ammonium sulfate aqueous two-phase system

Journal Pre-proof Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system Zhe-Ming Wu, Chang-Ling Hao, Tao Tong, Ren-Chao Zheng, Yu-Guo Zheng

PII:

S1359-5113(19)30947-X

DOI:

https://doi.org/10.1016/j.procbio.2019.10.023

Reference:

PRBI 11811

To appear in:

Process Biochemistry

Received Date:

23 June 2019

Revised Date:

2 October 2019

Accepted Date:

18 October 2019

Please cite this article as: Wu Z-Ming, Hao C-Ling, Tong T, Zheng R-Chao, Zheng Y-Guo, Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system, Process Biochemistry (2019), doi: https://doi.org/10.1016/j.procbio.2019.10.023

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Title:

Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system

Authors and Affiliation:

a

ro of

Zhe-Ming Wua,b, Chang-Ling Haoa,b, Tao Tonga,b, Ren-Chao Zhenga,b* and Yu-Guo Zhenga,b Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and

Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of

b

-p

China

Engineering Research Center of Bioconversion and Biopurification of Ministry of Education,

re

Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China

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*Corresponding author:

Tel: +86-571-88320391, Fax: +86-571-88320884, E-mail: [email protected]

Jo

ur

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Graphical abstract

Highlights 

An aqueous two-phase system (ATPS) containing acetone and (NH4)2SO4 was developed. Related phase behaviors and back-extraction were investigated.



High extraction recovery (92.11%) was achieved by optimized ATPS.



This is the first report for (S)-3-cyano-5-methylhexanoic acid separation.

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ro of



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ABSTRACT

(S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA) is the key chiral intermediate

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of pregabalin. In this paper, an aqueous two-phase system (ATPS) was developed to extract (S)-CMHA from nitrilase-catalyzed bioconversion broth. Inorganic salts and solvents

were

screened

na

hydrophilic

to

form

ATPS,

among

which

an

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acetone/ammonium sulfate ATPS was investigated in detail, including phase diagram, effect of phase composition and stability of (S)-CMHA. The maximum product

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recovery of 99.15% was obtained by an optimized ATPS system composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone with the removal of 99% cells and 86.27% proteins. The total (S)-CMHA yield reached 92.11% after back-extraction. The recycling use of ammonium sulfate was investigated, and 93.10% of salt in the salt-rich phase was recovered with the addition of methanol. The results demonstrated

the efficiency of the two-step extraction process for separation of (S)-CMHA.

Keywords:

Bioconversion,

Aqueous

two-phase

extraction,

Purification,

(S)-3-cyano-5-methylhexanoic acid

Introduction

ro of

1.

Pregabalin ((S)-(+)-3-aminomethyl-5-methylhexanoic acid) is a lipophilic 4-aminobutyric acid (GABA) analog and marketed as LyricaTM for the treatment of

-p

epilepsy [1, 2]. Although pregabalin was originally developed for the adjunctive therapy

re

of partial seizures, recent studies showed its effectiveness for the treatment of neuropathic pain, post-herpetic neuralgias and anxiety disorders [3, 4]. Due to its broad

lP

and high therapeutic activity, pregabalin grows as one of the top 20 best-selling drugs attaining a global sale of $5.065 billion in 2017. As the active pharmaceutical

na

ingredients of pregabalin is mainly in the (S)-enantiomer form, considerable chemical

ur

and biocatalytic routes have been developed for synthesis of (S)-pregabalin [5, 6]. (S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA), a key chiral intermediate of can

Jo

pregabalin,

be

easily

synthesized

by

kinetic

resolution

of

racemic

2-isobutyl-succinonitrile (IBSN) with nitrilase as catalysts (Fig. 1). This route has the advantage of higher atom economy than the first- and second-generation process for pregabalin developed by Pfizer [7]. A sustainable separation method for (S)-CMHA has become of great importance for efficient production of pregabalin with high overall

yield. However, separation and extraction of (S)-CMHA has not been reported yet. Aqueous two-phase systems (ATPS) are essentially composed of water in both phases, which combine two types of water soluble polymers or polymer-salt or two different salts

[8, 9]

. Classical ATPSs used for organic acid separation, such as the

polymer-polymer,

polymer-salt

and

salt-salt

ATPS

suffered

disadvantages including emulsion formation and high viscosity

[10]

from

several

. In recent years,

interest in separation of organic acids

ro of

novel ATPS based on hydrophilic solvent and inorganic salt has received considerable [11, 12]

, due to its excellent performance

including better sustainability, less extraction times and lower associated cost

[13-15]

.

-p

Furthermore, the phase-forming components can be easily recycled by evaporation. [16]

,

re

Numerous ATPSs have been extensively used in the extraction of organic acids

and clavulanic acid [19].

lP

such as succinic acid [11, 17], phenylsuccinic acid [18], gallic acid [9], chlorogenic acid [12]

na

In our previous work, a nitrilase from Arabis alpine was exploited to kinetically resolve racemic 2-isobutyl-succinonitrile (IBSN) to afford (S)-CMHA at a substrate

ur

loading of 1.2 M, with a 42% conversion and 99% ee (enantiomeric excess) [20, 21]. In this paper, an ATPS composed of acetone/ammonium sulfate was developed to

Jo

separate (S)-CMHA from the bioconversion broth (Fig. 2). The effects of the phase-forming components on partition of (S)-CMHA, pH of bioconversion broths, removal of cells and proteins were investigated. Under optimized conditions, the highest (S)-CMHA yield of 99.15% was obtained using an ATPS composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone, with the removal ratios for cells

and proteins at 99% and 86.27%, respectively. The total (S)-CMHA yield was 92.11% after a simple back-extraction. This study is the first report for the extraction of (S)-CMHA, and the obtained results demonstrated the developed ATPS system as an adequate strategy to extract and purify (S)-CMHA.

2.

Materials

ro of

2.1.

Materials and methods

(S)-CMHA and IBSN were kindly provided by Zhejiang Chiral Medicine Chemicals Co., Ltd. (Hangzhou, China). Kanamycin was obtained from Beijing

-p

Solarbio Science and Technology (Beijing, China). BCA Protein Assay Kit was

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purchased from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). Lactose was obtained from Sunchina (Xiamen) Trading Co., Ltd. Dipotassium Hydrogen

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Phosphate Trihydrate (K2HPO4·3H2O), Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), Magnesium sulfate (MgSO4), ammonium sulfate (NH4)2SO4 and

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sodium carbonate anhydrous (Na2CO3) were bought from Aladdin (Shanghai, China).

ur

Ethanol, acetone and other reagents were obtained from Huadong Medicine Group

Jo

Co., Ltd. (Hangzhou, China). All chemicals were of analytical grade.

2.2.

Cultivation of recombinant Escherichia coli expressing nitrilase

Recombinant E. coli BL21 (DE3)/pET28b (+)-AaNit were grown in 250-mL

shake-flasks containing 100 mL Luria-bertani (LB) medium containing kanamycin (50 μg/mL) (peptone 10 g/L, yeast extract 5 g/L，NaCl 10 g/L) at 37 oC until the

optical density at 600 nm reached ~0.6. Fed-batch fermentation was conducted in a 5-L stirred bioreactor (BIOTECH-5BG-7000A, Shanghai, China) containing 3-L medium (g/L): peptone 15, yeast extract 12, NaCl 10, glycerine 15, (NH4)2SO4 5, KH2PO4 1.36, K2HPO4·3H2O 2.28, MgSO4·7H2O 0.375, with inoculation volume of 10 % (v/v). The temperature and agitation rate were controlled at 37 oC and 450 rpm, respectively. After 3 h of cultivation, the expression of recombinant protein was

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induced by 10 g/L lactose for 12 h at 28 oC. The whole cells were collected by

centrifugation at 12000×g under 4 oC for 10 min, washed twice with NaHCO3 buffer

Preparation of bioconversion broth

re

2.3.

-p

(50 mM, pH 8.0) for biotransformation.

The whole cells of recombinant E. coli harboring nitrilase was suspended in 500

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mL of NaHCO3 buffer (50 mM, pH 8.0) with a cell concentration of 15 g/L. IBSN was added to the mixture at a final concentration of 100 g/L. The reaction mixtures

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were shaken at 30 oC and 200 rpm in 1-L flasks for 12 h, and the bioconversion broth

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was collected for further investigation. The conversion of IBSN was determined by

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gas chromatography (GC).

2.4.

Partition of (S)-CMHA in ATPS

ATPS for (S)-CMHA extraction were prepared by accurate weighing 2 g of

various inorganic salts (K2HPO4·3H2O, NaH2PO4·2H2O, MgSO4, Na2CO3 and (NH4)2SO4) in 10 mL graduated tubes. Six gram of centrifuged bioconversion broth

was then added to fully dissolve the salt. Subsequently, 2 g of hydrophilic organic solvents (methanol, ethanol, 1-propanol, 2-propanol and acetone) were added to the solutions to form a two-phase aqueous system. All system components were thoroughly shaken with a vortex mixer for 3 min and allowed to stand for 30 min at room temperature to reach equilibrium with complete separation of the phases

[22]

.

The respective volumes of the bottom and top phases were measured accurately by 10

performance liquid chromatography (HPLC).

Phase diagram of acetone/ammonium sulfate ATPS

-p

2.5.

ro of

mL cylinder. The concentrations of (S)-CMHA in both phases were analyzed by high

turbidity titration method

[23]

re

The experimental determination of the binodal curve was prepared by using the . A certain amount of ammonium sulfate solution was

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added in a test tube. And acetone was added drop by drop, until the solution turned turbid. Then, deionized water was added drop-wise until the mixture become clear

na

again. The above steps were repeated sequentially to plot the phase diagram. In order

ur

to investigate influence of (S)-CMHA on the phase diagram, the turbidity system was

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destroyed by using deionized water and (S)-CMHA standard solution, respectively.

2.6.

Effects of both ammonium sulfate and acetone concentrations on the partition of (S)-CMHA in ATPS

The effects of ammonium sulfate on the partition of (S)-CMHA from the bioconversion broth were investigated at a final concentration from 8% to 21% (w/w).

The effects of acetone concentrations were investigated at a final concentration from 15% to 45% (w/w). Furthermore, the removal ratios of the cells and proteins from the bioconversion broth under non-centrifugal conditions were also determined at the different condition.

2.7.

Analytical methods

albumin as the standard

[24]

ro of

The protein concentration was determined using BCA assay with bovine serum . The biomass concentration was determined by

absorbance at 650 nm using a spectrophotometer

[25]

. The conversion of IBSN was

-p

determined by GC (Shimadzu, Japan) equipped with FID detector and chiral capillary

re

column BGB-174 (BGB Analytik, Switzerland, 30m × 0.25 mm, 0.25 μm film thickness) using helium as the carrier gas. The detector temperature was set at 220 oC

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and column temperature was set at 170 oC. (S)-CMHA concentration in both the top and bottom phases was quantified by HPLC (Shimadzu LC-10A system, Japan)

na

equipped with a C18 column (5 mm × 250 mm × 4.6 mm) and an ultraviolet detector

ur

(SPD-10A VP Plus, Shimadzu, Japan) setting at a wavelength of 210 nm. Column temperature was maintained at 40 oC. The mobile phase consisted of 76% buffer

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(containing 0.58 g/L NH4H2PO4 and 1.83 g/L NaClO4, pH 1.8) and 24% acetonitrile and the flow rate was 1 mL/min. The phase ratio (R) and partition coefficient (K) was defined using the following

equation: R=

Vt Vb

(3)

Where Vt and Vb are the volumes of acetone-rich phase and salt-rich phase, respectively. K=

Ct

(4)

Cb

Where Ct and Cb are the (S)-CMHA concentrations in the acetone-rich phase and salt-rich phase, respectively. The extraction efficiency (E) was defined using the following equation: RK

×100%

(5)

RK+1

ro of

E=

Where R and K are the phase ratio and partition coefficient, respectively.

The cells and soluble proteins removal ratio (Y) was defined using the following

Pt

(6)

P

re

Y=

-p

equation:

Where Pt and P are the cells and soluble proteins concentrations in the acetone-rich

Selection of the ATPS

ur

3.1.

Results and discussion

na

3.

lP

phase and the bioconversion broth, respectively.

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In order to choose an optimal ATPS, five organic solvents and five inorganic salts were evaluated based on their phase demixing and formation. The partition experiments in ATPS were performed at a 4-g scale. After shaking the mixture, the extraction efficiency of (S)-CMHA was investigated after 30 min, 1 h, 3 h and 5 h standing. The results showed that no increase in the extraction efficiency of (S)-CMHA was observed after standing the mixture for 30 min. The phase- forming

ability of these ATPS was determined by the abilities of the inorganic salts and hydrophilic organic solvents to capture water molecules [11]. As shown in Table 1, methanol and ethanol were not selected due to their poor phase-forming ability and formation of precipitation with inorganic salts. The precipitation of salts in bottom phase might be attributed to the high hydrophilicity of alcohols, which absorb water molecules surrounding the salt molecules in the [11]

. The phase formation

ro of

bioconversion broth and thus leading to the precipitation

abilities of the other three solvents were determined in the following order: acetone>1-propanol>2-propanol. Meanwhile, the boiling point of acetone is 56.53 oC,

-p

which is lower than that of 1-propanol (97 oC) and 2-propanol (82.6 oC). As acetone

re

can be much more easily recycled by evaporation, it was chosen as the organic solvent of ATPS.

lP

The effects of salts showed that ammonium sulfate system exhibited excellent

na

performance with partition coefficient, and could easily promote to form ATPS with hydrophilic organic solvent. Sodium carbonate showed poor phase-forming ability

ur

with the tested organic solvents, probably due to its low solubility comparing to the other salts

[11]

. Salts with high solubility can promote organic solvent to form ATPS

Jo

due to the high affinity towards water molecules

[26]

. The highest partition coefficient

and (S)-CMHA yield were obtained by acetone/ammonium sulfate system, which was selected for further study in the following experiments. Generally, the ATPS formation depends on the ability of the components to capture water molecules. The screened acetone/ammonium sulfate system in this work was found to be in

agreement with those reported ATPS for organic acids extraction [11].

3.2.

Phase diagram of ATPS

In order to determine the optimal ratio of acetone and ammonium sulfate concentrations at different turbid points, the phase diagrams were drawn with 50 g/L (S)-CMHA standard solution and deionized water separately. As shown in Fig. 3, two

ro of

zones were delineated by a curve for acetone/ammonium sulfate ATPS. The two-phase zone was above the binodal curve while the homogeneous zone was in the opposite direction. It can be observed that the trend of the two lines was basically

-p

same, but the phase diagram of (S)-CMHA was found to be slightly lower than that of

re

the deionized water, indicating that (S)-CMHA in solution could facilitate the

3.3.

lP

formation of ATPS.

Effect of pH on yield of (S)-CMHA

na

As described in previous studies, the formation of ATPS was significantly

ur

influenced by pH, since it played an important role in affecting the hydrophilicity by altering the electric charge properties

[27, 28]

. Moreover, (S)-CMHA was chemically

Jo

unstable under acidic or alkaline conditions due to the hydrolysis of cyano group to amide group

[29, 30]

. Therefore, it was essential to investigate the stability of

(S)-CMHA under different pH conditions. As shown in Fig. 4, when pH values below the pKa of (S)-CMHA (pKa 3.8), there was a drastic decrease in (S)-CMHA yield. The results showed that (S)-CMHA

was less stable in a strongly acidic condition than alkaline or neutral conditions, and the stability of (S)-CMHA was increased along with the increase of solution pH. Thus, the effect of pH on the extraction of (S)-CMHA in ATPS under neutral and alkaline conditions was examined. As aqueous pH increased from 7 to 12, the yield of (S)-CMHA was almost unchanged with a recovery of approximately 99%, suggesting that pH under neutral and alkaline conditions has no significant influence on

ro of

(S)-CMHA extraction (Fig. 5). Moreover, with pH varying under alkaline condition, significant influences of pH on phase equilibrium and distribution behaviors have not

been obviously observed. Therefore, initial bioconversion broth pH 8.0 was chosen as

Selection of optimal acetone/ammonium sulfate ATPS

re

3.4.

-p

extraction pH for further examination.

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The partition behavior of (S)-CMHA in acetone/ammonium sulfate ATPS was investigated with the centrifuged bioconversion broth (Fig. 6). When the

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concentration of ammonium sulfate increased from 8% to 25%, the partition

ur

coefficient of (S)-CMHA increased accordingly. It can be explained by the fact that increase in the mass of ammonium sulfate can promote the salting-out effect

[30]

,

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leading to improvement of exclusion of (S)-CMHA. As shown in Fig. 6, the recovery of (S)-CMHA seemed to be much more easily affected by increased concentrations of ammonium sulfate rather than acetone, suggesting that the change in recovery is an integrated result of both the phase volume ratio and partition coefficient

[32]

. The

phase volume ratio decreased from 3.25 to 1.28 with the increasing concentrations of

ammonium sulfate, whereas it increased from 0.78 to 3.42 with the increasing concentrations of acetone from 15% (w/w) to 45% (w/w). These results agree with the previous research on the organic acids purification using acetone/ammonium sulfate ATPS system [33]. The highest partition coefficient was achieved with a value of 58.46 at a concentration of 20% (w/w) ammonium sulfate and 35% (w/w) acetone, while the highest recovery of (S)-CMHA was 99.15% at the concentration of 15% (w/w)

3.5.

ro of

ammonium sulfate and 35% (w/w) acetone.

Removal of cells and proteins

-p

Cells and soluble proteins usually existed in the bioconversion broth, which need

re

to be removed via centrifugation or adsorption in the downstream processing of bio-products recovery. Previous study reported that this step could be eliminated by

lP

ATPS, with the cells and soluble proteins removal ratio of 99.63% and 85.90%, respectively [34]. Thus, the bioconversion broth was directly used for ATPS extraction [35]

na

. Most of the denatured cells and proteins were aggregated at the interface between

ur

the top and bottom phases. As shown in Fig.7, the removal ratio of soluble proteins was slightly enhanced when the concentration of acetone or ammonium sulfate was

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increased. At the ammonium sulfate concentration of 18% (w/w) and acetone 40% (w/w), the highest removed ratios of cells and proteins was achieved with a value of 99% and 86.27%, respectively. The concentration of soluble proteins in the top phase decreased from 0.92 g/L to 0.18 g/L. Removal of cells and proteins in this work is much better than the previous reports, which used polyacrylamide and chitosan as

flocculating agents [36].

3.6.

Back-extraction of (S)-CMHA with hydrophobic organic reagents

After the extraction step with acetone/ammonium sulfate ATPS, (S)-CMHA could be aggregated in the acetone-rich top phase. However, since unreacted (R)-IBSN (about half of added IBSN) dissolved in the organic phase, (S)-CMHA still

ro of

needs to be separated from the top phase to a new water phase. In this work, a back-extraction step was introduced to further separate (S)-CMHA from (R)-IBSN in the acetone-rich top phase.

-p

The key criterion for selection of a back-extraction solvent for (S)-CMHA

re

contains (i) easy to be recycled and reused (ii) cheap and convenient to use for large-scale industrial production and (iii) immiscible with the water and miscible with

lP

acetone so that the organic phase can be separated conveniently. Taking these account for (S)-CMHA back-extraction, three hydrophobic organic reagents, dichloromethane,

na

ethyl acetate and methyl tert-butyl ether were utilized for back-extraction of [37]

. A new two-phase was formed from the

ur

(S)-CMHA from the acetone-rich phase

separated acetone-rich phase after addition of back-extraction solvents, and

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(S)-CMHA was aggregated in the water phase. In order to increase the back-extraction efficiency, various volumetric ratio (acetone-rich phase: organic reagent) were tested. As shown in Fig. 8, higher (S)-CMHA yield was obtained by using dichloromethane as back-extraction solvent than that of ethyl acetate and methyl tert-butyl ether. The highest back extraction yield reached 92.11% at a volumetric ratio of acetone-rich

phase to dichloromethane 1.0, achieving a purity of 98.43% of (S)-CMHA. Furthermore, the unreacted (R)-IBSN can be recycled by evaporation and a recovery yield of 95% was obtained. In this case, dichloromethane is easier to be evaporated and cheaper than the other two solvents, making it the best choice for industrial application.

Recycling of ammonium sulfate

ro of

3.7.

In order to maintain the process sustainability and minimize the waste produced

in the extraction process, ammonium sulfate should be recycled. The recycling of

-p

ATPS forming components is feasible. Organic solvents can be recycled by [38, 39]

. Thus,

re

evaporation, while salts can be recovered by extractive crystallization

methanol was added into the salt-rich bottom phase for crystallization [26]. Ammonium

lP

sulfate could be precipitated out and easily recovered through filtration. As illustrated in Fig. 9, the result showed that the increase in methanol concentration could boost

na

the precipitation of ammonium sulfate, achieving a recovery of 93.10% when the

ur

volume of methanol was three times that of the salt-rich bottom phase. The methanol

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can be easily recycled through a simple distillation due to its low boiling point.

4.

Conclusion

In this paper, several ATPSs composed of inorganic salts and hydrophilic

solvents were investigated for their feasibility in extraction of (S)-CMHA from bioconversion broth. Among the tested five hydrophilic solvents and salts, ammonium

sulfate and acetone were found to be the best ATPS for extraction of (S)-CMHA. The recovery of (S)-CMHA seemed to be more relevant to the increased concentrations of ammonium sulfate rather than acetone. The highest recovery of (S)-CMHA reached 99.15% at the concentration of 15% (w/w) ammonium sulfate and 35% (w/w) acetone. The effect of pH on stability of (S)-CMHA was also investigated, and the result indicated that (S)-CMHA was more stable under neutral and alkaline conditions. With

ro of

a back-extraction step using dichloromethane as solvent, the recovery of (S)-CMHA reached 92.11% with the removal ratio of 99% and 86.27% for cells and proteins,

respectively. These obtained results demonstrated the developed two-step procedure

-p

as a promising strategy in the large-scale extraction of (S)-CMHA.

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Conflict of interest

The authors declare that they have no conflicts of interest with

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the contents of this article

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Acknowledgments

This work was supported by the National Natural Science Foundation of China

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(No. 21978269), Natural Science Foundation of Zhejiang Province (No. LR19B060001) and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13096).

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Figure captions:

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Fig. 1. Enzymatic kinetic resolution of 2-isobutylsuccinonitrile (IBSN) using

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nitrilases.

Fig. 2. Illustration of whole route for recovery of (S)-3-cyano-5-methylhexanoic acid

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((S)-CMHA) from the bioconversion broth.

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Fig. 3. Phase diagrams of acetone/ammonium sulfate system. Symbols: 50 g/L (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) (●); deionized water (■).

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Fig. 4. Effects of pH on the stability of (S)-CMHA. 0.1 M HCl or NaOH were added into the 50 g/L (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) to adjust the pH. All

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mixtures were prepared by weight with an uncertainty of ±10-4 g. The different pHs were 1 (■), 3 (●), 5 (▲), 7 (▼), 9 (◆), 11 (◀). Error bars show the deviation from the mean values of duplicate measurements. Fig. 5. Effect of pH on the recovery of (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) from bioconversion broth. The aqueous two-phase system (ATPS)

consisted of 20% (w/w) ammonium sulfate and 30% (w/w) acetone. Error bars show the deviation from the mean values of duplicate measurements. Fig. 6. (A) Effect of ammonium sulfate on the partition coefficients and (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) yield. The concentrations of acetone (w/w) were 25% (●), 30% (■) and 35% (▲). (B) Effect of acetone on the partition coefficients and (S)-CMHA yield from the centrifuged bioconversion broth.

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Concentrations of ammonium sulfate (w/w) were 10% (●), 15% (■), and 20% (▲).

Fig. 7. (A) Effect of ammonium sulfate on the removal ratio of cells and proteins. The aqueous two-phase system (ATPS) consisted of 10%-18% (w/w) ammonium sulfate

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and 35% (w/w) acetone. (B) Effect of acetone on the removal ratio of cells and

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proteins. The ATPS consisted of 20%-40% (w/w) acetone and 15% (w/w) ammonium sulfate. Error bars show the deviation from the mean values of duplicate

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measurements.

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Fig. 8. Back-extraction of (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) using non-miscible organic solvents under different volumetric ratio. The volumetric ratio

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of acetone-rich phase to the organic reagent increased from 0.8 to 1. The non-miscible organic solvents were dichloromethane (●), ethyl acetate (▲), methyl tert-butyl ether

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(■). Error bars show the deviation from the mean values of duplicate measurements. Fig. 9. Recovery of ammonium sulfate by adding methanol. The volumetric ratio of methanol added to the ammonium sulfate solution increased from 0.6 to 3.2.

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45 40 35 30 25 20 15 10 5 0

5

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15

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Acetone concentration (%, w/w)

50

20

25

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Ammonium sulfate concentration (%, w/w)

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Fig. 3

(S)-CMA yield (%)

100 90 80 70

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10

20

30

40

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Time (min)

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Fig. 4

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80

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Fig. 6 P a r tit io n c o f fic ie n t ( K ) 40

20

P a r tit io n c o f fic ie n t ( K )

(S )-C M H A y ie ld (% ) 98

96

94

92

(S )-C M H A y ie ld (% )

(A )

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A m m o n iu m s u lfa t e (% , w /w ) 10

A c e to n e ( % , w /w )

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(B )

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Removal ratio (%)

100 90 80 70 60 50 10

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14

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Ammonium sulfate (%, w/w)

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(B)

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Removal ratio (%)

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35

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Acetone (%, w/w)

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Fig. 7

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(S)-CMA yield (%)

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1.6

Volumetric ratio (acetone-rich phase: organic reagent)

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90 80 70 60 50

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Recovery of ammonium sulfate (%)

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2.0

2.5

3.0

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Volumetric ratio (methanol: ammonium sulfate)

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Fig. 9

K2HPO4·3H2O

NaH2PO4·2H2O

(NH4)2SO4

MgSO4

Na2CO3

Methanol

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K

Ethanol

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Table 1 Partitions of (S)-CMHA in different organic solvents/salt systems

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-

-

K

5.73

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E (%)

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E (%)

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92.11

1-propanol

-

K

18.20

12.03

25.75

3.57

E (%)

93.83

91.09

91.20

83.36

2-propanol

-

K

20.87

21.09

21.39

5.21

E (%)

87.67

90.41

86.64

75.78

Acetone

-

-

K

13.86

25.84

6.64

E (%)

92.10

95.96

90.70

a

Each system contains 2.0 g organic solvents, 2.0 g salt and 6 g centrifugated

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bioconversion broth.