parapsilosis

Biocatalytic reduction of aromatic imines by resting cells of C. parapsilosis ATCC 7330 gave the corresponding secondary aromatic amines (ee: >99% and yield: up to 80%) [48]. The bioreduction of 2-methyl-1-pyrroline (2-MPN) was carried out with glucose as cosubstrate using Streptomyces sp. GF3587 and 3546 resulted in (R)-2-methylpyrrolidine (R-2-MP) with >99% ee and (S)-2-MP (ee: 92%) respectively in the presence of glucose [138]. β-Carboline imine reduction catalyzed by Saccharomyces bayanus is also reported [139] (Table 6), wherein a reversal in the enantioselectivity from 'S' to 'R' was observed with increasing chain lengths in the substrates.

6. Oxidoreductases from Candida parapsilosis as biocatalysts

In addition to the whole resting cells of C. parapsilosis, oxidoreductases from this yeast have been reportedly used for specific functional group transformations leading to valuable enantiopure molecules (Table 4). For instance, a novel ketopantoyl lactone reducing enzyme was observed in the cell lysate of C. parapsilosis IFO 0708 [136]. In this study under optimal conditions, C. parapsilosis IFO 0708 catalyzed the bioreduction of ketopantoyl lactone to D-(−)pantoyl lactone good yield (~76%) and ee (up to 87%). Another enzyme namely, ketopantoyllactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was isolated and crystallized from C. parapsilosis IFO 0708 cells. This NADPH dependent enzyme catalyzed the bioreduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone [140]. In another study, the cell free extract of C. parapsilosis IFO 0708 35

was employed in the deracemization (stereoinversion) of (RS)-1,2-pentanediol. The stereoinversion proceeded via oxidation of (R)-1,2-pentanediol to 1-hydroxy-2-pentanone, by an NAD+ dependent (R)-specific alcohol dehydrogenase, followed by the reduction of the ketone to (S)-1,2-pentanediol by a NADPH dependent (S)-specific keto-1-alcohol reductase [52]. The above strain produced (S)-l,2-pentanediol (27.9 g/L) from the racemate (30 g/L) in 24 h (molar yield 93%, ee ~100%). Peters et al. reported a novel NADH-dependent carbonyl reductase (135 kDa) from C. parapsilosis DSM 70125 which reduced different carbonyl compounds such as ketoesters, diketones, cyclic ketones, alkyl and aryl ketones and aliphatic aldehydes. Among the substrates studied, ethyl 4-chloro 3-oxobutanoate, halo ketones and aliphatic aldehydes showed higher activity compared to alcohol dehydrogenases from Thermoanaerobium brockii and Pseudomonas sp. The enzyme also showed higher activity towards the oxidation of the (S)isomers of methyl 3-hydroxybutanoate, 2-butanol and phenyl ethanol to produce methyl 3oxobutanoate, 2-butanone and acetophenone respectively. The purified enzyme showed activity in a relatively broad pH (6.5-9) and temperature (36⁰-42 ⁰C) range. In the reduction reactions, the products obtained followed Prelog's rule [141]. The preparative scale bioreduction of methyl 3oxobutanoate using the enzyme coupled with formate dehydrogenase produced the corresponding (S)-hydroxy ester (ee: 99%), a chiral precursor for different pheromones and antibiotics [142].

The carbonyl reductase from C. parapsilosis (CpCR) carried out the asymmetric reduction of various aromatic ketones to produce their corresponding optically pure (S)-alcohols i.e. (S)-phenyl ethanol and (S)-1-(2-naphthyl)-ethanol, (S)-(E)-4-phenyl-3-en-2-ol in excellent ee (>99%) and yields (up to 77%) [143, 144]. It also catalyzed the asymmetric reduction of differently substituted acetylenic ketones to the corresponding propargylic alcohols up to 99% ee [145] (Scheme 28).

36

Scheme 28. CpCR catalyzed the asymmetric reduction of substituted acetylenic ketones

One of the problems of conventional biotransformations is the low solubility of organic substrates in aqueous media. Microemulsions are good alternatives for such less water soluble substrates; but these microemulsions have low stability and activity and are not very practical solutions. The phase behavior of the microemulsion, Marlipal O13-60 and its effect on the activity and stability of the carbonyl reductase from Candida parapsilosis (CpCR) showed that CpCR in the microemulsion had higher activity towards 2-butanone to form optically pure 2butanol (ee: ~100%; conversion: 60%) [146].

Hidalgo et al. isolated and characterized two conjugated polyketone reductases (CPRs) from C. parapsilosis IFO 0708 which showed high sequence similarity with enzymes of the aldo-keto reductase superfamily (AKR). The CPRs preferred ketopantoyl lactone and isatin as substrates compared to various aldehydes and ketones (minadione, p-nitrobenzaldehyde, pyridine-3-aldehyde, and ethyl-4-chloro acetoacetate) [147]. Yamamura et al. reported the crystal structure of the conjugated polyketone reductase C2 (CPR-C2) from C. parapsilosis IFO 0708 which catalyzed the bioreduction of ketopantoyl lactone to D-pantoyl lactone [148]. Matsuyama et al. 2001, characterized the (S)-1,3-BDO dehydrogenase (CpSADH), a novel secondary alcohol dehydrogenase from C. parapsilosis IFO 1396 and overexpressed in E. coli. The CpSADH activity of the recombinant E. coli strain was found to be twice that of whole cells of C. parapsilosis [84].

37

Simon et al. reported the chemoenzymatic preparation of enantiomerically enriched syn 1-aryl-3-methylisochroman derivatives present in various natural products. The bioreduction of different aryl ketones produced 1-aryl-2-propanols (ee: up to >99%) using alcohol dehydrogenases from Candida parapsilosis, Thermoanaerobium sp., Lactobacillus kefir among others. The pure 1-aryl-2-propanols on Oxa-Pictet-Spengler reaction produce the 1-aryl-3methylisochroman derivatives [149]. Different aryl ketones including fluoro-substituted acetophenone were reduced to the corresponding (S)-alcohols [ee: up to >99%; conversion: up to >99%] [150].

A novel short-chain carbonyl reductase (molecular weight: 37.5 kDa) from C. parapsilosis ATCC 28474 with unusual stereoselectivity for the (R)-enantiomer was reported for the biocatalytic reduction of different ketones (alkyl and aromatic ketones) and was found to be selective towards short-chain and medium-chain alkyl ketones. The NADH-dependent carbonyl reductase catalyzed the asymmetric reduction of β-hydroxyacetophenone to produce (R)-PED with ~100% ee [151]. The same authors reported another short chain alcohol dehydrogenase (molecular weight: 31 kDa) from C. parapsilosis CCTCC M203011 catalyzed the reduction of 2-hydroxy acetophenone (HAP) to anti-Prelog (S)-1-phenyl-1,2-ethanediol (PED) (ee: >99% [152] (Scheme 29).

Scheme 29. Asymmetric reduction of 2-hydroxy acetophenone using purified enzymes from different strains of C. parapsilosis

Yang et al. purified a NAD(H) dependent secondary alcohol dehydrogenase (molecular weight: 30 kDa) from C. parapsilosis CCTCC M203011 that catalyzed the asymmetric reduction of α-hydroxy acetophenone (HAP) and ethyl 4-chloro-3-oxobutanoate to form (S)-1-phenyl-1,2ethanediol (PED) (ee: ~100%) and (R)-ethyl 4-chloro-3-hydroxybutanoate (ee: 94%) respectively [153]. Guo et al. reported the isolation and purification of aldoketo reductases from C. 38

parapsilosis CCTCC M203011 for the reduction of various aryl ketones and aliphatic/aryl ketoesters to form optically pure product alcohols (ee: up to >99%) [154]. A novel NADPHdependent (S)- specific carbonyl reductase II (SCR II) was purified from C. parapsilosis to study the reduction of acetophenone and β-hydroxy acetophenone to produce the (R)-phenyl ethanol and (S)-PED with >99 ee. The mutant strains SCR II-A220D and SCR II E228S produced the same products with improved yield up to 80 % for (R)-phenyl ethanol, while a reduced yield was found for (S)-PED [155].

Later, another (S)-specific carbonyl reductase SCRII belonging to the short-chain dehydrogenase/reductase family was purified from C. parapsilosis CCTCC M203011. The catalytic activity of this enzyme was found to be similar to SCR which reduced HAP to (S)-PED (anti-Prelog product) with ~100% ee and 98% yield [156]. There are a number of reductases with anti-Prelog stereo preference but not as many as those reported which follow Prelog's rule. The bioinformatic analysis based on sequence-similarity with an anti-Prelog stereospecific alcohol dehydrogenase from C. parapsilosis reported three homologous carbonyl reductases (SCR1, SCR2 and SCR3) and an alcohol dehydrogenase (CpADH) [157]. These SCRs exhibited higher catalytic activities for producing (S)-PED from HAP with NADPH as the coenzyme. The carbonyl reductase SCRII along with glucose 6-phosphate dehydrogenase was coexpressed in Pichia pastoris. The recombinant enzyme SCRII was used as the catalyst for the enantioselective reduction of HAP to produce (S)-PED (ee: >99%; yield: 96%); the yield is ~20% higher compared to that obtained from E. coli BL21/SCRII. The P. pastoris/SCRIIG cells were recycled up to ten times when used with 50 gL−1 of glucose as co-substrate to give (S)PED in >99% ee and >85% yield respectively [158]. An improvement in the protein expression of (R)-specific carbonyl reductase (RCR) was reported using 6× histidine-tagged RCR clone from C. parapsilosis CCTCC M203011 expressed in Pichia pastoris GS115 under AOX1 (methanol-inducible

promoter).

This

reductase

catalyzed

the

reduction

of

2-

hydroxyacetophenone (HAP) to (R)-PED, an important chiral synthon with broad industrial applications. The RCR purified through one-step Ni2+ affinity chromatography showed a nearly 3-fold improvement in specific activity (1.35 U/mg), and the product (R)-PED (ee: ~95%; yield: 87%) formed shows improved ee and yield by 18% and 43%, respectively as compared to E. coli expressed RCR [159]. 39

Another recombinant short-chain dehydrogenase/reductase enzyme from C. parapsilosis CDC317 (CPE) showed optimal activity at pH 5.5 and 40 oC towards the biocatalytic reduction of COBE to produce (S)- CHBE (ee: > 99%; yield: 91%) [160] (Scheme 30).

Scheme 30. Asymmetric reduction of COBE using purified enzyme from C. parapsilosis CDC317

Recently, an NADH-dependent (R)-carbonyl reductase from C. parapsilosis CCTCC M203011 expressed in E. coli was reported for catalyzing the biocatalytic reduction of 2-HAP to (R)-PED employing 2-propanol (10% v/v) and glycerol (8% v/v) as sacrificial cosubstrates. The (R)-PED product was obtained in high optical purity (>99.9% ee) and ~85% conversion, which were nearly 2- and 11-fold higher than those without adding cosubstrate, respectively. The NADH-dependent (R)-carbonyl reductase from Candida parapsilosis (RCR) was purified, crystallized and diffracted to 2.15 Å resolution in the presence of NAD+ [161].

Another biocatalytic method for the preparation of (S)-PED from (R)-PED using engineered E. coli expressing RCR and SCR was reported by Zhang and coworkers. The study discussed the introduction of pyridine nucleotide transhydrogenases (PNTs) into E. coli RSAB strain which showed an increase in the intracellular NAD+ and NADH concentrations without change in the growth properties of the cells. The presence of these transdehydrogenases rebalanced the cofactor pathway as NAD+ was involved in RCR catalyzed oxidiation of (R)-PED to HAP and SCR (NADPH-dependent) catalyzed the bioreduction of HAP to (S)-PED. The PNT introduced RSAB strain produced (S)-PED in ~97% ee, and ~95% yield in 7-fold lesser reaction time (6 h) [162] . It was also observed that the asymmetric reduction of HAP by (R)-carbonyl reductase (RCR) in the presence of NADH was slow and resulted in low yields which could be due to insufficient regeneration of cofactors. To improve the cofactor recycling pathway in the E. 40

coli system, the enzyme-coupling system with RCR and GDH (glucose dehydrogenase) was designed using Shine-Dalgarno sequence and aligned spacing sequence. The developed system showed excellent catalytic properties such that the reaction time was reduced to half of its initial time to form (R)-PED (ee: >99% & yield: >99%) [163]. In a recent report, an (S)-specific carbonyl reductase from C. parapsilosis DSMZ 70125 (CpCR2) was used to reduce ethyl levulinate to (S)-ethyl-4-hydroxypentanoate (4HPOEt) with high ee (>99%) using 2-propanol as a cosubstrate involving a substrate-coupled cofactor regeneration system [164] (Scheme 31).

Scheme 31. Asymmetric reduction of ethyl levulinate using CpCR2 from C. parapsilosis DSMZ 70125

Ansorge-Schumacher and coworkers studied the structure of (S)-specific carbonyl reductase (CpCR2), a medium-chain reductase from Candida parapsilosis, for the reduction of prochiral ketones. The role of zinc in the catalysis was studied and using saturation mutagenesis insights were reported to improve the catalytic efficiency of CpCR2 [165]. Recently, CpCR2 was used for the enantio-and regio-selective reduction of α-diketones to produce (S)- α-hydroxy ketones (ee: up to 95% and yields up to 47%), which are chiral synthons for antidepressants and fungicides. In this study, the specificity of the enzyme is restricted to carbonyl groups at C-2 position due to its small substrate binding pocket, which accounts for the regioselective reduction of asymmetric diketones [166] (Scheme 32).

Scheme 32. Asymmetric reduction of α-diketones using CpCR2 from C. parapsilosis 41

The structural determinants of the zinc-dependent CpCR2 were analyzed in silico substrate docking and mutational studies. The results indicated that C57A variant showed an increase (27 fold) in specific activity for 4'-acetamidoacetophenone and CPCR2 B16-(C57A, L119A) showed improvement (45 fold) in kcat/Km value, thus highlighting the importance of these residues in the enzyme activity studies [167]. Alcohol dehydrogenase from Candida parapsilosis (CpCR2; EC 1.1.1.1) is a versatile biocatalyst for the asymmetric reduction of several carbonyl substrates. In this study, using quantum mechanical/molecular mechanical (QM/MM) calculations, the mechanism of hydride and proton transfer steps during carbonyl reduction was investigated using zinc-dependent CpCR2 (PDB ID 4C40). Using the x-ray structure, the two different conformers of Glu 66 and catalytic zinc of the enzyme are discussed. The study highlights the movement of zinc ion and glutamine which account for the exchange of ligand and catalytic process of MDRs [168]. Kosjek et al. reported the bioreduction of racemic ,-unsaturated carbonyl substrates using various alcohol dehydrogenases including C. parapsilosis

alcohol

dehydrogenase

to

produce

chiral

allylic

alcohol

with

high

diastereoselectivity and enantioselectivity [169]. In another study, 2-acetyl naphthalene, a lipophilic ketone was reduced to the product alcohol 1-(2-naphthyl)ethanol (ee: >99%, conversion: 97%) with a 147-fold increase in solubility using C. parapsilosis carbonyl reductase in aqueous -cyclodextrins [170] (Scheme 33).

Scheme 33. Asymmetric reduction of 2-acetyl naphthalene using C. parapsilosis carbonyl reductase

The steady state kinetics using stereoselective oxidation of (S)-PED as the model reaction was studied using alcohol dehydrogenase (NAD+ dependent) from C. parapsilosis CCTCC M203011. The study revealed that the oxidation follows Theorell-Chance BiBi mechanism 42

[171]. Later, the kinetic studies for deracemization via stereoinversion were carried out using the enzymes CpADH for oxidation and CpCR for the reduction steps to produce (S)-PED. The kinetic data obtained from the rate experiments proved that oxidation as well as reduction proceeds via Theorell-Chance BiBi mechanism. The kinetic and thermodynamic parameters when compared at varying temperatures and pH values showed that the oxidation reaction was thermodynamically unfavourable and restricted at high concentration of the substrate. This was overcome by using liquid-solid phase (extractive) biocatalytic methods at 50g/L of substrate to form the (S)-alcohol with high ee (~92%) and yield (~83%) [172]. Yamamoto et al. studied the purification and characterization of NAD+ dependent-(S)-1,3-butanediol dehydrogenase from C. parapsilosis IFO 1396, which stereospecifically oxidized the (S)-enantiomer of racemic 1,3butanediol to produce the ketone. In addition, the same enzyme catalyzed the oxidation of several aliphatic secondary alcohols and reduction of aliphatic aldehydes and ketones [173].

7. Genetically engineered carbonyl reductases from Candida parapsilosis as biocatalyst

Apart from the potential advantages like eco-friendly operations, simple work-up, reduced production of waste, the overexpression of carbonyl reductases in E. coli cells has helped in the biocatalytic conversion of ketones that are unstable in the aqueous media. There are numerous reports on the cloning and overexpression of enzymes from C. parapsilosis [16, 151, 174, 175]. Table 4 shows the details of reductase which have been engineered

from C.

parapsilosis. Recently, a recombinant carbonyl reductase (NAD(P)H-dependent) from C. parapsilosis ATCC 7330 was purified, its crystal structure studied using X-ray crystallography and diffracted up to 1.86Å resolution. The enzyme catalyzed the biocatalytic reduction of ethyl 4-phenyl-2-oxobutanoate to its (R)-enantiomer (ee: >99%), a chiral precursor for the synthesis of ACE inhibitors [45] (Scheme 34).

Scheme 34. Asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate using a carbonyl reductase from C. parapsilosis ATCC 7330 43

Zhang et al. reported a short chain carbonyl reductase from C. parapsilosis to catalyze the HAP reduction to (S)-PED. Using site directed mutagenesis, several SCR mutants were designed i.e. Ser67Asp, His68Asp and Pro69Asp in different combinations. All the three positions were located in close proximity of the coenzyme binding pocket. All these SCR mutants showed a shift towards (R)-selectivity in HAP reduction. The S67D/H68D mutant gave (R)-PED (ee: 97%; yield: 85%) using NADH as cofactor. The kinetic studies of this double mutant showed an increase (10 fold) in kcat/Km value in the presence of NADH, while it decreased 20-fold when NADPH was used as the cofactor [175]. Zhang and coworkers studied codon optimization of the CprCR from C. parapsilosis CCTCC M203011 which increased the protein productivity. In the biocatalytic reduction of HAP (substrate concentration of 5g/L) to (R)-PED, the variant showed a high molar conversion/yield (86%) and ee (94%) which is increased by 36% and 16% respectively as compared to the wild type [176]. In biocatalysis, the approach of using a recombinant enzyme is a productive, beneficial, efficient and convenient system to prepare several optically pure alcohols on an industrial scale. For instance, the recombinant E. coli strain expressing CpSADH gave (R)-1, 3-BDO (ee: 93%; yield: 95%) from the racemic alcohol [91] and it also catalyzed the reduction of COBE to (R)-CHBE [yield: 36 g/L, (95% ); ee: 99%] in 17 h [177] (Scheme 35). Under optimized conditions, recombinant E. coli cells expressing CpSADH yielded (R)-1, 3-BDO [ee: 95%; yield: 48% (72 g/L)] from its racemate [178].

Scheme 35. Asymmetric reduction of 1,3-butanediol and ethyl 4-chloro-3-oxobutanoate using recombinant strain expressing CpSADH

44

Asymmetric reduction of acetophenone and 3-butyn-2-one using lyophilized recombinant cells expressing NADH-dependent CpCR produced the corresponding (S)-alcohols (ee: >99%) [179]. Jakoblinnert and coworkers identified the positions of amino acids involved in the recognition of the substrates in carbonyl reductase from Candida parapsilosis (wtCpCR2) and carried out saturation mutagenesis. The asymmetric reduction of 2-methyl cyclohexanone was carried out using the wild type enzyme and the mutants, where the wtCPCR2 predominantly produced the trans (1R, 2R) isomer and the mutant CpCR2-L119M also preferably gave the same product. The difference in selectivity among the wild type and the mutant strain was observed with the cis-products (1R, 2S and 1S, 2R); the mutant strain produced 3-fold higher amount of (1R, 2S) isomer compared to the wild type. The L119M substitution also exhibited improved activity towards bioreduction of substituted (up to > 5-fold) and unsubstituted cyclohexanone (>4-fold) [180]. In order to increase the activity in biphasic reaction media, the carbonyl reductase from C. parapsilosis DSM 70125 (CpCR2) was manipulated genetically. The site-directed and saturation mutagenesis were carried out and the engineered variant CpCR2(A275N, L276Q) exhibited improved activity (1.5-fold), increased stability in the biphasic reaction medium (1.5-fold), and enhanced thermal resistance (T50 = +2.7 oC). This study also revealed that positions 275 and 276, located in the dimer interface proximal to the active site, influences the stability and activity respectively. Site-saturation of positions 275 and 276 resulted in the variant CpCR2-(A275S, L276Q) resulted in increased activity (1.4-fold), improved stability at the interface (1.5-fold) and higher temperature resistance (T50 = +5.2 ◦C) [181]. Nie et al. studied the biocatalytic reduction of (HAP) to (R)-PED (ee: 95%; yield: 93%) in the presence of 2-propanol using recombinant RCR expressed in E. coli under optimized reaction conditions [182] (Scheme 36). In another study, the recombinant E. coli cells expressing a (S)specific carbonyl reductase (SCR) from C. parapsilosis CCTCC M203011 were used for the bioreduction of HAP to produce the anti-Prelog product (S)-PED (ee: >99%) (Scheme 36). The crystal structure was determined which presents insight about the specificity of the coenzyme in SCR and other short-chain dehydrogenases/reductases [174].

45

Scheme 36. Asymmetric reduction of 2-hydroxyacetophenone using carbonyl reductases overexpressed in E. coli

Cell-free extracts of recombinant E. coli overexpressing NADPH-dependent carbonyl reductase SCR1 from C. parapsilosis CCTCC M203011 was used in a biphasic system for the biocatalytic reduction of aryl ketones. The system involves the use of enzymes present in the cell-free extract for the regeneration of NADPH (enzyme-coupled approach). and using this catalytic system, a variety of aryl ketones were reduced to their corresponding enantiopure alcohols with high ee (>99%) and yields (up to 91%) [183]. In addition, it offers high yield compared to whole cells and is easier to handle unlike isolated enzymes.

Conclusions

This review presents the importance of C. parapsilosis as a versatile biocatalyst for deracemization reactions for a variety of sec. alcohols [diols (aliphatic and aromatic), α-, and βhydroxy esters, α -hydroxy propargylic esters, allylic alcohols, aryl ethanols, aliphatic β-hydroxy esters] and asymmetric reduction of prochiral ketones [aryl (ketones, ketoesters and amides) aliphatic (keto esters & nitro ketones), lactones, diketones, oxoaldehydes], other carbonyl compounds and even imines. These biotransformations were carried out with high chemo-, regioand stereospecificity using the whole cells, immobilized cells, partially purified/cell-free extracts, purified enzymes, recombinant and engineered proteins from C. parapsilosis.

In conclusion, this review not only summarizes the biotransformations useful in organic chemistry but also describes (a) several biocatalytic routes which avoid routine organic protection-deprotection steps for the synthesis of multifunctional chiral molecules , (b) synthesis

46

of chiral molecules with high enantiopurity and (c) a scope to further explore the use of the biocatalyst for the synthesis of value added chemicals and pharmaceutical intermediates.

List of abbreviations

Candida sp.: Candida species,

C. parapsilosis: Candida parapsilosis, FDH: Formate

dehydrogenase, PDH: phenylalanine dehydrogenase, ACE: angiotensin-converting enzyme, NEP: neutral endopeptidase, KR: kinetic resolution, BDO: 1,3-butanediol, PED: 1-phenyl-1,2ethanediol, CpSADH: secondary alcohol dehydrogenase from C. parapsilosis, ee: enantiomeric excess, CpCR: carbonyl reductase from C. parapsilosis, AKR: aldo-keto reductase, CPR: conjugated polyketone reductases, CprCR: (R)-specific carbonyl reductase from C. parapsilosis CCTCCM203011, SCR: (S)-specific carbonyl reductase, RCR: (R)-specific carbonyl reductase, COBE: 4-chloro-3-oxobutanoate ethyl ester/ ethyl-4-chloro-3-oxobutanoate, CHBE: (S)-4chloro-3-hydroxybutanoate ethyl ester/ ethyl-4-chloro-3-hydroxybutanoate, PNTs: pyridine nucleotide

transhydrogenases,

HAP:

2-hydroxyacetophenone,

4HPOEt:

(S)-ethyl-4-

hydroxypentanoate, ILs: ionic liquids, TMSB: 4-(trimethylsilyl)-3-butyn-2-one, {(S)-TMSBOL: (S)-4-(trimethylsilyl)-3-butyn-2-ol, hexafluorophosphate,

IL:

ionic

C4MIM·PF6: liquid,

(R)-1-TMSE:

1-butyl-3-methylimidazolium (R)-1-trimethylsilylethanol,

(C2OHMIM.NO3): 1-(2-hydroxyl)ethyl-3-methylimidazolium nitrate.

Acknowledgements

Sowmyalakshmi V acknowledges BRNS, DAE for the fellowship. R. Preetha acknowledges Department of Science and Technology (DST, India) for WOS-A fellowship. S.K. Padhi acknowledges UGC, New Delhi for the Start-Up research grant.

References

[1] J. Tao, G.Q. Lin, A. Liese, Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing, John Wiley & Sons 2009. 47

[2] J. Tao, J.-H. Xu, Biocatalysis in development of green pharmaceutical processes, Curr. Opin. Chem. Biol. 13 (2009) 43-50. [3] D. Gamenara, P. Dominguez de Maria, Candida spp. redox machineries: An ample biocatalytic platform for practical applications and academic insights, Biotechnol. Adv. 27 (2009) 278-285. [4] R. Sharma, Y. Chisti, U.C. Banerjee, Production, purification, characterization, and applications of lipases, Biotechnol. Adv. 19 (2001) 627-662. [5] A. Singh, M. Mukhopadhyay, Overview of Fungal Lipase: A Review, Appl. Biochem. Biotechnol. 166 (2012) 486-520. [6] P. Soni, G. Kaur, A.K. Chakraborti, U.C. Banerjee, Candida viswanathii as a novel biocatalyst for stereoselective reduction of heteroaryl methyl ketones: a highly efficient enantioselective synthesis of (S)-α-(3-pyridyl)ethanol, Tetrahedron: Asymmetry 16 (2005) 24252428. [7] D.J. Pollard, K. Telari, J. Lane, G. Humphrey, C. McWilliams, S. Nidositko, P. Salmon, J. Moore, Asymmetric reduction of α, β-unsaturated ketone to (R) allylic alcohol by Candida chilensis, Biotechnol. Bioeng. 93 (2006) 674-686. [8] P. Soni, H. Kansal, U.C. Banerjee, Purification and characterization of an enantioselective carbonyl reductase from Candida viswanathii MTCC 5158, Process Biochem. 42 (2007) 16321640. [9] H. Kansal, U.C. Banerjee, Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system, Bioresour. Technol. 100 (2009) 1041-1047. [10] R. Patil, L. Banoth, A. Singh, Y. Chisti, U.C. Banerjee, Enantioselective bioreduction of cyclic alkanones by whole cells of Candida Species, Biocatal. Biotransform. 31 (2013) 123-131. [11] M. Hall, B. Hauer, R. Stuermer, W. Kroutil, K. Faber, Asymmetric whole-cell bioreduction of an α,β-unsaturated aldehyde (citral): competing prim-alcohol dehydrogenase and C–C lyase activities, Tetrahedron: Asymmetry 17 (2006) 3058-3062.

48

[12] Y. Chen, H. Lin, X. Xu, S. Xia, L. Wang, Preparation the key Intermediate of angiotensinconverting enzyme (ACE) Inhibitors: High enantioselective production of ethyl (R)-2-hydroxy4-phenylbutyrate with Candida boidinii CIOC21, Adv. Synth. Catal. 350 (2008) 426-430. [13] S. Fukunaga, S. Yuno, M. Takahashi, S. Taguchi, Y. Kera, S. Odani, R.-H. Yamada, Purification and properties of d-glutamate oxidase from Candida boidinii 2201, J. Ferment. Bioeng. 85 (1998) 579-583. [14] D.B. Gonçalves, A.F. Batista, M.Q.R.B. Rodrigues, K.M.V. Nogueira, V.L. Santos, Ethanol production from macaúba (Acrocomia aculeata) presscake hemicellulosic hydrolysate by Candida boidinii UFMG14, Bioresour. Technol. 146 (2013) 261-266. [15] M. Kataoka, A. Hoshino-Hasegawa, R. Thiwthong, N. Higuchi, T. Ishige, S. Shimizu, Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production, Enzyme Microb. Technol. 38 (2006) 944-951. [16] A. Matsuyama, H. Yamamoto, Y. Kobayashi, Practical application of recombinant wholecell biocatalysts for the manufacturing of pharmaceutical intermediates such as chiral alcohols, Org. Process Res. Dev. 6 (2002) 558-561. [17] L.S. Chen, S.M. Mantovani, L.G. de Oliveira, M.C.T. Duarte, A.J. Marsaioli, 1,2Octanediol deracemization by stereoinversion using whole cells, J. Mol. Catal. B: Enzym. 54 (2008) 50-54. [18] H. Tamakawa, S. Ikushima, S. Yoshida, Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain, J. Biosci. Bioeng. 113 (2012) 73-75. [19] P. Soni, U.C. Banerjee, Biotransformations for the production of the chiral drug (S)Duloxetine catalyzed by a novel isolate of Candida tropicalis, Appl. Microbiol. Biotechnol. 67 (2005) 771-777. [20] P. Soni, U.C. Banerjee, Enantioselective reduction of acetophenone and its derivatives with a new yeast isolate Candida tropicalis PBR-2 MTCC 5158, Biotechnol. J 1 (2006) 80-85. [21] M. Sakamoto, M. Hamada, T. Higashi, M. Shoji, T. Sugai, Elaboration on the access to (S)4-(4-methylbenzyl)oxy-3-hydroxybutanenitrile, a key intermediate for statins, by combining the kinetic resolution of racemate and the recycle of undesired enantiomer, J. Mol. Catal. B: Enzym. 64 (2010) 96-100. 49

[22] C. Hiraoka, M. Matsuda, Y. Suzuki, S. Fujieda, M. Tomita, K.-i. Fuhshuku, R. Obata, S. Nishiyama, T. Sugai, Screening, substrate specificity and stereoselectivity of yeast strains, which reduce sterically hindered isopropyl ketones, Tetrahedron: Asymmetry 17 (2006) 3358-3367. [23] M. Wada, H. Kawabata, A. Yoshizumi, M. Kataoka, S. Nakamori, Y. Yasohara, N. Kizaki, J. Hasegawa, S. Shimizu, Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae, J. Biosci. Bioeng. 87 (1999) 144-148. [24] J. Ren, W. Dong, B. Yu, Q. Wu, D. Zhu, Synthesis of optically active α-bromohydrins via reduction of α-bromoacetophenone analogues catalyzed by an isolated carbonyl reductase, Tetrahedron: Asymmetry 23 (2012) 497-500. [25] C. Gruber, S. Krahulec, B. Nidetzky, R. Kratzer, Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: Stereoselectivity, cell activity, in situ substrate supply and product removal, Biotechnol. J. 8 (2013) 699-708. [26] Simone L. Pival, M. Klimacek, B. Nidetzky, The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site, Biochem. J 421 (2009) 43-49. [27] M. Vogl, R. Kratzer, B. Nidetzky, L. Brecker, Candida tenuis xylose reductase catalysed reduction of acetophenones: the effect of ring-substituents on catalytic efficiency, Org. Biomol. Chem. 9 (2011) 5863-5870. [28] M. Vogl, R. Kratzer, B. Nidetzky, L. Brecker, Candida tenuis xylose reductase catalyzed reduction of aryl ketones for enantioselective synthesis of active oxetine derivatives, Chirality 24 (2012) 847-853. [29] B. Nidetzky, K. Brüggler, R. Kratzer, P. Mayr, Multiple forms of xylose reductase in Candida intermedia:  Comparison of their functional properties using quantitative structure−activity relationships, steady-state kinetic analysis, and pH studies, J. Agric. Food. Chem. 51 (2003) 7930-7935. [30] E.J. Tomotani, P.V.d. Arruda, M. Vitolo, M.d.G.d.A. Felipe, Obtaining partial purified xylose reductase from Candida guilliermondii, Braz. J. Microbiol. 40 (2009) 631-635.

50

[31] G.A. Khoury, H. Fazelinia, J.W. Chin, R.J. Pantazes, P.C. Cirino, C.D. Maranas, Computational design of Candida boidinii xylose reductase for altered cofactor specificity, Protein Sci. 18 (2009) 2125-2138. [32] X. Guo, R. Zhang, Z. Li, D. Dai, C. Li, X. Zhou, A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan, Bioresour. Technol. 128 (2013) 547552. [33] I.S.M. Rafiqul, A.M.M. Sakinah, Production of xylose reductase from adapted Candida tropicalis grown in sawdust hydrolysate, Biocatal. Agric. Biotechnol. 3 (2014) 227-235. [34] Y. Bai, S.-T. Yang, Production and separation of formate dehydrogenase from Candida boidinii, Enzyme Microb. Technol. 40 (2007) 940-946. [35] M.H. Kim, S. Park, Y.H. Kim, K. Won, S.H. Lee, Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates, J. Mol. Catal. B: Enzym. 97 (2013) 209-214. [36] C.G.C.M. Netto, M. Nakamura, L.H. Andrade, H.E. Toma, Improving the catalytic activity of formate dehydrogenase from Candida boidinii by using magnetic nanoparticles, J. Mol. Catal. B: Enzym. 84 (2012) 136-143. [37] M. Chartrain, C. Roberge, J. Chung, J. McNamara, D. Zhao, R. Olewinski, G. Hunt, P. Salmon, D. Roush, S. Yamazaki, T. Wang, E. Grabowski, B. Buckland, R. Greasham, Asymmetric bioreduction of (2-(4-nitro-phenyl)-N-(2-oxo-2-pyridin-3-yl-ethyl)-acetamide) to its corresponding (R) alcohol [(R)-N- (2-hydroxy-2-pyridin-3-yl-ethyl)-2-(4-nitro-phenyl)acetamide] by using Candida sorbophila MY 1833, Enzyme Microb. Technol. 25 (1999) 489496. [38] J.Y.L. Chung, G.-J. Ho, M. Chartrain, C. Roberge, D. Zhao, J. Leazer, R. Farr, M. Robbins, K. Emerson, D.J. Mathre, J.M. McNamara, D.L. Hughes, E.J.J. Grabowski, P.J. Reider, Practical chemoenzymatic synthesis of a 3-pyridylethanolamino β3 adrenergic receptor agonist, Tetrahedron Lett. 40 (1999) 6739-6743. [39] R.L. Hanson, J.M. Howell, T.L. LaPorte, M.J. Donovan, D.L. Cazzulino, V. Zannella, M.A. Montana, V.B. Nanduri, S.R. Schwarz, R.F. Eiring, S.C. Durand, J.M. Wasylyk, W.L. Parker, M.S. Liu, F.J. Okuniewicz, B.-C. Chen, J.C. Harris, K.J. Natalie Jr, K. Ramig, S. Swaminathan, V.W. Rosso, S.K. Pack, B.T. Lotz, P.J. Bernot, A. Rusowicz, D.A. Lust, K.S. Tse, J.J. Venit, L.J. 51

Szarka, R.N. Patel, Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius, Enzyme Microb.Technol. 26 (2000) 348-358. [40] R.N. Patel, Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug, Biomol. Eng 17 (2001) 167-182. [41] Y.-C. He, Z.-C. Tao, X. Zhang, Z.-X. Yang, J.-H. Xu, Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH-dependent reductase from E. coli CCZU-K14, Bioresour. Technol. 161 (2014) 461-464. [42] M.-K. Kwak, M. Ku, S.-O. Kang, NAD+-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans, FEBS Lett. 588 (2014) 1144-1153. [43] T. Johanson, N.S. Parachin, P. Adamis, M.-F. Gorwa-Grauslund, Identification of a Candida sp. reductase behind bicyclic exo-alcohol production, J. Mol. Catal. B: Enzym. 59 (2009) 286-291. [44] M. Kataoka, A. Kotaka, R. Thiwthong, M. Wada, S. Nakamori, S. Shimizu, Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound, J. Biotechnol. 114 (2004) 1-9. [45] N. Aggarwal, P.K. Mandal, N. Gautham, A. Chadha, Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330, Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 69 (2013) 313-315. [46] Q. Hu, Y. Xu, Y. Nie, Enhancement of Candida parapsilosis catalyzing deracemization of (R,S)-1-phenyl-1,2-ethanediol to its (S)-enantiomer by a highly productive "two-in-one" resinbased in situ product removal strategy, Bioresour. Technol. 101 (2010) 8461-8463. [47] A. Matsuyama, Y. Kobayashi, H. Ohnishi, Microbial production of optically active 1,3butanediol from the racemate, Biosci. Biotechnol. Biochem. 57 (1993) 685-6. [48] T. Vaijayanthi, A. Chadha, Asymmetric reduction of aryl imines using Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry 19 (2008) 93-96. [49] T. Sivakumari, A. Chadha, Candida parapsilosis ATCC 7330 mediated oxidation of aromatic (activated) primary alcohols to aldehydes, RSC Adv. 5 (2015) 91594-91600. 52

[50] T. Sivakumari, R. Preetha, A. Chadha, Enantioselective oxidation of secondary alcohols by Candida parapsilosis ATCC 7330, RSC Adv. 4 (2014) 2257-2262. [51] T. Sivakumari, A. Chadha, Regio- and enantio-selective oxidation of diols by Candida parapsilosis ATCC 7330, RSC Adv. 4 (2014) 60526-60533. [52] J. Hasegawa, M. Ogura, S. Tsuda, S.-i. Maemoto, H. Kutsuki, T. Ohashi, High-yield production of optically active 1,2-diols from the corresponding racemates by microbial stereoinversion Agric. Biol. Chem. 54 (1990) 1819-1827. [53] A. Goswami, K.D. Mirfakhrae, R.N. Patel, Deracemization of racemic 1,2-diol by biocatalytic stereoinversion, Tetrahedron: Asymmetry 10 (1999) 4239-4244. [54] Y. Nie, Y. Xu, X.Q. Mu, Highly enantioselective conversion of racemic 1-phenyl-1,2ethanediol by stereoinversion involving a novel cofactor-dependent oxidoreduction system of Candida parapsilosis CCTCC M203011, Org. Process Res. Dev. 8 (2004) 246-251. [55] Y. Nie, Y. Xu, Q.S. Hu, R. Xiao, A new strategy to improve the efficiency and sustainability of Candida parapsilosis catalyzing deracemization of (R,S)-1-phenyl-1,2ethanediol under non-growing conditions: increase of NADPH availability, J. Microbiol. Biotechnol. 19 (2009) 65-71. [56] Y. Nie, Y. Xu, T.F. Lv, R. Xiao, Enhancement of Candida parapsilosis catalyzing deracemization of (R,S)-1-phenyl-1,2-ethanediol: agitation speed control during cell cultivation, J. Chem. Technol. Biotechnol. 84 (2009) 468-472. [57] M.-w. Xu, X.-q. Mu, Y. Xu, Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system, Bioprocess Biosyst. Eng. 33 (2010) 367-373. [58] S.M. Amrutkar, L. Banoth, U.C. Banerjee, One-pot synthesis of (R)-1-(1-naphthyl)ethanol by stereoinversion using Candida parapsilosis, Tetrahedron Lett. 54 (2013) 3274-3277. [59] T. Kaliaperumal, S.N. Gummadi, A. Chadha, Candida parapsilosis ATCC 7330 can also deracemise 1-arylethanols, Biocatal. Biotransform. 29 (2011) 262-270.

53

[60] W. Adam, K. Peters, E.M. Peters, V.R. Stegmann, Hydroxy-Directed Regio- and Diastereoselective [2+2] Photocycloaddition (Paternò−Büchi Reaction) of Benzophenone to Chiral Allylic Alcohols, J. Am. Chem. Soc. 122 (2000) 2958-2959. [61] D. Titu, A. Chadha, Enantiomerically pure allylic alcohols: preparation by Candida parapsilosis ATCC 7330 mediated deracemisation, Tetrahedron: Asymmetry 19 (2008) 16981701. [62] T. Saravanan, S. Jana, A. Chadha, Utilization of whole cell mediated deracemization in a chemoenzymatic synthesis of enantiomerically enriched polycyclic chromeno[4,3-b] pyrrolidines, Org. Biomol. Chem. 12 (2014) 4682-4690. [63] A. Chadha, B. Baskar, Biocatalytic deracemisation of α-hydroxy esters: high yield preparation of (S)-ethyl 2-hydroxy-4-phenylbutanoate from the racemate, Tetrahedron: Asymmetry 13 (2002) 1461-1464. [64] B. Baskar, N.G. Pandian, K. Priya, A. Chadha, Deracemisation of aryl substituted αhydroxy esters using Candida parapsilosis ATCC 7330: Effect of substrate structure and mechanism, Tetrahedron 61 (2005) 12296-12306. [65] R. Wohlgemuth, Development of Sustainable Biocatalytic Reduction Processes for Organic Chemists, Synthetic Methods for Biologically Active Molecules, Wiley-VCH Verlag GmbH & Co. KGaA2013, pp. 1-25. [66] V. Thangavel, A. Chadha, Preparation of optically pure (3E,5E)-alkyl-2-hydroxy-6arylhexa-3,5-dienoates by Candida parapsilosis ATCC 7330 mediated deracemisation of the racemates, Tetrahedron 63 (2007) 4126-4133. [67] T. Saravanan, A. Chadha, Biocatalytic deracemization of alkyl-2-hydroxy-4-arylbut-3ynoates using whole cells of Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry 21 (2010) 2973-2980. [68] T. Vaijayanthi, A. Chadha, Preparation of enantiomerically pure (3E)-alkyl-4-(hetero-2-yl)2-hydroxybut-3-enoates by Candida parapsilosis ATCC 7330 mediated deracemisation and determination of the absolute configuration of (3E)-ethyl-4-(thiophene-2-yl)-2-hydroxybut-3enoate, Tetrahedron: Asymmetry 18 (2007) 1077-1084.

54

[69] S.K. Padhi, N.G. Pandian, A. Chadha, Microbial deracemization of aromatic β-hydroxy acid esters, J. Mol. Catal. B: Enzym. 29 (2004) 25-29. [70] V. Ratovelomanana-Vidal, C. Girard, R. Touati, J.P. Tranchier, B.B. Hassine, J.P. Genêt, Enantioselective hydrogenation of β-keto esters using chiral diphosphine-ruthenium complexes: Optimization for academic and industrial purposes and synthetic applications, Adv. Synth. Catal. 345 (2003) 261-274. [71] P.J. Jervis, B.M. Kariuki, L.R. Cox, Stereoselective synthesis of 2,4,5-trisubstituted tetrahydropyrans using an intramolecular allylation strategy, Org. Lett. 8 (2006) 4649-4652. [72] D. Titu, A. Chadha, Preparation of optically pure alkyl 3-(hetero-2-yl)-3hydroxypropanoates by Candida parapsilosis ATCC 7330 mediated deracemisation, J. Mol. Catal. B: Enzym. 52–53 (2008) 168-172. [73] T. Saravanan, R. Selvakumar, M. Doble, A. Chadha, Stereochemical preference of Candida parapsilosis ATCC 7330 mediated deracemization: E- versus Z-aryl secondary alcohols, Tetrahedron: Asymmetry 23 (2012) 1360-1368. [74] S. Venkataraman, A. Chadha, Biocatalytic deracemization of aliphatic β-hydroxy esters: Improving the enantioselectivity by optimization of reaction parameters, J. Ind. Microbiol. Biotechnol. 42 (2015) 173-180. [75] S. Venkataraman, A. Chadha, Preparation of enantiomerically enriched (S)-ethyl 3-hydroxy 4,4,4-trifluorobutanoate using whole cells of Candida parapsilosis ATCC 7330, J. Fluorine Chem. 169 (2015) 66-71. [76] T. Matsuda, R. Yamanaka, K. Nakamura, Recent progress in biocatalysis for asymmetric oxidation and reduction, Tetrahedron: Asymmetry 20 (2009) 513-557. [77] K. Nakamura, R. Yamanaka, T. Matsuda, T. Harada, Recent developments in asymmetric reduction of ketones with biocatalysts, Tetrahedron: Asymmetry 14 (2003) 2659-2681. [78] P. Mahajabeen, A. Chadha, One-pot synthesis of enantiomerically pure 1,2-diols: Asymmetric reduction of aromatic α-oxo aldehydes catalyzed by Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry 22 (2011) 2156-2160.

55

[79] B. Baskar, N.G. Pandian, K. Priya, A. Chadha, Asymmetric reduction of alkyl 2-oxo-4arylbutanoates and -but-3-enoates by Candida parapsilosis ATCC 7330: assignment of the absolute configuration of ethyl 2-hydroxy-4-(p-methylphenyl)but-3-enoate by 1H NMR, Tetrahedron: Asymmetry 15 (2004) 3961-3966. [80] T. LÜ, Y. Xu, X. Mu, Y. Nie, Promotion effect of xylose co-substrate on stability of catalytic system for asymmetric redox of (R,S)-1- phenyl-1,2-ethanediol to Its (S)-enantiomer by Candida parapsilosis, Chin. J. Catal. 28 (2007) 446-450. [81] T. Mahapatra, N. Jana, S. Nanda, Stereoselective desymmetrization of 2,2bishydroxymethyl-1-tetralones by iodocyclization, synthesis of novel enantiopure [6.6.5] tricyclic framework and chemoenzymatic diversity generation, Adv. Synth. Catal. 353 (2011) 2152-2168. [82] R. Bhuniya, S. Nanda, Enantiomeric scaffolding of -tetralone and related scaffolds by EKR (Enzymatic Kinetic Resolution) and stereoselective ketoreduction with ketoreductases, Org. Biomol. Chem. 10 (2012) 536-547. [83] P. Mahajabeen, A. Chadha, Regio- and enantioselective reduction of diketones: preparation of enantiomerically pure hydroxy ketones catalysed by Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry 26 (2015) 1167-1173. [84] A. Matsuyama, H. Yamamoto, N. Kawada, Y. Kobayashi, Industrial production of (R)-1,3butanediol by new biocatalysts, J. Mol. Catal. B: Enzym. 11 (2001) 513-521. [85] J. Peters, T. Zelinski, M.-R. Kula, Studies on the distribution and regulation of microbial keto ester reductases, Appl. Microbiol. Biotechnol. 38 (992) 334-340. [86] T. Kaliaperumal, S.N. Gummadi, A. Chadha, Synthesis of both enantiomers of ethyl-4chloro-3-hydroxbutanoate from a prochiral ketone using Candida parapsilosis ATCC 7330, Tetrahedron: Asymmetry 22 (2011) 1548-1552. [87] T. Kaliaperumal, S. Kumar, S. Gummadi, A. Chadha, Asymmetric synthesis of (S)-ethyl-4chloro-3-hydroxybutanoate using Candida parapsilosis ATCC 7330, J. Ind. Microbiol. Biotechnol. 37 (2010) 159-165.

56

[88] S. Venkataraman, R.K. Roy, A. Chadha, Asymmetric reduction of alkyl-3-oxobutanoates by Candida parapsilosis ATCC 7330: Insights into solvent and substrate optimisation of the biocatalytic reaction, Appl. Biochem. Biotechnol. 171 (2013) 756-770. [89] S. Venkataraman, A. Chadha, Enantio- & chemo-selective preparation of enantiomerically enriched aliphatic nitro alcohols using Candida parapsilosis ATCC 7330, RSC Adv. 5 (2015) 73807-73813. [90] S. Stella, A. Chadha, Biocatalytic reduction of α-keto amides to (R)-α-hydroxy amides using Candida parapsilosis ATCC 7330, Catal. Today 198 (2012) 345-352. [91] H. Yamamoto, N. Kawada, A. Matsuyama, Y. Kobayashi, Cloning and expression in Escherichia coli of a gene coding for a secondary alcohol dehydrogenase from Candida parapsilosis, Biosci. Biotechnol. Biochem. 63 (1999) 1051-1055. [92] H. Wu, Y. Liang, J. Shi, X. Wang, D. Yang, Z. Jiang, Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres, Mater. Sci. Eng. C 33 (2013) 1438-1445. [93] J.M. Guisan, Immobilization of Enzymes and Cells, 2 ed., Humana Press2006. [94] E. Battistel, D. Bianchi, P. Cesti, C. Pina, Enzymatic resolution of (S)-(+)-Naproxen in a continuous reactor, Biotechnol. Bioeng. 38 (1991) 659-664. [95] S.M. Thomas, R. DiCosimo, V. Nagarajan, Biocatalysis: applications and potentials for the chemical industry, Trends Biotechnol. 20 (2002) 238-242. [96] M.B. Cassidy, H. Lee, J.T. Trevors, Environmental applications of immobilized microbial cells: a review, J. Ind. Microbiol. 16 (1996) 79-101. [97] S.K. Padhi, D. Titu, N.G. Pandian, A. Chadha, Deracemisation of β-hydroxy esters using immobilised whole cells of Candida parapsilosis ATCC 7330: Substrate specificity and mechanistic investigation, Tetrahedron 62 (2006) 5133-5140. [98] S.K. Padhi, A. Chadha, Deracemization of aromatic β-hydroxy esters using immobilized whole cells of Candida parapsilosis ATCC 7330 and determination of absolute configuration by 1 H NMR, Tetrahedron: Asymmetry 16 (2005) 2790-2798.

57

[99] B.-B. Zhang, W.-Y. Lou, M.-H. Zong, H. Wu, Efficient synthesis of enantiopure (S)-4(trimethylsilyl)-3-butyn-2-ol via asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one with immobilized Candida parapsilosis CCTCC M203011 cells, J. Mol. Catal. B: Enzym. 54 (2008) 122-129. [100] W.-Y. Lou, L. Chen, B.-B. Zhang, T.J. Smith, M.-H. Zong, Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells, BMC Biotechnol. 9 (2009) 90. [101] B.-B. Zhang, J. Cheng, W.-Y. Lou, P. Wang, M.-H. Zong, Efficient anti-Prelog enantioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based biphasic systems, Microb. Cell Fact. 11 (2012) 108. [102] B.-B. Zhang, W.-Y. Lou, W.-J. Chen, M.-H. Zong, Efficient asymmetric reduction of 4(trimethylsilyl)-3-butyn-2-one by Candida parapsilosis cells in an ionic liquid-containing system, PLoS One 7 (2012) e37641. [103] X.Q. Mu, Y. Xu, Y. Nie, J. Ouyang, Z.H. Sun, Candida parapsilosis CCTCC M203011 and the optimization of fermentation medium for stereoinversion of (S)-1-phenyl-1,2-ethanediol, Process Biochem. 40 (2005) 2345-2350. [104] K. Nakamura, M. Fujii, Y. Ida, Stereoinversion of arylethanols by Geotrichum candidum, Tetrahedron: Asymmetry 12 (2001) 3147-3153. [105] X. Ju, H.-L. Yu, J. Pan, D.-Z. Wei, J.-H. Xu, Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011, Appl. Microbiol. Biotechnol. 86 (2010) 83-91. [106] N.W. Fadnavis, S.K. Vadivel, M. Sharfuddin, Chemoenzymatic synthesis of (4S,5R)-5hydroxy-γ-decalactone, Tetrahedron: Asymmetry 10 (1999) 3675-3680. [107] C.C. Gruber, I. Lavandera, K. Faber, W. Kroutil, From a racemate to a single enantiomer. Deracemization by stereoinversion, Adv. Synth. Catal. 348 (2006) 1789-1805. [108] C.V. Voss, C.C. Gruber, W. Kroutil, Deracemization of secondary alcohols through a concurrent tandem biocatalytic oxidation and reduction, Angew. Chem. Int. Ed. 47 (2008) 741745. 58

[109] C.V. Voss, C.C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil, Orchestration of concurrent oxidation and reduction cycles for stereoinversion and deracemisation of secalcohols, J. Am. Chem. Soc. 130 (2008) 13969-13972. [110] M. Amidjojo, D. Weuster-Botz, Asymmetric synthesis of the chiral synthon ethyl (S)-4chloro-3-hydroxybutanoate using Lactobacillus kefir, Tetrahedron: Asymmetry 16 (2005) 899901. [111] B.N. Zhou, A.S. Gopalan, F. VanMiddlesworth, W.R. Shieh, C.J. Sih, Stereochemical control of yeast reductions. 1. Asymmetric synthesis of L-carnitine, J. Am. Chem. Soc. 105 (1983) 5925-5926. [112] A.C. Dahl, M. Fjeldberg, J.Ø. Madsen, Baker's yeast: Improving the D-stereoselectivity in reduction of 3-oxo esters, Tetrahedron: Asymmetry 10 (1999) 551-559. [113] R.N. Patel, C.G. McNamee, A. Banerjee, J.M. Howell, R.S. Robison, L.J. Szarka, Stereoselective reduction of β-keto esters by Geotrichum candidum, Enzyme Microb. Technol. 14 (1992) 731-738. [114] J.S. Yadav, S. Nanda, P.T. Reddy, A.B. Rao, Efficient enantioselective reduction of ketones with Daucus carota root, J. Org. Chem. 67 (2002) 3900-3903. [115] Z.-H. Yang, R. Zeng, G. Yang, Y. Wang, L.-Z. Li, Z.-S. Lv, M. Yao, B. Lai, Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue, J. Ind. Microbiol. Biotechnol. 35 (2008) 1047-1051. [116] J.-Y. He, Z.-H. Sun, W.-Q. Ruan, Y. Xu, Biocatalytic synthesis of ethyl (S)-4-chloro-3hydroxy-butanoate in an aqueous-organic solvent biphasic system using Aureobasidium pullulans CGMCC 1244, Process Biochem. 41 (2006) 244-249. [117] W. Zhang, Y. Ni, Z. Sun, P. Zheng, W. Lin, P. Zhu, N. Ju, Biocatalytic synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate with Candida krusei SW2026: A practical process for high enantiopurity and product titer, Process Biochem. 44 (2009) 1270-1275. [118] B. Baskar, S. Ganesh, T.S. Lokeswari, A. Chadha, Highly stereoselective reduction of 4aryl-2-oxo but-3-enoic carboxylic esters by plant cell culture of Daucus carota, J. Mol. Catal. B: Enzym. 27 (2004) 13-17.

59

[119] K. Ishihara, K. Iwai, H. Yamaguchi, N. Nakajima, K. Nakamura, T. Ohshima, Stereoselective reduction of α- and β-keto esters with aerobic thermophiles, Bacillus strains, Biosci. Biotechnol. Biochem. 60 (1996) 1896-1898. [120] K. Ishihara, H. Nagai, K. Takahashi, M. Nishiyama, N. Nakajima, Stereoselective reduction of α-keto ester and α-keto amide with marine actinomycetes, Salinispora strains, as novel biocatalysts, Biochem. Insights 4 (2011) 29-33. [121] K. Ishihara, N. Nakajima, Stereoselective reduction of carbonyl compounds using the cellfree extract of the earthworm, Lumbricus rubellus, as a novel source of biocatalyst, Biosci. Biotechnol. Biochem. 70 (2006) 3077-3080. [122] K. Ishihara, N. Nakajima, H. Yamaguchi, H. Hamada, Y.s. Uchimura, Stereoselective reduction of keto esters with marine microalgae, J. Mol. Catal. B: Enzym. 15 (2001) 101-104. [123] K. Ishihara, H. Yamaguchi, N. Adachi, H. Hamada, N. Nakajima, Stereocontrolled Reduction of  and & -Keto Esters with Micro Green Algae, Chlorella Strains, Biosci. Biotechnol. Biochem. 64 (2000) 2099-2103. [124] K. Ishihara, H. Yamaguchi, H. Hamada, N. Nakajima, K. Nakamura, Stereocontrolled reduction of α-keto esters with thermophilic actinomycete, Streptomyces thermocyaneoviolaceus IFO 14271, J. Mol. Catal. B: Enzym. 10 (2000) 429-434. [125] P.D. Gennaro, S. Bernasconi, F. Orsini, E. Corretto, G. Sello, Multienzymatic preparation of 3-[(1R)-1-hydroxyethyl]benzoic acid and (2S)-hydroxy(phenyl)ethanoic acid, Tetrahedron: Asymmetry 21 (2010) 1885-1889. [126] J.D. Carballeira, E. Álvarez, M. Campillo, L. Pardo, J.V. Sinisterra, Diplogelasinospora grovesii IMI 171018, a new whole cell biocatalyst for the stereoselective reduction of ketones, Tetrahedron: Asymmetry 15 (2004) 951-962. [127] C.P. Mangone, E. N.Pereyra, S. Argimón, S. Moreno, A. Baldessari, Chemo- and stereoselective reduction of β-keto esters by spores and various morphological forms of Mucor rouxii, Enzyme Microb. Technol. 30 (2002) 596-601. [128] N. Mochizuki, T. Sugai, H. Ohta, Biochemical reduction of 3-oxoalkanoic esters by a bottom-fermentation yeast, Saccharomyces cerevisiae IFO 0565, Biosci. Biotechnol. Biochem. 58 (1994) 1666-70. 60

[129] N.A. Salvi, S. Chattopadhyay, Rhizopus arrhizus mediated asymmetric reduction of alkyl 3-oxobutanoates, Tetrahedron: Asymmetry 15 (2004) 3397-3400. [130] S.R. Wallner, I. Lavandera, S.F. Mayer, R. Öhrlein, A. Hafner, K. Edegger, K. Faber, W. Kroutil, Stereoselective anti-Prelog reduction of ketones by whole cells of Comamonas testosteroni in a ‘substrate-coupled’ approach, J. Mol. Catal. B: Enzym. 55 (2008) 126-129. [131] K. Nakamura, Y. Inoue, J. Shibahara, S. Oka, A. Ohno, Asymmetric reduction of β- and γnitro ketones by bakers' yeast, Tetrahedron Lett. 29 (1988) 4769-4770. [132] K. Nakamura, Y. Inoue, J. Shibahara, A. Ohno, Stereochemical control in microbial reduction. 11. Enantioselective reduction of β- and γ-nitroketones with bakers' yeast, Bull. Inst. Chem. Res. Kyoto Univ. 67 (1989) 99-106. [133] T. Hafner, H.U. Reissig, (S)-5-Nitro-2-pentanol by reduction with baker's yeast, Liebigs Ann. Chem. (1989) 937-8. [134] A. Guarna, E.G. Occhiato, L.M. Spinetti, M.E. Vallecchi, D. Scarpi, Baker's yeast reduction of prochiral γ-nitro ketones: enantioselective synthesis of (S)-4-nitro alcohols, Tetrahedron 51 (1995) 1775-88. [135] G. Fantin, M. Fogagnolo, M.E. Guerzoni, E. Marotta, A. Medici, P. Pedrini, Microbial and enzymic approaches to chiral β- and γ-nitro alcohols, Tetrahedron: Asymmetry 3 (1992) 947-52. [136] S. Shimizu, H. Hata, H. Yamada, Reduction of ketopantoyl lactone to D-(-)-pantoyl lactone by microorganisms, Agric. Biol. Chem. 48 (1984) 2285-2291. [137] H. Hata, S. Shimizu, H. Yamada, Enzymic production of D-(-)-pantoyl lactone from ketopantoyl lactone, Agric. Biol. Chem. 51 (1987) 3011-16. [138] K. Mitsukura, M. Suzuki, K. Tada, T. Yoshida, T. Nagasawa, Asymmetric synthesis of chiral cyclic amine from cyclic imine by bacterial whole-cell catalyst of enantioselective imine reductase, Org. Biomol. Chem. 8 (2010) 4533-4535. [139] M. Espinoza-Moraga, T. Petta, M. Vasquez-Vasquez, V.F. Laurie, L.A.B. Moraes, L.S. Santos, Bioreduction of β-carboline imines to amines employing Saccharomyces bayanus, Tetrahedron: Asymmetry 21 (2010) 1988-1992.

61

[140] H. Hata, S. Shimizu, S. Hattori, H. Yamada, Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase, BBA - Gen. Subjects 990 (1989) 175-181. [141] V. Prelog, Specification of the stereospecificity of some oxidoreductases by diamond lattice sections, Pure Appl. Chem. 9 (1964) 119-30. [142] J. Peters, T. Minuth, M.-R. Kula, A novel NADH-dependent carbonyl reductase with an extremely broad substrate range from Candida parapsilosis: Purification and characterization, Enzyme Microb. Technol. 15 (1993) 950-958. [143] T. Zelinski, A. Liese, C. Wandrey, M.-R. Kula, Asymmetric reductions in aqueous media: enzymatic synthesis in cyclodextrin containing buffers, Tetrahedron: Asymmetry 10 (1999) 1681-1687. [144] S. Rissom, J. Beliczey, G. Giffels, U. Kragl, C. Wandrey, Asymmetric reduction of acetophenone in membrane reactors: comparison of oxazaborolidine and alcohol dehydrogenase catalysed processes, Tetrahedron: Asymmetry 10 (1999) 923-928. [145] T. Schubert, W. Hummel, M.-R. Kula, M. Müller, Enantioselective synthesis of both enantiomers of various propargylic alcohols by use of two oxidoreductases, Eur. J. Org. Chem. 2001 (2001) 4181-4187. [146] B. Orlich, H. Berger, M. Lade, R. Schomacker, Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration, Biotechnol. Bioeng. 70 (2000) 638-46. [147] A.-R.G.D. Hidalgo, M.A. Akond, K. Kita, M. Kataoka, S. Shimizu, Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis, Biosci., Biotechnol., Biochem. 65 (2001) 2785-2788. [148] A. Yamamura, S. Maruoka, J. Ohtsuka, T. Miyakawa, K. Nagata, M. Kataoka, N. Kitamura, S. Shimizu, M. Tanokura, Expression, purification, crystallization and preliminary Xray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, Acta Cryst. F 65 (2009) 1145-1148.

62

[149] R.C. Simon, E. Busto, N. Richter, F. Belaj, W. Kroutil, Chemoenzymatic synthesis of enantiomerically pure syn-configured 1-aryl-3-methylisochroman derivatives, Eur. J. Org. Chem. 2014 (2014) 111-121. [150] S. Broussy, R.W. Cheloha, D.B. Berkowitz, Enantioselective, ketoreductase-based entry into pharmaceutical building blocks: Ethanol as tunable nicotinamide reductant, Org. Lett. 11 (2009) 305-308. [151] Y. Nie, Y. Xu, M. Yang, X.Q. Mu, A novel NADH-dependent carbonyl reductase with unusual stereoselectivity for (R)-specific reduction from an (S)-1-phenyl-1,2-ethanediolproducing micro-organism: purification and characterization, Lett. Appl. Microbiol. 44 (2007) 555-562. [152] Y. Nie, Y. Xu, X.Q. Mu, H.Y. Wang, M. Yang, R. Xiao, Purification, characterization, gene cloning, and expression of a novel alcohol dehydrogenase with anti-Prelog stereospecificity from Candida parapsilosis, Appl. Environ. Microbiol. 73 (2007) 3759-3764. [153] M. Yang, Y. Xu, X. Mu, R. Xiao, Purification and characterization of a novel carbonyl reductase with high stereo-selectivity, Front. Chem. Eng. China 1 (2007) 404-410. [154] R. Guo, Y. Nie, X.Q. Mu, Y. Xu, R. Xiao, Genomic mining-based identification of novel stereospecific aldo-keto reductases toolbox from Candida parapsilosis for highly enantioselective reduction of carbonyl compounds, J. Mol. Catal. B: Enzym. 105 (2014) 66-73. [155] R. Zhang, B. Zhang, Y. Xu, Y. Li, M. Li, H. Liang, R. Xiao, Efficient (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration, PLoS One 8 (2013) e83586/1-e83586/10, 10 pp. [156] R. Zhang, Y. Geng, Y. Xu, W. Zhang, S. Wang, R. Xiao, Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity, Bioresour. Technol. 102 (2011) 483-489. [157] Y. Nie, R. Xiao, Y. Xu, G.T. Montelione, Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones, Org. Biomol. Chem. 9 (2011) 4070-4078.

63

[158] Y. Geng, R. Zhang, Y. Xu, S. Wang, C. Sha, R. Xiao, Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol, Biocatal. Biotransform. 29 (2011) 172-178. [159] R. Zhang, Y. Xu, R. Xiao, S. Wang, B. Zhang, Improved production of (R)-1-phenyl-1,2ethanediol using Candida parapsilosis (R)-carbonyl reductase expressed in Pichia pastoris, Process Biochem. 46 (2011) 709-713. [160] Q. Wang, L. Shen, T. Ye, D. Cao, R. Chen, X. Pei, T. Xie, Y. Li, W. Gong, X. Yin, Overexpression and characterization of a novel (S)-specific extended short-chain dehydrogenase/reductase from Candida parapsilosis, Bioresour. Technol. 123 (2012) 690-694. [161] S. Wang, Y. Nie, X. Yan, T.-P. Ko, C.-H. Huang, H.-C. Chan, R.-T. Guo, R. Xiao, Crystallization and preliminary X-ray diffraction analysis of (R)-carbonyl reductase from Candida parapsilosis, Acta Cryst. F 70 (2014) 800-2. [162] R. Zhang, Y. Xu, R. Xiao, B. Zhang, L. Wang, Efficient one-step production of (S)-1phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli, Microb. Cell Fact. 11 (2012) 167-167. [163] X. Zhou, R. Zhang, Y. Xu, H. Liang, J. Jiang, R. Xiao, Coupled (R)-carbonyl reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical selectivity, Process Biochem. 50 (2015) 1807-1813. [164] K. Goetz, A. Liese, M. Ansorge-Schumacher, L. Hilterhaus, A chemo-enzymatic route to synthesize (S)-γ-valerolactone from levulinic acid, Appl. Microbiol. Biotechnol. 97 (2013) 38653873. [165] H. Man, C. Loderer, M.B. Ansorge-Schumacher, G. Grogan, Structure of NADHdependent carbonyl reductase (CPCR2) from Candida parapsilosis provides insight into mutations that improve catalytic properties, ChemCatChem 6 (2014) 1103-1111. [166] C. Loderer, M.B. Ansorge-Schumacher, Enzyme-catalysed regio- and enantioselective preparative scale synthesis of (S)-2-hydroxy alkanones, RSC Adv. 5 (2015) 38271-38276. [167] C. Loderer, G.V. Dhoke, M.D. Davari, W. Kroutil, U. Schwaneberg, M. Bocola, M.B. Ansorge-Schumacher, Investigation of structural determinants for the substrate specificity in the

64

zinc-dependent alcohol dehydrogenase CPCR2 from Candida parapsilosis, ChemBioChem 16 (2015) 1512-1519. [168] G.V. Dhoke, M.D. Davari, U. Schwaneberg, M. Bocola, QM/MM calculations revealing the resting and catalytic states in zinc-dependent medium-chain dehydrogenases/reductases, ACS Catal. 5 (2015) 3207-3215. [169] B. Kosjek, D.M. Tellers, M. Biba, R. Farr, J.C. Moore, Biocatalytic and chemocatalytic approaches to the highly stereoselective 1,2-reduction of an α,β-unsaturated ketone, Tetrahedron: Asymmetry 17 (2006) 2798-2803. [170] T. Zelinski, M.-R. Kula, Asymmetric enzymic reduction of lipophilic ketones in aqueous solution containing cyclodextrins, Biocatal. Biotransform. 15 (1997) 57-74. [171] X.Q. Mu, Y. Xu, M. Yang, Z.H. Sun, Steady-state kinetics of the oxidation of (S)-1phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011, J. Mol. Catal. B: Enzym. 43 (2006) 23-28. [172] X.Q. Mu, Y. Xu, M. Yang, Z.H. Sun, New kinetic and thermodynamic approach of onepot synthesis of chiral alcohol by oxidoreductase from Candida parapsilosis, Process Biochem. 46 (2011) 233-239. [173] H. Yamamoto, A. Matsuyama, Y. Kobayashi, N. Kawada, Purification and characterization of (S)-1, 3-butanediol dehydrogenase from Candida parapsilosis, Biosci. Biotechnol. Biochem. 59 (1995) 1769-1770. [174] R. Zhang, G. Zhu, W. Zhang, S. Cao, X. Ou, X. Li, M. Bartlam, Y. Xu, X.C. Zhang, Z. Rao, Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity, Protein Sci. 17 (2008) 1412-1423. [175] R. Zhang, Y. Xu, Y. Sun, W. Zhang, R. Xiao, Ser67Asp and His68Asp substitutions in Candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction, Appl. Environ. Microbiol. 75 (2009) 2176-2183. [176] R. Zhang, Y. Xu, Y. Geng, S. Wang, Y. Sun, R. Xiao, Improved Production of (R)-1phenyl-1,2-ethanediol by a codon-optimized R-specific carbonyl reductase from Candida parapsilosis in Escherichia coli, Appl. Biochem. Biotechnol. 160 (2010) 868-878.

65

[177] H. Yamamoto, A. Matsuyama, Y. Kobayashi, Synthesis of ethyl (R)-4-chloro-3hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase, Biosci. Biotechnol. Biochem. 66 (2002) 481-483. [178] H. Yamamoto, A. Matsuyama, Y. Kobayashi, Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)specific secondary alcohol dehydrogenase, Biosci. Biotechnol. Biochem. 66 (2002) 925-927. [179] A. Jakoblinnert, R. Mladenov, A. Paul, F. Sibilla, U. Schwaneberg, M.B. AnsorgeSchumacher, P.D. de Maria, Asymmetric reduction of ketones with recombinant E. coli whole cells in neat substrates, Chem. Commun. 47 (2011) 12230-12232. [180] A. Jakoblinnert, J. Wachtmeister, L. Schukur, A.V. Shivange, M. Bocola, M.B. AnsorgeSchumacher, U. Schwaneberg, Reengineered carbonyl reductase for reducing methyl-substituted cyclohexanones, Protein Eng. Des. Sel. 26 (2013) 291-298. [181] A. Jakoblinnert, A. van den Wittenboer, A.V. Shivange, M. Bocola, L. Heffele, M. Ansorge-Schumacher, U. Schwaneberg, Design of an activity and stability improved carbonyl reductase from Candida parapsilosis, J. Biotechnol. 165 (2013) 52-62. [182] Y. Nie, Y. Xu, H.Y. Wang, N. Xu, R. Xiao, Z.H. Sun, Complementary selectivity to (S)-1phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone, Biocatal. Biotransform. 26 (2008) 210-219. [183] Z. Yan, Y. Nie, Y. Xu, X. Liu, R. Xiao, Biocatalytic reduction of prochiral aromatic ketones to optically pure alcohols by a coupled enzyme system for cofactor regeneration, Tetrahedron Lett. 52 (2011) 999-1002. [184] M.M. Musa, K.I. Ziegelmann-Fjeld, C. Vieille, J.G. Zeikus, R.S. Phillips, Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus, J. Org. Chem. 72 (2007) 30-34. [185] K. Nakamura, Y. Inoue, T. Matsuda, A. Ohno, Microbial deracemization of 1-arylethanol, Tetrahedron Lett. 36 (1995) 6263-6266.

66

[186] K. Kita, M. Kataoka, S. Shimizu, Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis, J. Biosci. Bioeng. 88 (1999) 591-598. [187] C. He, D. Chang, J. Zhang, Asymmetric reduction of substituted α- and β-ketoesters by Bacillus pumilus Phe-C3, Tetrahedron: Asymmetry 19 (2008) 1347-1351. [188] M. Bertau, D. Scheller, Equilibrium-dependent hydration of ethyl 4,4,4-trifluoroacetoacetate in aqueous solutions and consequences for the whole-cell biotransformation with Saccharomyces cerevisiae, Enzyme Microb. Technol. 32 (2003) 491-497. [189] J. He, X. Mao, Z. Sun, P. Zheng, Y. Ni, Y. Xu, Microbial synthesis of ethyl (R)-4,4,4trifluoro-3-hydroxybutanoate by asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate in an aqueous-organic solvent biphasic system, Biotechnol. J 2 (2007) 260-265. [190] S.S.d.S. Oliveira, L.R.S. Dias, N.C. Barbosa, M.L. Bello, F.J.M. Novaes, M.d.C.K.V. Ramos, F.R.d. Aquino Neto, S.B. Fiaux, Enantioselective bioreduction of ethyl 4,4,4-trihalide-3oxobutanoate by Kluyveromyces marxianus, Tetrahedron Lett. 54 (2013) 3067-3070. [191] A. Guerrero, F. Raja, Preparation of both enantiomers of ethyl 4,4,4-trifluoro-3-hydroxy butanoate by enantioselective microbial reduction, Bioorg. Med. Chem. Lett. 1 (1991) 675-8. [192] T. Tsujigami, T. Sugai, H. Ohta, Microbial asymmetric reduction of α-hydroxyketones in the anti-Prelog selectivity, Tetrahedron: Asymmetry 12 (2001) 2543-2549. [193] D.H. Dao, Y. Kawai, K. Hida, S. Hornes, K. Nakamura, A. Ohno, M. Okamura, T. Akasaka, Stereochemical Control in Microbial Reduction. 30. Reduction of Alkyl 2-Oxo-4phenylbutyrate as Precursors of Angiotensin Converting Enzyme (ACE) Inhibitors, Bull. Chem. Soc. Jpn. 71 (1998) 425-432.

67

Table 1 Deracemization of secondary alcohols using whole cells of C. parapsilosis % ee (absolute

Substrates (racemic) / C. parapsilosis strain

References

configuration) /% yield

Aliphatic 1, 2-diols: 99-~100 a/ 56-100

[52]

C. parapsilosis IFO 0708 Aromatic 1, 2-diols

1-98 (S) / C. parapsilosis CCTCC M 203011; ATCC 10232, 22019,7330; CECT 10211, 10304, 10434, 1037; AS 2. 491 , 590, 1497; CICC 1627; IFO 0708

41-92

C. parapsilosis IFO 0708

~99(S)/ 100

68

[54]

[52]

C. parapsilosis ATCC 7330

>99(S) /86

[73]

80 (R)/ NRb

[53]

~100 (R)/ 88

[58]

C. parapsilosis ATCC 52820 Aromatic alcohols

C. parapsilosis MTCC 1965 89 - >99 (R) / 60- [59] C. parapsilosis ATCC 7330

78

C. parapsilosis ATCC 7330

> 99 (R) / 76

69

[73]

Allylic alcohols

76->99 (R) / 68- [61]

C. parapsilosis ATCC 7330

79 a

Absolute configuration varies with functional group;

b

Not Reported

70

Table 2 Deracemization of α and β-hydroxy esters using whole cells of C. parapsilosis ATCC 7330 %ee

(absolute

configuration)/

Substrates (racemic)

References

% yield

α-hydroxy esters:

>99 (S)/ 70-85

[63]

03- >99 (S)a/ 52 – 93

[64]

42- >99 (S) /

[66]

51- 80

71

32 - >99 (S) /

[67]

61-81

>99 (S) / 78

[73]

90 ->99 (S) /52-79

[68]

15- 99 (S)/ 62- 75

[69]

β-hydroxy esters:

72

9 - >99 (S)/ 10- 75 Immobilized cells

64->99 (R or S)/

[97]

[74]

32-71

92-99 (S) / 58-75 Immobilized cells

73

[72]

>99 (S) / 78; Whole cells

[73]

>99 (S) / 28

[98]

Immobilized cells a

Absolute configuration not reported

74

Table 3 Asymmetric reduction of prochiral ketones and imines using whole cells of C. parapsilosis ATCC 7330 % ee ; Substrates reduction

used

for

asymmetric Products

obtained

after

reduction

asymmetric

Absolute configuration/

References

% yield 90->99(S)/ 39-70 [78]

62 (R)/39

93-99 (S)a/ 58-71 [79] 90-97(S)a,b /52-65 b

75

>99(R/S)c /94-96

41->99 (S) /49.5-73

95->99 (R)/5580

[86]

[88]

[48]

72->99 (S)/55-75 [59]

a c

Absolute configuration not report for all substrates done, bFor Immobilized cells, Absolute configuration depends on the type of carbon source present in the medium

76

Table 4 Asymmetric reduction of prochiral ketones using whole cells/purified enzymes of C. parapsilosis

Substrates

Product

C. parapsilosis

% ee Absolute

Strain/

configuration/

enzyme used

77

References

% yield

IFO 1396

98 (S)/60

[84]

NBRC 1396

97 (1S, 2R)/68

[81]

NBRC 1396

99 (1R, 2R)/76

[82]

NBRC 1396

99 (1R, 2R)/75

[82]

NBRC 1396

99 (1R, 2R)/80

[82]

NBRC 1396

98 (1R, 2R)/74

[82]

NBRC 1396

CCTCC M203011 (Immobilized)

98 (1R, 2R)/72 >99 (S)/81 >99 (S)/98* >99 (R)/99**

[99-102]

>99 (S)/95***

DSM 70125/ Carbonyl

[82]

[142] 99(S)/NR

Reductased Carbonyl Reductase d Carbonyl Reductase d

>99 (S)/77

[143]

>99 (S)/70

[143]

Carbonyl Reductase 49->99 (S)/ 20-100

78

[145]

Carbonyl

49 (S)/100

Reductase

67(S)/9076 (S)/65

[145]

62 (R)/ <5

CDC317/SDR

99 (S)/91

[160]

CCTCCM

99 (R)/NR

[153]

~100 (R)/NR

[151]

94(R)/ 86

[176]

RCRf

95 (R)/ 87

[159]

RCRf

>99 (R)/99

[163]

CpADH

~100 (S)/NR

[153]

SCRII e

>99 (S)/96

[155]

SCR e,g,h

~100 (S)/98

[156]

203011 / CR d

ATCC 28474/CR d CCTCCM 203011 / CR d

(E.coli expressed)

79

CCTCC M 203011/

>99 (S)/

carbonyl

up to 91

[183]

reductase g,h DSM

70125/ 99 (S)/98

CR lyophilized (E.

coli

[179]

expressed)

a

CpAR2

>99 (S)/ NR

CpAR4

>99 (R)/NR

In ionic liquid (IL: [C4MIM] PF6), b In water immiscible IL [(C4MIM·PF6)/buffer], c In ionic liquid (IL: (C2OHMIM.NO3), dPurified

carbonyl reductase, e (S)-specific carbonyl reductase, f(R)-specific carbonyl reductase g

[154]

E. coli over-expressed

h

Cell-free extracts

80

Table-5 C. parapsilosis mediated deracemization versus other microbial catalysts Deracemization reported by C. parapsilosis Substrates

Other microbes % ee Absolute

Strain/ % ee Absolute

configuration)/

configuration/

Organism/Substrate*

% yield /Reaction time/

% yield /Reaction time /References

References

95 (S)/73 a

IFO 0708/ ~100 (S)/93

Candida maltosa ATCC 20275

48 h [52]

Geotrichum candidum IFO 4601

95 (R)/43

24 h [52] IFO 1396/95 (R)/50[84] Aliphatic diols

Candida

inconspicua

IFO 97 (S)/50

0621Pichia opuntiae IFO 10025

96 (S)/50

Kluyveromyces lactis IFO 1267

99 (S)/45 [84]

CCTCC M203011/

Geotrichum candidum IFO5767

98 (S)/92(S)a

~100/(S)/69 [104]

Aromatic diols

[54] ATCC 52820/ 80(R)/ NRb [53]

Candida boidinii Pichia methanolica

ee up to ~100 [53] ee up to ~100 [53]

81

ee up to 60 Hansenula polymorpha

[53]

ATCC 7330/ 99 (S), n= 0, 2 70-90 [63]

23 (S) /42

ATCC 7330/ ee: 91-95 (S)

R=CH3, C2H5

yield: 66-70

Pseudomonas sp. ECU1011

12 h, pH-7, 10 mM (sub con.) [105]

ee: 50-99 (S) yield: 55-85 [64] 99 (S)/up to 79 [68]

NRb

ATCC 7330/ α-hydroxy esters

42 - >99 (S)/ 51–80 [66] ATCC 7330/ >99(S)/up to 81

82

[67] ATCC 7330/ up to

NRb

>99 (S)/62-70 [69]

NRb

ATCC 7330/

β-hydroxy esters

up to >99 (S)/ 65-75 [69]

a

99 (R)/69 [74]

G. candidum IFO5767

ATCC 7330/

W110A

10-99(R) /68-79

dehydrogenase

[61]

from Thermoanaerobacter

secondary

97 (R)/65 [104]

alcohol 99 (R)/50 (conversion) [184]

Phenylethanols)

ethanolicus

(Substituted

1-arylethanols

Allylic alcohol

ATCC 7330/

(S)-PED yield

ATCC 7330/

Geotrichum candidum

72-99(S)/60-95 [59]

IFO 5767 Dipoduscus magnusii IFO 4600

b

Not Reported 83

99 (R)/96

90 (R)/78 [104, 185]

Table-6 Comparison of asymmetric reductions reported by using C. parapsilosis and other whole cell micro organisms as the biocatalyst

Substrate

Asymmetric reduction reported using Candida parapsilosis

Aliphatic compounds

Strain/ % ee

Other biocatalysts Organism/Substrate*

% ee

(Absolute configuration)/

(Absolute configuration)/

% yield / References

% yield / References

IFO 1396/

C. arborea IAM 4147

98(S)/60 [84]

Issatchenkia

scutulata

99 (R)/37 IFO

99 (R)/48

10070

[84]

CCTCC M203011/

Rhodotorula sp. AS2.22

>99 (S)/14

99 (S)/ 81.3

Geotrichum candidum

[99]

>99 (S)/5 [99]

ATCC 7330

Lactobacillus kefir

>99/ 97 [110]

>99/up to 96

Geotrichum candidiumSC 5469

95 (S)/85 [113]

[86, 87]

Saccharomyces cerevisiae IFO 92/(S)/28 0206; Trichosporon cutaneum 98 (S)/30 IFO 1198

84

[186]

ATCC 7330

Saccharomyces cerevisiae

Aliphatic compounds

up to >99/up to 72

84-97(S)/up to 66 [128] 70->99 (S)/15-68 [129]

[88]

Rhizopus arrhizus

ATCC 7330

Bacillus pumilus Phe-C3

94(R)/83[187]

84 (S)/60

Saccharomyces cerevisiae

62 (R)/35 [188]

[75]

Saccharomyces uvarum sw58

76 (R)/56d [189]

Kluyveromyces marxianus

29 (R)/81 [190]

Candida utilis

59 (S)/34 [191]

Comamonas testosteroni

>99 (S)/47 [130]

Aspergillus niger IFO 4415

78 (R)/78 a; 41/22 (R) b

ATCC 7330 59 (S)/76 [89] IFO 0708/ 90 (R)/59a; 76 (R)/49b

Compounds

Aromatic

Sporobolomyces holasticus IFO 88 (R)/76 IFO 0585/

1032

81(R)/ 79a; 68 (R)/ 42b

Byssochlamys fulva IFO 7901

88 (R)/76

[136]

[136]

ATCC 7330

>99(S)/91

>99(S)/up to 70 [78]

[192]

85

Yamadazyma

farinosa

IFO

10896 ATCC 7330

C. krusei SW2026

>99 (R)/95

[117]67/ (R)/up to 86

>99 (S)/71

Saccharomyces cerevisiae

[193]

Daucus carota

97->99(R)/62-73

[79] ATCC 7330 95-98(S)/58-64 [79]

[118]

Imines

ATCC 7330/95->99% (R)/ Streptomyces sp. GF3587 Streptomyces sp. GF 3546 55-80%

99 (R)/ 73 92 (S)/ 67 [138]

50-94(S or R)/45-68` Saccharomyces bayanus a c

Substrate concentration 30 mg/ml in the reaction mixture, d

Molecular yield, conversion

86

b

[139]

Substrate concentration 60 mg/ml in the reaction mixture,

Graphical Abstract

Highlights

 The yeast, Candida parapsilosis is used for: o

redox reactions of organic compounds to generate of optically pure chiral synthons

o

preparation of optically pure secondary alcohols via deracemisation

o

asymmetric reduction of carbonyl compounds and imines

o

carrying out oxidative kinetic resolution reactions and oxidation

 Purified enzymes of the yeast are used to understand the mechanism of redox reactions