Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

ELSEVIER

FEMS

Microbiology

Letters

129 (1995)

57-62

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270 Alan J. Blakey a, John Colby a,*, Edwin Williams

b, Catherine O’Reilly

a

a School of Health Sciences, Uniuersiry of Sunderland, Sunderland SRI 3SD, UK b Microbial Technology Group, Universily of Newcastle, Newcastle-upon-Tyne NE1 7RU, UK Received

28 February

199.5; revised

30 March

1995; accepted 30 March

1995

Abstract

An acetonitrile-utilisingbacteriumAJ270 hasbeenisolatedfrom soil, identifiedasa Rhodococcus sp. andshownto be distinct from all the recognisedspeciesof the genus.It grows well on 32 of 36 aliphatic,aromaticand hetero-aromatic nitriles testedand is capableof rapid growth on high concentrations(0.25-0.38 M) of acetonitrile,benzonitrile and 3-cyanopyridine.The nitrile hydrataseof Rho&coccusAJ270 is stableon storagefor 18 monthsat - 20” C, hasactivity againsta very broad range of nitriles and dinitriles and is able to catalyse regio-specific and stereo-specific nitrile biotransformations.

The suitability

of AJ270 as a robust and versatile biocatalyst is discussed.

Keywords:Nitrile hydratase;Rhodococcus;Chiralbiotransformation

1. Introduction Microorganisms that can grow on nitriles hydrolyse these substratesto the corresponding carboxylate by one of two enzymic mechanisms[l]. Nitrilases achieve this conversion in one step (Reaction 11, whereas a combination of nitrile hydratase and amidaseare required for the alternative route (Reactions 2 and 3). RCN + 2H,O + RCOOH + NH,

(1)

RCN + H,O + RCONH, + Hz0

(2)

RCONH, + H,O + RCOOH + NH,

(3)

* Corresponding

author.

0378-1097/95/$09.50 SSDIO378-1097(95)00135-2

Fax:

+ 44 (191) 515 2502

0 1995 Federation

of European

Microbiological

The hydrolysis of nitriles by microbial nitrile hydratase has been exploited for the commercial production of acrylamide 121.Recently attention has been directed towards regio- and/or stereo-selectivenitrile hydrolyses by, for example, Breuibacterium imperiale B222 [3], Pseudomonasspp. [4], certain Rhodococcusspp. [5,6] and the immobilised preparation from Rhodococcus sp. CH5 formerly supplied by Novo Nordisk under the product numbers SP361 and SP409 [7,8]. Much of this interest has been directed towards the production of optically active pharmaceuticalcompoundsand other potentially useful products [9,10]. This paper describesthe isolation of Rhodococcus AJ270, a new nitrile hydratase-containing strain with the ability to hydrolyse and utilise for growth a wide range of nitriles and dinitriles, and its usefulnessfor Societies.

All rights reserved

A.J. Blakey

58

carrying out regio-specific biotransformations.

2. Materials

et al. /FEMS

and stereo-specific

Microbiology

nitrile

and methods

2.1. Materials

All chemicals were purchased from Sigma, Aldrich or British Drug Housesplc.

Letters

129 (1995) 57-62

butyronitrile was determinedby incubating 1 g of the substrate (added in 0.1-g aliquots at 24-h intervals) with 50 mg dry weight of cells in 50 ml of buffer A. The reaction was stopped after 284 h and the reaction mixture extracted with CH,Cl,. The extract was analysedby IR spectroscopy and the optical rotation of the product determined by polarimetry at 25“ C using a sodium lamp and glucose solutions as standards. 2.4. Analysis

2.2. Growth

of Rhodococcus

The mineral medium used was mineral base E [ 111.Nitriles, amidesand carboxylic acidswere tested as growth substratesat 0.1% w/v in 250-ml shake flasks containing 50 ml of medium. When nitriles were tested as nitrogen sources,the ammonium sulphate solution was omitted and 6 mM glucose was included as carbon source. Growth was estimatedby measuringthe OD,,, of cultures after 24 h incubation and scored as follows: -, OD,,, < 0.1; +, OD,,, > 0.1; + +, OD,,, > 0.5; + + +, OD,,, > 1.0. Bacteria for nitrile hydratase assayswere grown at 30” C in 2-l shake flasks containing 500 ml of mineral medium supplementedwith 0.25 M acetonitrile. Cultures were harvested by centrifugation in late log phase(after about 20 h), washed twice with 0.1 M potassiumphosphatebuffer, pH 7.2 (buffer A) and re-suspendedin the samebuffer. 2.3. Determination

of nitrile

for the products

of nitrile

hydrolysis

AJ270

hydratase

activity

Reactions were carried out at 30” C with shaking. Reaction mixtures (usually 1.5 ml) contained washed bacterial suspension(l-100 mg dry weight l- ’ >, 50 mM nitrile and 100 mM buffer A. Samples were removed at lo-min intervals for analysis by GC, GC-MS or HPLC as appropriate. Nitrile hydratase activities in Table 1 are expressedas relative activities with the rate for acetonitrile (4.3 mmol min-’ (mg dry weight))‘) set at 100%. Regio-specific hydrolysis of dinitriles was tested in 1.5 ml reaction mixtures containing 50 pmol of dinitrile and the reactions were allowed to go to completion which took about 2 h in each case.The NH: releasedwas estimated using the Sigma colour reagent. The stereo-selective hydrolysis of (R,S)-2-phenyl-

GC analysis for the amide and acid products of the simpler nitriles was accomplishedon columns of Porapak Q and PS. Samples incorporated propionic acid or butyric acid as internal standard. HPLC analysis of the amide and acid products of aromatic and hetero-aromatic nitriles was done on a Cl8 reverse phase column using CH,CN/potassium phosphate pH 2.8 (1:4) as solvent. GC-MS using a Hewlett-Packard 5890 seriesII GC coupled to a Trio 2000 MS was used to confirm the identity of some hydrolysis products. A 50-m open tubular capillary column coated with OVl was used.

3. Results 3.1. Isolation

and properties

of Rhodococcus

AJ270

Rhodococcus AJ270 was isolated on 25 mM acetonitrile mineral agar plates using an Anderson sampler. The source was an air-dried soil sample collected from an obsolete industrial site on the banks of the river Tyne. A chemotaxonomic study of the wall mycolic acids and meso-diaminopimelic acid content of AJ270 [12] assigned the strain to the genus Rhodococcus [13]. Numerical taxonomy, based largely on fluorogenic enzyme assaysand nutritional tests and using UPGMA (unweighted pair group method with arithmetic averages) cluster analysis, demonstratedthat AJ270 is significantly different to all of the currently recognised species within the genera Rhodococcus, Gordona and Tsukamurella [12]. The optimum temperature for growth on acetonitrile was 30” C and rapid growth was observed at acetonitrile concentrations between 25 mM and 1.25 M with the best growth rate ( /.L= 1.54 h-‘) ob-

A.J. Blakey

served at 0.38 M. The best growth of aromatic and hetero-aromatic benzonitrile and 3-cyanopyridine) 0.25 M, the highest concentration limited solubility of the substrates. 3.2. Growth

et al. /FEMS

Microbiology

rates on a variety nitriles (including were observed at tested due to the

substrate range

Some 36 aliphatic, aromatic and hetero-aromatic nitriles and dinitriles’ were tested for their ability to

Table 1 Nitriles as substrates

for growth

and for nitrile C source

Aliphatic nitriles Acetonitrile Chloroacetonitrile Methoxyacetonitrile Propionitrile n-Butyronitrile Valeronitrile lsobutyronitrile Pivalonitrile Isovaleronitrile Lactonitrile Acrylonitrile Crotononitrile Methylacrylonitrile Malononitrile * Succinonitrile * Glutaronitrile * Adiponitrile * 2Xyanoacetamide Aromatic nitriles Benzonitrile Phenylacetonitrile Mandelonitrile Cinnamonitrile 2-Aminobenzonitrile 3-Aminobenzonitrile 4-Aminobenzonitrile 2-Chlorobenzonitrile 2-Nitrobenzonitrile 2-Methoxybenzonitrile 2-Cyanophenol p-Tolunitrile 1,2-Dicyanobenzene 1,3-Dicyanobenzene Hetero-aromatic 2-Pyridylacetonitrile 2Kyanopyridine 3-Cyanopyridine 4Xyanopyridine

and dinitriles

hydratase

N source

NH activity 100 272 28 256 182 87 272 259 272 21 69 187 123 92 95 49 95 82

*

++ +++ +++

59

support the growth of Rhodococcus AJ270 when provided as carbon and energy source or as nitrogen source (Table 1). All except malononitrile*, cinnamonitrile, mandelonitrile and 2-cyanophenol were nitrogen sources. All except these four substrates, lactonitrile, acrylonitrile, 1,2-dicyanobenzene * and 2-nitrobenzonitrile were carbon and energy sources. Eleven amides were tested as carbon and energy sources. Acetamide, propionamide, n-butyramide, chloroacetamide, methacrylamide, isobutyramide,

*

+++ +++ +++ +++ +++ +++

* * nitriles

129 (1995) 57-62

(NH)

+ + ++ ++ +++ +++ +++ +++ +

and dinitriles

Letters

++ ++ ++ +++ +++ + + + ++ ++ +++

75 169 279 90 72 74 77 5 172 8 23 23 15 15 241 223 233 218

(% of CH,CN

rate)

60

A.J. Blakey

et al. / FEMS

Microbiology

benzamide, 2-aminobenzamide and nicotinamide supported growth, whereas lactamide and acrylamide did not. Acetic acid, propionic acid, n-butyric acid, isobutyric acid, lactic acid, benzoic acid, 2-aminobenzoic acid and nicotinic acid were all good carbon and energy sources. 3.3. Nitrile hydratase Rhodococcus AJ270

activity

of acetonitrile-grown

Highest rates of acetonitrile hydrolysis were observed at 30” C and pH 7.0 although > 65% of the rate at pH 7 was observed over the pH range 5-9. Cell pellets stored at - 20” C retained about 80% of their original activity after 18 months. The substrate range of the nitrile hydratase is shown in Table 1. Malononitrile * , cinnamonitrile, 2-cyanophenol and mandelonitrile, none of which supported growth either ascarbon and energy or as nitrogen source,were all hydrolysed; 2-chlorobenzonitrile and 2methoxybenzonitrile were the poorest substratesfor hydrolysis. All the other nitriles were hydrolysed at a rate 2 10% of that observed with acetonitrile. In all cases,amide was observed as product with or without the correspondingcarboxylic acid, indicating the presenceof a nitrile hydratase. The results presented here do not exclude the possibility of a nitrilase also being presentin AJ270. However, as yet unpublished work in our laboratories involving the probing of AJ270 DNA with a nitrilase-specific DNA probe strongly suggeststhat there is no nitrilase gene in this Rhodococcus strain. Aliphatic nitriles with 3-5 carbon atomswere the best substrates and methyl-branched nitriles were generally better substratesthan their straight-chained homologues.Most aromatic nitriles were poorer subTable 2 Regio-specific Aliphatic

nitrile

dinitrile

Malonitrile Adiponitrile Fumaronitrile 1,2-Dicyanobenzene 1,3-Dicyanobenzene

hydrolysis NH; 48 46 98 97 99

by Rhodococcus released ( fimol)

AJ270

Letters

129 (1995)

57-62

strates with mandelonitrile, phenylacetonitrile and 2-nitrobenzonitrile being exceptions. The heteroaromatic nitriles tested were all good substrates. In an experiment to demonstratethat nitrile hydra lysis was amenableto scale-up, 1 g dry weight of AJ270 was incubated at 30” C with 0.75 mol of 3-cyanopyridine in 50 ml of buffer A and samples removed for HPLC analysis at 6-h intervals. The nitrile was stoichiometrically converted to nicotinamide over 144 h and then the amide more slowly converted to nicotinic acid. No nitrile was detectable after 168 h. 3.4. Regiospecific

hydrolysis

of dinitriles

Of the five dinitriles tested (Table 2), two of the aliphatic nitriles, namely malononitrile and adiponitrile, yielded half of the NH,f expected if both cyanide groups were fully hydrolysed, suggesting that only one end of the molecule had been attacked. Even prolonged incubation for up to 12 h failed to produce more NH:. In each case, the possibility that the remaining nitrile had been hydrolysed as far as the amide was excluded by estimation of the remaining substrateby GC and HPLC. 3.5. Stereospecific butyronitrile

hydrolysis

of

(R,S)-phenyl-

A Rhodococcus AJ270 cell suspensionwas incubated with 1 g of (R,S)-2-phenylbutyronitrile for 284 h as described in Materials and methods. IR analysis of the extracted product showed that the corresponding amide was produced; no carboxylic acid was found. The optical rotation of the extracted 2-phenylbutyramide demonstratedthat the (R)-( + >form predominated in 83% enantiomeric excess.

4. Discussion

Selectivity Regio-specific Regio-specific Non-selective Non-selective Non-selective

Cell suspensions were incubated with 50 pmol of dinitrile for 2 h and then the release of NH: was monitored. Full details are given in Materials and methods.

Rhodococcus AJ270 grew well on a wide range of aliphatic, aromatic and hetero-aromatic nitriles and rapidly hydrolysed all but 2 of the 36 nitriles tested, producing the corresponding amide or mixture of amide and carboxylic acid. AJ270 grew extremely rapidly (mean doubling time 27 min) on 0.38 M acetonitrile and concentrations up to 1.25 M could be

A.J. Blakey

et al./FEMS

Microbiology

used so that high cell densities were readily achieved. We have demonstrated that the biotransformations can be readily scaled up, that the biocatalyst is stable on long-term storage and that cell suspensions are able to catalyse regio-specific and stereo-specific nitrile hydrolyses. These properties suggest that Rhodococcus AJ270 should prove a robust and versatile biocatalyst.

Letters

[5]

[6]

[7]

Acknowledgements [8]

The authors wish to thank Professor Otto MethCohn for help with chiral analyses and Professor Mike Goodfellow for advice on the taxonomy of rhodococci and related genera.

References Asano, Y., Tani, Y. and Yamada, H. (19801 A new enzyme ‘nitrile hydratase’ which degrades acetonitrile in combination with amidase. Agr. Biol. Chem. 44, 2251-2252. [2] Kobayashi, M., Nagasawa, T. and Yamada, H. (1992) Enzymic synthesis of acrylamide: a success story not yet over. Trends Biotechnol. 10, 402-408. [3] Bianchi, D., Bosetti, A., Cesti, P., Fransozi, Cl. and Spezia, S. (1991) Stereoselective microbial hydrolysis of 2aryoxypropionitriles. Biotechnol. Lett. 13, 241-244. [4] Layh, N., Stolz, A., Forster, S., Effenberger, F. and Knackmuss, H.J. (1992) Enantioselective hydrolysis of O-acetyl-

191

[lo]

[l]

[ll]

1121

[13]

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mandelonitrile to 0acetylmandelic acid by bacterial nitrilases. Arch. Microbial. 158, 405-411. Gilligan, T., Yamada, H. and Nagasawa, T. (1993) Production of (S)-( + )-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by a combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328. Appl. Microbial. Biotechnol. 39, 720-725. Gradley, M.L., Deverson, C.J.F. and Knowles, C.J. (1994) Asymmetric hydrolysis of (R,S)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216. Arch. Microbial. 161, 246-251. Crosby, J.A., Parratt, J.S. and Turner, N.J. (1992) Enzymatic hydrolysis of prochiral dinitriles. Tetrahedron Asym. 3, 1543-1546. Beard, T., Cohen, M.A., Paratt, J.S., Turner, N.J., Crosby, J. and Moilliet, J. (1993) Stereoselective hydrolysis of nitriles and amides under mild conditions using a whole cell catalyst. Tetrahedron Asym. 4, 1085-1104. Bhalla, T.C., Miura, A., Wakamoto, A., Ohba, Y. and Furuhashi, K. (1992) Asymmetric hydrolysis of cr-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous Pa-34. Appl. Microbial. Biotechnol. 37, 184190. Yamamoto, K., Fujimatsu, I. and Komatsu, K.-I. (1992) Purification and characterisation of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile. J. Ferm. Bioeng. 72, 425-430. Owens, J.D. and Keddie, R.M. (1969) The nitrogen nutrition of soil and herbage coryneform bacteria. J. Appl. Bacterial. 32, 338-347. Blakey, A.J. (1994) Isolation of nitrile utilizing microorganisms and physiological and biochemical investigation of Rhodococcus nov. sp. AJ270 and investigation of its nitrile hydratase. PhD Thesis, University of Sunderland, UK. Goodfellow, M. and Alderson, G. (19771 The actinomycete genus Rhodococcus: a home for the ‘rhodochrous’ complex. J. Gen. Microbial. 100, 99-122.