Enzymatic characterization of a recombinant carbonyl reductase from Acetobacter sp. CCTCC M209061

Abstract

Background

Acetobacter sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from Acetobacter sp. CCTCC M209061 needs to be investigated.

Results

A CR from Acetobacter sp. CCTCC M209061 (AcCR) was cloned and expressed in E. coli. AcCR was purified and characterized, finding that AcCR as a dual coenzyme-dependent short-chain dehydrogenase/reductase (SDR) was more preferred to NADH for biocatalytic reactions. The AcCR was activated and stable when the temperature was under 35 °C and the pH range was from 6.0 to 8.0 for the reduction of 4′-chloroacetophenone with NADH as coenzyme, and the optimal temperature and pH were 45 °C and 8.5, respectively, for the oxidation reaction of isopropanol with NAD⁺. The enzyme showed moderate thermostability with half-lives of 25.75 h at 35 °C and 13.93 h at 45 °C, respectively. Moreover, the AcCR has broad substrate specificity to a range of ketones and ketoesters, and could catalyze to produce chiral alcohol with e.e. >99% for the majority of tested substrates following the anti-Prelog rule.

Conclusions

The recombinant AcCR exhibited excellent enantioselectivity, broad substrate spectrum, and highly stereoselective anti-Prelog reduction of prochiral ketones. These results suggest that AcCR is a powerful catalyst for the production of anti-Prelog alcohols.

Keywords

Carbonyl reductase / Acetobacter sp. / Chiral alcohols / Enzymatic characterization

References

[1] Adler JJ, Singh PK, Patist A, Rabinovich YI, Shah DO, Moudgil BM. Correlation of particulate dispersion stability with the strength of self-assembled surfactant films. Langmuir, 2000, 16: 7255-7262.

[2] Birolli WG, Ferreira IM, Alvarenga N, Santos DDA, de Matos IL, Comasseto JV, Porto ALM. Biocatalysis and biotransformation in Brazil: an overview. Biotechnol Adv, 2015, 33: 481-510.

[3] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-254.

[4] Chen R, Liu X, Wang JL, Lin JP, Wei DZ. Cloning, expression, and characterization of an anti-Prelog stereospecific carbonyl reductase from Gluconobacter oxydans DSM2343. Enzyme Microb Technol, 2015, 70: 18-27.

[5] Cheng J, Lou W, Zong M. Biocatalytic asymmetric oxidation of racemic 1-(4-methoxyphenyl) ethanol using immobilized Acetobacter sp CCTCC M209061 cells in organic solvent-containing biphasic system. Chem J Chin Univ Chin, 2014, 35: 1529-1535.

[6] Cui ZM, Zhang JD, Fan XJ, Zheng GW, Chang HH, Wei WL. Highly efficient bioreduction of 2-hydroxyacetophenone to (S)- and (R)-1-phenyl-1,2-ethanediol by two substrate tolerance carbonyl reductases with cofactor regeneration. J Biotechnol, 2017, 243: 1-9.

[7] Dako E, Jankowski CK, Bernier A-M, Asselin A, Simard RE. A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris. Int J Food Microbiol, 2008, 126: 186-194.

[8] Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, Ladenstein R, Jornvall H, Oppermann U. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem, 2002, 277: 25677-25684.

[9] Grosch J-H, Loderer C, Jestel T, Ansorge-Schumacher M, Spieß AC. Carbonyl reductase of Candida parapsilosis—stability analysis and stabilization strategy. J Mol Catal B Enzym, 2015, 112: 45-53.

[10] Guo R, Nie Y, Mu XQ, Xu Y, Xiao R. 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, 2014, 105: 66-73.

[11] Gutierrez MC, Ferrer ML, Yuste L, Rojo F, del Monte F. Bacteria incorporation in deep-eutectic solvents through freeze-drying. Angew Chem Int Ed, 2010, 49: 2158-2162.

[12] He S, Wang Z, Zou Y, Chen S, Xu X. Purification and characterization of a novel carbonyl reductase involved in oxidoreduction of aromatic β-amino ketones/alcohols. Process Biochem, 2014, 49: 1107-1112.

[13] He YC, Zhang DP, Di JH, Wu YQ, Tao ZC, Liu F, Zhang ZJ, Chong GG, Ding Y, Ma CL. Effective pretreatment of sugarcane bagasse with combination pretreatment and its hydrolyzates as reaction media for the biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by whole cells of E. coli CCZU-K14. Bioresour Technol, 2016, 211: 720-726.

[14] Itoh N. Use of the anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols. Appl Microbiol Biotechnol, 2014, 98: 3889-3904.

[15] Jörnvall H, Hedlund J, Bergman T, Oppermann U, Persson B. Superfamilies SDR and MDR: from early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities. Biochem Biophys Res Commun, 2010, 396: 125-130.

[16] Kaluzna IA, Rozzell JD, Kambourakis S. Ketoreductases: stereoselective catalysts for the facile synthesis of chiral alcohols. Tetrahedron Asymmetry, 2005, 16: 3682-3689.

[17] Kavanagh KL, Jornvall H, Persson B, Oppermann U. Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci, 2008, 65: 3895-3906.

[18] Leuchs S, Greiner L. Alcohol dehydrogenase from Lactobacillus brevis: a versatile robust catalyst for enantioselective transformations. Chem Biochem Eng Q, 2011, 25: 267-281.

[19] Li BJ, Li YX, Bai DM, Zhang X, Yang HY, Wang J, Liu G, Yue JJ, Ling Y, Zhou DS, Chen HP. Whole-cell biotransformation systems for reduction of prochiral carbonyl compounds to chiral alcohol in Escherichia coli. Sci Rep, 2014, 4: 6750.

[20] Li A, Ye L, Wu H, Yang X, Yu H. Characterization of an excellent anti-Prelog short-chain dehydrogenase/reductase EbSDR8 from Empedobacter brevis ZJUY-1401. J Mol Catal B Enzym, 2015, 122: 179-187.

[21] Liu X, Chen R, Yang Z, Wang J, Lin J, Wei D. Characterization of a putative stereoselective oxidoreductase from Gluconobacter oxydans and its application in producing ethyl (R)-4-chloro-3-hydroxybutanoate ester. Mol Biotechnol, 2014, 56: 285-295.

[22] Luo X, Wang YJ, Zheng YG. Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461. Enzym Microb Technol, 2015, 77: 68-77.

[23] Ma CW, Zhang L, Dai JY, Xiu ZL. Characterization and cofactor binding mechanism of a novel NAD(P)H-dependent aldehyde reductase from Klebsiella pneumoniae DSM2026. J Microbiol Biotechnol, 2013, 23: 1699-1707.

[24] Masud U, Matsushita K, Theeragool G. Molecular cloning and characterization of two inducible NAD(+)-adh genes encoding NAD(+)-dependent alcohol dehydrogenases from Acetobacter pasteurianus SKU1108. J Biosci Bioeng, 2011, 112: 422-431.

[25] Montfort L, Frenette G, Sullivan R. Sperm-zona pellucida interaction involves a carbonyl reductase activity in the hamster. Mol Reprod Dev, 2002, 61: 113-119.

[26] Musa MM, Phillips RS. Recent advances in alcohol dehydrogenase-catalyzed asymmetric production of hydrophobic alcohols. Catal Sci Technol, 2011, 1: 1311.

[27] Nakajin S, Minamikawa N, Baker ME, Toyoshima S. An NADPH-dependent reductase in neonatal pig testes that metabolizes androgens and xenobiotics. Biol Pharm Bull, 1998, 21: 1356-1360.

[28] Ni Y, Li CX, Zhang J, Shen ND, Bornscheuer UT, Xu JH. Efficient reduction of ethyl 2-oxo-4-phenylbutyrate at 620 g/L⁻¹ by a bacterial reductase with broad substrate spectrum. Adv Synth Catal, 2011, 353: 1213-1217.

[29] Persson B, Kallberg Y. Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs). Chem Biol Interact, 2013, 202: 111-115.

[30] Persson B, Kallberg Y, Oppermann U, Jörnvall H. Coenzyme-based functional assignments of short-chain dehydrogenases/reductases (SDRs). Chem Biol Interact, 2003, 143–144: 271-278.

[31] Qian XL, Pan J, Shen ND, Ju X, Zhang J, Xu JH. Efficient production of ethyl (R)-2-hydroxy-4-phenylbutyrate using a cost-effective reductase expressed in Pichia pastoris. Biochem Eng J, 2014, 91: 72-77.

[32] Richter N, Hummel W. Biochemical characterisation of a NADPH-dependent carbonyl reductase from Neurospora crassa reducing alpha- and beta-keto esters. Enzyme Microb Technol, 2011, 48: 472-479.

[33] Sengupta D, Naik D, Reddy AR. Plant aldo-keto reductases (AKRs) as multi-tasking soldiers involved in diverse plant metabolic processes and stress defense: a structure-function update. J Plant Physiol, 2015, 179: 40-55.

[34] Singh A, Bhattacharyya MS, Banerjee UC. Purification and characterization of carbonyl reductase from Geotrichum candidum. Process Biochem, 2009, 44: 986-991.

[35] Takeuchi M, Kishino S, Park S-B, Kitamura N, Ogawa J. Characterization of hydroxy fatty acid dehydrogenase involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a. J Mol Catal B Enzym, 2015, 117: 7-12.

[36] Tang TX, Liu Y, Wu ZL. Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49. J Mol Catal B Enzym, 2014, 105: 82-88.

[37] Wang XT, Yue D-M, Zong MH, Lou WY. Use of ionic liquid to significantly improve asymmetric reduction of ethyl acetoacetate catalyzed by Acetobacter sp. CCTCC M209061 cells. Ind Eng Chem Res, 2013, 52: 12550-12558.

[38] Wang YJ, Liu XQ, Luo X, Liu ZQ, Zheng YG. Cloning, expression and enzymatic characterization of an aldo-keto reductase from Candida albicans XP1463. J Mol Catal B Enzym, 2015, 122: 44-50.

[39] Weckbecker A, Hummel W. Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir. Biocatal Biotransform, 2009, 24: 380-389.

[40] Wei P, Xu P, Wang XT, Lou WY, Zong MH. Asymmetric reduction of ethyl acetoacetate catalyzed by immobilized Acetobacter sp CCTCC M209061 cells in hydrophilic ionic liquid hybrid system. Biotechnol Bioprocess Eng, 2015, 20: 324-332.

[41] Wei P, Liang J, Cheng J, Zong M-H, Lou W-Y. Markedly improving asymmetric oxidation of 1-(4-methoxyphenyl) ethanol with Acetobacter sp CCTCC M209061 cells by adding deep eutectic solvent in a two-phase system. Microb Cell Fact, 2016, 15: 5.

[42] Xu Q, Xu X, Huang H, Li S. Efficient synthesis of (R)-2-chloro-1-phenylethol using a yeast carbonyl reductase with broad substrate spectrum and 2-propanol as cosubstrate. Biochem Eng J, 2015, 103: 277-285.

[43] Ye Q, Yan M, Yao Z, Xu L, Cao H, Li Z, Chen Y, Li S, Bai J, Xiong J, Ying H, Ouyang P. A new member of the short-chain dehydrogenases/reductases superfamily: purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis. Biores Technol, 2009, 100: 6022-6027.

[44] Zhang BB, Lou WY, Zong MH, Wu H. 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, 2008, 54: 122-129.

[45] Zhang R, Geng Y, Xu Y, Zhang W, Wang S, Xiao R. Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity. Biores Technol, 2011, 102: 483-489.

[46] Zhang Y, Ujor V, Wick M, Ezeji TC. Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052. Anaerobe, 2015, 33: 124-131.

[47] Zheng YG, Yin HH, Yu DF, Chen X, Tang XL, Zhang XJ, Xue YP, Wang YJ, Liu ZQ. Recent advances in biotechnological applications of alcohol dehydrogenases. Appl Microbiol Biotechnol, 2017, 101: 987-1001.

Funding

the National Natural Science Foundation of China(21336002)

the Fundamental Research Funds for the Chinese Universities(2015PT002)

the Program of State Key Laboratory of Pulp and Paper Engineering(2017ZD05)

the Open Funding Project of the State Key Laboratory of Bioreactor Engineering