Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Article abstract of DOI:10.1080/10826068.2015.1045611

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5?g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

Full text of DOI:10.1080/10826068.2015.1045611

This article was downloaded by: [University of Sussex Library]
On: 11 August 2015, At: 02:53
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place,
London, SW1P 1WG
Preparative Biochemistry and Biotechnology
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lpbb20
Highly Selective Anti-Prelog Synthesis of Optically
Active Aryl Alcohols by Recombinant Escherichia coli
Expressing Stereospecific Alcohol Dehydrogenase
Ming Lia, Yao Niea, Xiao Qing Mua, Rongzhen Zhanga & Yan Xuab
a School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of
Education, Jiangnan University, Wuxi, China
b State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China
Accepted author version posted online: 15 Jul 2015.
Click for updates
To cite this article: Ming Li, Yao Nie, Xiao Qing Mu, Rongzhen Zhang & Yan Xu (2015): Highly Selective Anti-Prelog Synthesis of
Optically Active Aryl Alcohols by Recombinant Escherichia coli Expressing Stereospecific Alcohol Dehydrogenase, Preparative
Biochemistry and Biotechnology
To link to this article: http://dx.doi.org/10.1080/10826068.2015.1045611
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service
to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting,
typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of
the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Highly selective anti-Prelog synthesis of optically active aryl alcohols by
recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase
Ming Li1, Yao Nie1, Xiao Qing Mu1, Rongzhen Zhang1, Yan Xu1,2
1School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of
Education, Jiangnan University, Wuxi, China, 2State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi, China
Address correspondence to Prof. Yao Nie and Prof. Yan Xu, School of Biotechnology and
Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan
University, 214122, Wuxi, China. Email: ynie@jiangnan.edu.cn and
yxu@jiangnan.edu.cn
Abstract
Biocatalytic asymmetric synthesis has been widely used for preparation of optically
active chiral alcohol as the important intermediates and precursors of active
pharmaceutical ingredients. However, the available whole-cell system involving
anti-Prelog specific alcohol dehydrogenase is yet limited. Recombinant Escherichia coli
system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida
parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction
of aryl ketones to anti-Prelog configurated alcohols. Using 2-hydroxyacetophenone as the
substrate, reaction factors including pH, cell status, and substrate concentration had
obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be
an available auxiliary substrate for intracellular cofactor regeneration, by which
(S)-1-phenyl-1,2-ethanediol was achieved with the optical purity of 97%e.e. and yield of
1

89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the
recombinant cells towards different aryl ketones was investigated, and most of the
corresponding chiral alcohol products were obtained with the optical purity over 95%e.e.
Therefore, the whole-cell system involving recombinant stereospecific alcohol
dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective
anti-Prelog synthesis of optically active aryl alcohols and would be promising in
pharmaceutical industry.
KEYWORDS: Alcohol dehydrogenase, anti-Prelog reduction, aryl ketone, asymmetric
synthesis, optically active alcohol, active pharmaceutical ingredient
INTRODUCTION
Microbial oxidoreductive systems involving stereospecific oxidoreductases has gained
increasing relevance in organic synthesis for the production of optically active
alcohols,[1,2] such as 1-phenyl-1,2-ethanediol (PED) and its derivatives, the valuable and
versatile chiral building blocks for synthesis of pharmaceuticals, agrochemicals, and
pheromones, etc.[3,4] The asymmetric reduction of prochiral carbonyl compounds to their
corresponding alcohols is potentially one of the most useful ways of introducing chirality
in a molecule. For those chiral auxiliaries production from their corresponding prochiral
ketones, alcohol dehydrogenases have the inherent advantages over chemo-catalysts in
terms of their highly chemo-, enantio- and regioselectivity.[5,6] These features make
2

stereospecific alcohol dehydrogenases very interesting from both scientific and industrial
perspectives.
Currently, however, most enzymes catalyzing reductions generally follow the Prelog’s
rule in the sense of the stereochemistry outcomes, and another type of biocatalysts with a
complementary selectivity are yet limited.[7] Only several microorganisms were found to
possess anti-Prelog selectivity.[8] Especially for optically active PED production, only
Geotrichum sp. was reported to catalyze anti-Prelog reduction of 2-hydroxyacetophenone
to (S)-PED (91%e.e., yield 62%) with substrate concentration of 2.72 g/L.[9] Therefore,
the application of enzymes catalyzing anti-Prelog type reaction would be valuable for
filling the demands of asymmetric synthesis to produce chiral alcohols in different
configuration.
The key issue to biocatalytic process architecture defined in a particular case is whether
the catalyst is to be used as a whole cell or an isolated enzyme. For cofactor-dependent
alcohol dehydrogenases, the alternative whole-cell process will prove the preferred
option.[10,11] Moreover, whole cell biocatalysts are usually more stable due to the
protective cell matrix envelope for the enzyme.[12] Although whole cells typically contain
multiple oxidoreductases, leading to mixed stereoselectivity and side reactions by other
competing enzymes, the interference of other enzymes could be avoided by expressing
3

the desired enzyme in a recombinant system, and an easy way for cofactor regeneration
can be afforded in organism cells.[13]
In this work, recombinant Escherichia coli system expressing the identified anti-Prelog
stereospecific alcohol dehydrogenase from Candida parapsilosis (CPADH) was adopted
to create a whole-cell system for catalyzing the reduction of prochiral carbonyl
compounds to anti-Prelog configurated alcohols in high optical purity.[14,15] Additionally,
the factors of the recombinant cell-mediated reaction system were optimized and the
availability of the recombinant cells involving stereospecific alcohol dehydrogenase was
investigated towards kinds of aryl ketones.
EXPERIMENTAL
Materials
2-Hydroxyacetophenone (1a) and its derivatives including
o-chloro-2-hydroxyacetophenone (2a), m-chloro-2-hydroxyacetophenone (3a),
p-chloro-2-hydroxyacetophenone (4a), p-methoxy-2-hydroxyacetophenone (5a),
p-nitro-2-hydroxyacetophenone (6a), and acetophenone (7a), and the corresponding
chiral alcohols (1b-7b) were purchased from ACROS Co. Ltd. or Sigma-Aldrich
Chemical Co. Coenzymes including NAD(P)H and NAD(P)+ were purchased from
Sigma-Aldrich Chemical Co. All other chemicals used in this work were of analytical
grade and commercially available.
4

Preparation Of Recombinant Cells For Synthesis
Recombinant E. coli BL21(DE3)(pETCPADH) expressing CPADH was constructed and
cultivated as described previously.[15] After cultivation, the cells harvested by
centrifugation at 10000 rpm for 30 min and washed twice with physiological saline
solution were used as the wet cells. The lyophilized cells of recombinant E. coli were
prepared by Freeze Dry System/Freezone 4.5 (Labconco Co.) and stored at 4 ºC for
further use.
Synthesis Of Chiral Aryl Alcohols By Recombinant E. Coli
The reaction mixture in 2 mL was comprised of 0.1 M potassium phosphate buffer (pH
7.0), 2 mg substrate (1a-7a), and 0.2 g wet cells (equivalent to 0.03 g dry cell weight) or
0.05 g lyophilized cells of the recombinant E. coli transformant. For the substrates not
completely soluble in potassium phosphate buffer, 100 μL of isopropanol was applied as
co-solvent. To investigate the effects of auxiliary substrates, 10 mg of xylose, arabinose,
glucose, fructose, lactose, sucrose, maltose, or 10 μL of isopropanol was added to the
reaction mixture respectively. The reactions were carried out at 30 °C for 48 h with
shaking at 200 rpm. The reaction mixture was extracted with 3 volumes of ethyl acetate
and the organic layer was subjected to chiral HPLC analysis.
5

Analysis Of Chiral Aryl Alcohols Products
The reaction products were analyzed by chiral HPLC (HP 1100, Agilent, USA) equipped
with Chiralcel OB-H column or OD-H column (4.6 mm × 250 mm; Daicel Chemical Ind.
Ltd., Japan). Enantiomers were eluted with hexane and isopropanol at 0.5 mL/min. The
effluent was monitored at 215 nm and the areas under each peak were integrated to
calculate conversion, yield, and enantiomeric excess (e.e.) percentage values.[14,16] The
detailed conditions for analysis of substrates and chiral alcohol products were shown in
Table 1. All the values shown in this study were averaged from three replicates with
standard deviations.
RESULTS AND DISCUSSION
Effects Of Reaction System Components And Conditions
The whole cells of recombinant E. coli harboring the overexpressed CPADH were
applied for catalytic asymmetric reduction of 2-hydroxyacetophenone (1a). For
biocatalytic systems involving stereospecific enzymes, reaction pH generally has an
influence on the stereochemistry of enzymatic reactions.[17] Thus the effect of reaction pH
on the reduction of 1a using recombinant E. coli cells was investigated with the pH
values ranging from 4.0 to 9.0 (Figure 1). The stereospecificity of this CPADH-harboring
whole-cell system was shown to be pH-dependent, which afforded higher optical purity
(97%e.e.) and yield (83%) of (S)-1b at pH 7.0. However, the optimum pH of such
whole-cell system catalyzing asymmetric reduction was different to that of isolated
6

CPADH,[15] which might be due to the difference of micro environmental pH between in
vitro and in vivo in the reaction system with respect to the tuning of cell. Using whole
cells as biocatalyst, the cell status including wet cells or lyophilized cells would have an
influence on the biocatalytic performance and efficiency. It has been reported that
lyophilization of cells might have an influence on the permeability of the cell wall or
membrane and even the protein structure of intracellular enzymes.[18] Therefore,
compared with wet cells, lyophilized cells were applied in the reaction system to
investigate the effect of cell status on reaction efficiency by whole cells. Lyophilized cells
of the recombinant E. coli only afforded the corresponding chiral alcohol product with
optical purity of 72%e.e. and did not exhibit significant improvement of catalytic
performance, compared with wet cells, which might be due to the change of steric
structure and molecular rigidity of the stereospecific enzyme when processed by freeze
drying.[19]
In asymmetric reduction of 1a, substrate inhibition occurred with the recombinant E. coli
cell-mediated reaction. The apparent conversions and yields decreased at elevated
substrate concentrations, while the space-time yield (expressed as mg of (S)-1b produced
per liter of reaction solution per hour) increased, reaching a maximum at the substrate
concentration of 3 g/L (Figure 2). This result indicated that the activity of the key enzyme
in whole cells might be affected upon long-term exposure to reaction surroundings
involving non-naturally organic compounds. However, the stereoselectivity of the
7

enzyme still remained at elevated substrate concentrations and optically active (S)-1b was
obtained.
To improve the reaction efficiency at higher substrate concentrations, the amount of the
biocatalyst was increased accordingly. Using 20% (w/v) recombinant E. coli cells, (S)-1b
was obtained from 1a 3 g/L in a yield of 78.2%. Thus, applying more cells to the reaction
would be helpful to facilitate the asymmetric reduction. Nevertheless, overloading of the
cells in reaction would make the mixture slimy and breed negative influence to the
system homogenization and substance transference in reaction. Besides, for
cofactor-requiring oxidoreductions, the substrate inhibition might also result from
insufficient cofactor regeneration.[20]
Co-Substrate-Coupled Cofactor Regeneration
From the cofactor requirement point of view, feasible cofactor regeneration would be
necessary for improvement of the stereospecific oxidoreduction efficiency.[20] For
whole-cell system using viable cells, cofactor-related cellular machinery and metabolic
pathway in cell would be feasible for cofactor regeneration and thus co-substrate-coupled
approach was further considered to take advantage of cellular machinery for cofactor
regeneration. Compared with other sugars and alcohols commonly used for
substrate-coupled cofactor regeneration, addition of xylose or arabinose significantly
improved the reaction efficiency of asymmetric reduction of 1a to (S)-1b in an enhanced
8

yield over 85% (Figure 3). Then the effect of xylose concentration on the conversion by
E. coli cells was further investigated. Addition of xylose more than 5 g/L would inhibit
the reaction to some extent, which might result from substrate inhibition of xylose to the
cellular metabolism. When 5 g/L xylose was used, (S)-1b was afforded with the optical
purity of 97%e.e. in an increased yield of 89% with a higher substrate concentration of 5
g/L by 10% (w/v) recombinant E. coli wet cells. Concerning pentose metabolism in cells,
the fact that xylose drove the equilibrium of NADPH-requiring reduction towards the
desired product indicated that in vivo xylose metabolism would take place in the pentose
phosphate pathway, and simultaneously NADPH, which facilitates the asymmetric
reduction to produce (S)-1b, could be regenerated via pentose metabolism route and used
for CPADH catalyzing asymmetric reduction of 1a.[21]
Asymmetric Synthesis Of Chiral Aryl Alcohols
For enzymatic reactions, the structural variety of substrates generally have a significant
influence on the activity and stereoselectivity of enzymes by reason that the functional
groups in substrate affect the substrate bonding and orientation in enzyme and the steric
interactions between substrate and the active sites of enzyme.[22] Therefore, the feasibility
of recombinant E. coli bearing CPADH was investigated concerning its substrate
spectrum towards aryl ketones with different chemical structures (Table 2). For
substituted 2-hydroxyacetophenones bearing chloro at various position of the phenyl ring
(2a-4a), the ortho-chloro (2a) substitution was a somewhat poor substrate for the
9

recombinant E. coli cells, indicating that the substituent at ortho position might have a
steric effect on the hydrogen attack from electron donator NADPH to the carbonyl group
and significant influence on the reactivity of the functional enzyme. Another trend
observed for substrates with a single substitution at para position on the phenyl ring was
that electron-withdrawing groups provided for better efficiency than electron-donating
groups. p-Chloro-substituted 4a and p-nitro-substituted 6a were more suitable for
CPADH catalyzing asymmetric reduction than p-methoxy-substituted
2-hydroxyacetophenone (5a). These results indicated that the enzyme selectivity
markedly depends on the nature, i.e. the electronic property and the position of the
substituent group.[9] With anti-Prelog selectivity, CPADH expressed in recombinant E.
coli catalyzed asymmetric reduction of various 2-hydroxy aryl ketones in anti-Prelog type,
affording optical active aryl diols in (S)-configuration, whereas (R)-antipode for
acetophenone (7a) due to the switch in CIP priority.
CONCLUSION
In this study, the recently cloned and overexpressed CPADH in E. coli was employed for
the highly enantioselective reduction of a series of substituted 2-hydroxy aryl ketone
derivatives to anti-Prelog configurated chiral alcohols. In addition, the enzyme was
suggested to distinguish subtle steric and electronic differences in aryl ketone substrates,
performing different enantioselectivities and catalytic activities to aryl ketones with
various substituents. Besides the influences of reaction conditions, otherwise, available
10

pentoses were discovered as potent auxiliary substrates to improve the bioreduction
efficiency. From these consequences, this whole-cell system involving recombinant
stereospecific alcohol dehydrogenase was proposed as an efficient biocatalyst for
enantioselective reduction and the symmetry breaking potential makes it attractive tool
for synthesis of optically active aryl alcohols.
FUNDING
Financial supports from the National Key Basic Research and Development Program of
China (973 Program) (2011CB710800), the National High Technology Research and
Development Program of China (863 Program) (2011AA02A209 and 2011AA02A210),
the National Natural Science Foundation of China (NSFC) (21376107 and 21336009),
the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program
(GDW20133200113), the Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions, and the Jiangsu province
"Collaborative Innovation Center for Advanced Industrial Fermentation" industry
development program are greatly appreciated.
REFERENCES
1. Panke, S.; Held, M.; Wubbolts, M. Trends and Innovations in Industrial Biocatalysis
for the Production of Fine Chemicals. Curr. Opin. Biotechnol. 2004, 15, 272–279.
11

2. Jing, K.; Xu, Z.; Liu, Y.; Jiang, X.; Peng, L.; Cen, P. Efficient Production of
Recombinant Aldehyde Reductase and its Application for Asymmetric Reduction of Ethyl
4-Chloro-3-Oxobutanoate to Ethyl (R)-4-Chloro-3-Hydroxybutanoate. Prep. Biochem.
Biotechnol. 2005, 35, 203–215.
3. Liese, A.; Karutz, M.; Kamphui, J.; Wandrey, C.; Kragl, U. Resolution of
1-Phenyl-1,2-Ethanediol by Enantioselective Oxidation Overcoming Product Inhibition
by Continuous Extraction. Biotechnol. Bioeng. 1996, 51, 544–550.
4. Soyer, A.; Bayraktar, E.; Mehmetoglu, Ü. Optimization of Lipase-Catalyzed
Enantioselective Production of 1-Phenyl 1-Propanol Using Response Surface
Methodology. Prep. Biochem. Biotechnol. 2010, 40, 389–404.
5. Jeromin, G.E. Superabsorbed Alcohol Dehydrogenase – a New Catalyst for
Asymmetric Reductions. Biotechnol. Lett. 2009, 31, 1717–1721.
6. Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Recent Advances in the Biocatalytic
Reduction of Ketones and Oxidation of Sec-Alcohols. Curr. Opin. Chem. Biol. 2004, 8,
120–126.
7. Liang, P.; Qin, B.; Mu, M.; Zhang, X.; Jia, X.; You, S. Prelog and Anti-Prelog
Stereoselectivity of Two Ketoreductases from Candida glabrata. Biotechnol. Lett. 2013,
35, 1469–1473.
8. Hu, J.; Xu, Y. Anti-Prelog Reduction of Prochiral Carbonyl Compounds by
Oenococcus oeni in a Biphasic System. Biotechnol. Lett. 2006, 28, 1115–1119.
12

9. Wei, Z.L.; Lin, G.Q.; Li, Z.Y. Microbial Transformation of 2-Hydroxy and 2-Acetoxy
Ketones with Geotrichum sp. Bioorg. Med. Chem. 2000, 8, 1129–1137.
10. Burton, S.G.; Cowan, D.A.; Woodley, J.M. The Search for the Ideal Biocatalyst.
Nature Biotechnol. 2002, 20, 37–45.
11. Rocha, L.C.; Ferreira, H.V.; Pimenta, E.F.; Berlinck, R.G.S.; Seleghim, M.H.R.;
Javaroti, D.C.D.; Sette, L.D.; Bonugli, R.C.; Porto, A,L.M. Bioreduction of
a-Chloroacetophenone by Whole Cells of Marine Fungi. Biotechnol. Lett. 2009, 31,
1559–1563.
12. Yang, G.S.; Xu, J.Y.; Ou, Z.M.; Yao, S.J. Asymmetric Reduction of
(S)-3-Chloro-1-Phenylpropanol from 3-Chloropropiophenone by Preheated Immobilized
Candida utilis. Biotechnol. Lett. 2009, 31, 1879–1883.
13. Schroer, K.; Tacha, E.; Lütz, S. Process Intensification for Substrate-Coupled Whole
Cell Ketone Reduction by In Situ Acetone Removal. Org. Process. Res. Dev. 2007, 11,
836–841.
14. Nie, Y.; Xu, Y.; Mu, X.Q. Highly Enantioselective Conversion of Racemic
1-Phenyl-1,2-Ethanediol by Stereoinversion Involving a Novel Cofactor-Dependent
Oxidoreduction System of Candida parapsilosis CCTCC M203011. Org. Process Res.
Dev. 2004, 8, 246–251.
15. Nie, Y.; Xu, Y.; Mu, X.Q.; Wang, H.Y.; Yang, M.; Xiao, R. Purification,
Characterization, Gene cloning, and Expression of a Novel Alcohol Dehydrogenase with
13

Anti-Prelog Stereospecificity from Candida parapsilosis. Appl. Environ. Microbiol. 2007,
73, 3759–3764.
16. Nie, Y.; Xiao, R.; Xu, Y.; Montelione, G.T. Novel Anti-Prelog Stereospecific
Carbonyl Reductases from Candida parapsilosis for Asymmetric Reduction of Prochiral
Ketones. Org. Biomol. Chem. 2011, 9, 4070–4078.
17. Secundo, F.; Phillips, R.S. Effects of pH on Enantiospecificity of Alcohol
Dehydrogenases from Thermoanaerobacter ethanolicus and Horse Liver. Enzyme Microb.
Technol. 1996, 19, 487–492.
18. Tattini, V.Jr.; Parra, D.F.; Polakiewicz, B.; Pitombo, R.N.M. Effect of Lyophilization
on the Structure and Phase Changes of PEGylated-Bovine Serum Albumin. Int. J. Pharm.
2005, 304, 124–134.
19. Ru, M.T.; Dordick, J.S.; Reimer, J.A.; Clark, D.S. Optimizing the Salt-Induced
Activation of Enzymes in Organic Solvents: Effects of Lyophilization Time and Water
Content. Biotechnol. Bioeng. 1999, 63, 233–241.
20. Cai, P.; An, M.; Xu, L.; Xu, S.; Hao, N.; Li, Y.; Guo, K.; Yan, M. Development of a
Substrate-Coupled Biocatalytic Process Driven by an NADPH-Dependent Sorbose
Reductase from Candida albicans for the Asymmetric Reduction of Ethyl
4-Chloro-3-Oxobutanoate. Biotechnol. Lett. 2012, 34, 2223–2227.
21. Nie, Y.; Xu, Y.; Hu, Q.S.; Xiao, R. A New Strategy to Improve the Efficiency and
Sustainability of Candida parapsilosis Catalyzing Deracemization of
14

(R,S)-1-Phenyl-1,2-Ethanediol under Non-Growing Conditions: Increase of NADPH
Availability. J. Microbiol. Biotechnol. 2009, 19, 65–71.
22. Zhou, S.; Zhang, S.C.; Lai, D.Y.; Zhang, S.L.; Chen, Z.M. Biocatalytic
Characterization of a Short-Chain Alcohol Dehydrogenase with Broad Substrate
Specificity from Thermophilic Carboxydothermus hydrogenoformans. Biotechnol. Lett.
2013, 35, 359–365.
15

Table 1. Analysis of substrates and chiral alcohol products by HPLC
Condition
Retention time (min)
OB-H column, hexane/isopropanol (90:10, v/v)
OD-H column, hexane/isopropanol (90:10, v/v)
OD-H column, hexane/isopropanol (90:10, v/v)
OD-H column, hexane/isopropanol (90:10, v/v)
OD-H column, hexane/isopropanol (90:10, v/v)
OD-H column, hexane/isopropanol (85:15, v/v)
OB-H column, hexane/isopropanol (90:10, v/v)
15.0 (R-1b), 18.3 (S-1b), 27.1 (1a)
12.8 (R-2b), 13.7 (S-2b), 22.5 (2a)
14.1 (R-3b), 15.5 (S-3b), 24.1 (3a)
14.3 (R-4b), 18.7 (S-4b), 22.0 (4a)
12.5 (R-5b), 13.7 (S-5b), 23.7 (5a)
14.2 (R-6b), 18.5 (S-6b), 23.3 (6a)
16.4 (S-7b), 18.0 (R-7b), 19.4 (7a)
16

Table 2. Recombinant E. coli-Catalyzed Anti-Prelog Synthesis of Optically Active Aryl
Alcohols
Substrate Product Conversion (%) Optical purity (%e.e.)
1a
2a
3a
4a
5a
6a
7a
(S)-1b
(S)-2b
(S)-3b
(S)-4b
(S)-5b
(S)-6b
(R)-7b
83±2
58±3
61±1
81±2
69±1
78±3
82±2
97±1
81±2
95±3
96±2
91±3
95±4
97±2
Note. Reactions were carried out at 30 °C for 48 h in 2 mL 0.1 M potassium phosphate
buffer (pH 7.0) comprising 2 mg substrate (1a-7a) and 0.2 g wet cells of the recombinant
E. coli transformant. For the substrate of 7a, switch of stereo-configuration of the
corresponding product was defined according to the CIP priority.
17

Figure 1. Effects of reaction pH on product optical purity (◆) and yield (□) of 1a
reduction by recombinant E. coli. The reactions were carried out using recombinant E.
coli wet cells (10%, w/v) with 1a 1 g/L in 2 mL 0.1 M acetic acid buffer (pH 4.0-6.0),
potassium phosphate buffer (pH 6.0-8.0), or Tris-HCl buffer (pH 8.0-9.0) at 30 °C for 48
h.
18

Figure 2. Effects of substrate concentration (1a) on conversion (◆), yield (▲), and
space-time yield (■). The space-time yield was expressed as the amount of (S)-1b (mg)
produced per liter of reaction solution per hour by recombinant E. coli wet cells (10%,
w/v) in 2 mL 0.1 M potassium phosphate buffer (pH7.0) at 30 °C for 48 h.
19

Figure 3. Effects of different sugars and alcohols as auxiliary substrates on product
optical purity (□) and yield (■) of 1a reduction by recombinant E. coli. The reactions
were carried out using recombinant E. coli wet cells (10%, w/v) with 1a 3 g/L in 2 mL 0.1
M potassium phosphate buffer (pH7.0) at 30 °C for 48 h.
20