Citation: SHI Hongling, WANG Wenjie, LI Xiang, JIAO Zhujin, LIU Yue, TANG Cunduo, KAN Yunchao, YAO Lunguang. Enantioselective Biosynthesis of L-Phenylglycine via Cascade Biocatalysis of D-Mandelate Dehydrogenase and L-Leucine Dehydrogenase[J]. Chinese Journal of Applied Chemistry, ;2020, 37(2): 168-174. doi: 10.11944/j.issn.1000-0518.2020.02.190189 shu

Enantioselective Biosynthesis of L-Phenylglycine via Cascade Biocatalysis of D-Mandelate Dehydrogenase and L-Leucine Dehydrogenase

SHI Hongling ^a,‡ ,
WANG Wenjie ^a ,
LI Xiang ^a ,
JIAO Zhujin ^a ,
LIU Yue ^b ,
TANG Cunduo ^a,b,, ,
KAN Yunchao ^a ,
YAO Lunguang ^a

a.
Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Jiangnan University, Wuxi, Jiangsu 214122, China
b.
State Key Laboratory of Automotive Biofuel Technology, Nanyang, Henan 473061, China

Corresponding author: TANG Cunduo, tcd530@126.com

^‡

Received Date: 8 July 2019
Revised Date: 22 October 2019
Accepted Date: 20 November 2019

Fund Project: the State Key Laboratory of Motor Vehicle Biofuel Technology KFKT2018003Supported by the National Natural Science Foundation of China(No.3190110303, No.31870917), the State Key Laboratory of Motor Vehicle Biofuel Technology(No.KFKT2018003), and the Special Funded Projects of Nanyang Normal University(No.2018QN004)the National Natural Science Foundation of China 31870917the Special Funded Projects of Nanyang Normal University 2018QN004the National Natural Science Foundation of China 3190110303

Figures(7)

L-Phenylglycine is an important class of chiral non-natural amino acids, and can be used to synthesize a variety of important pharmaceutical intermediate. Exploiting the green synthesis process of phenylacetone acid has significant economic value. In this study, novel and highly active D-mandelate dehydrogenase (LhDMDH) and L-leucine dehydrogenase (EsLeuDH) are coupled to achieve bio-transformation of D-mandelic acid into L-phenylglycine on the premise of coenzyme circulation, and this reaction only requires a lower concentration of coenzyme. By optimizing the transformation conditions including added amount of enzyme, β-nicotinamide adenine dinucleotide (NAD⁺) concentration, NH₄⁺ concentration and substrate concentration, we obtain the most economical condition:200 mmol/L D-mandelic acid, 6.5 kU/L enzyme, 0.1 mmol/L NAD ⁺, 0.5 mol/L NH₄⁺ and 30℃ for 12 h. The product yield and enantionmeric excess (e.e.) value can reach more than 98% and 99% under the most economical condition, respectively. This transformation has large industrialization potential, and lays a solid foundation for achieving large-scale biosynthesis of L-phenylglycine.
- L-Phenylglycine,
- D-mandelate dehydrogenase,
- L-leucine dehydrogenase,
- biocatalysis,
- cascade reaction
1. [1]
  Yasukawa K, Asano Y. Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi-enzyme System[J]. Adv Synth Catal, 2012,354(17):3327-3332. doi: 10.1002/adsc.201100923
2. [2]
  Fan C W, Xu G C, Ma B D. A Novel D-Mandelate Dehydrogenase Used in Three Enzyme Cascade Reaction for Highly Efficient Synthesis of Non-natural Chiral Amino Acids[J]. J Biotechnol, 2015,195:67-71. doi: 10.1016/j.jbiotec.2014.10.026
3. [3]
  Wang Z M, Kolb H C, Sharpless K B. Large-Scale and Highly Enantioselective Synthesis of the Taxol C-13 Side Chain Through Asymmetric Dihydroxylation[J]. J Org Chem, 1994,59(17):5104-5105. doi: 10.1021/jo00096a072
4. [4]
  Han S W, Shin J S. One-Pot Preparation of d-Amino Acids Through Biocatalytic Deracemization Using Alanine Dehydrogenase and ω-Transaminase[J]. Catal Lett, 2018,148(12):3678-3684. doi: 10.1007/s10562-018-2565-3
5. [5]
  Resch V, Fabian W M F, Kroutil W. Deracemisation of Mandelic Acid to Optically Pure Non-natural L-Phenylglycine via a Redox-Neutral Biocatalytic Cascade[J]. Adv Synth Catal, 2010,352(6):993-997. doi: 10.1002/adsc.200900891
6. [6]
  FAN Changwei. Gene Mining of D-Mandelate Dehydrogenase and Its Application in the Synthesis of L-Phenylglycine by Multi-enzyme Conjugation[D]. East China University of Science and Technology, 2014(in Chinese).
7. [7]
  Bornscheuer U T, Huisman G W, Kazlauskas R J. Engineering the Third Wave of Biocatalysis[J]. Nature, 2012,485(7397):185-194. doi: 10.1038/nature11117
8. [8]
  TANG Cunduo, SHI Hongling, HE Zihan. Green Biosynthesis of Phenylglyoxylic Acid by Biotransformation Using Recombinant Escherichia Coli Whole Cells[J]. CIESC J, 2018,69(6):2627-2631.
9. [9]
  Tang C D, Shi H L, Xu J H. Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel D-Mandelate Dehydrogenase with High Catalytic Activity[J]. J Agric Food Chem, 2018,66(11):2805-2811. doi: 10.1021/acs.jafc.7b05835
10. [10]
  Gokhale D V, Bastawde K B, Patil S G. Chemoenzymatic Synthesis of D-Phenylglycine Using Hydantoinase of Pseudomonas Desmolyticum Resting Cells[J]. Enzyme Microb Technol, 1996,18(5):353-357. doi: 10.1016/0141-0229(95)00127-1
11. [11]
  Wiyakrutta S, Meevootisom V. A Stereo-Inverting D-Phenylglycine Aminotransferase from Pseudomonas Stutzeri ST-201:Purification, Characterization and Application for D-Phenylglycine Synthesis[J]. J Biotechnol, 1997,55(3):193-203. doi: 10.1016/S0168-1656(97)00075-8
12. [12]
  Mast Y J, Wohlleben W, Schinko E. Identification and Functional Characterization of Phenylglycine Biosynthetic Genes Involved in Pristinamycin Biosynthesis in Streptomyces Pristinaespiralis[J]. J Biotechnol, 2011,155(1):63-67. doi: 10.1016/j.jbiotec.2010.12.001
13. [13]
  Li D, Zeng Z, Yang J. Mandelate Racemase and Mandelate Dehydrogenase Coexpressed Recombinant Escherichia Coli in the Synthesis of Benzoylformate[J]. Biosci Biotechnol Biochem, 2013,77(6):1236-9. doi: 10.1271/bbb.121012
14. [14]
  Chen F F, Liu Y Y, Zheng G W. Asymmetric Amination of Secondary Alcohols by Using a Redox-Neutral Two-Enzyme Cascade[J]. ChemCatChem, 2015,7(23):3838-3841. doi: 10.1002/cctc.201500785
15. [15]
  TANG Cunduo, SHI Hongling, MA Yue. Gene Mining, Expression and Characterization of Novel R-Mandelate Dehydrogenases[J]. China Biotechnol, 2018,38(2):30-37.
16. [16]
  Gong X M, Qin Z, Li F L. Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor[J]. ACS Catal, 2019,9(1):147-153.
17. [17]
  Zhou J, Wang Y, Xu G. Structural Insight into Enantioselective Inversion of an Alcohol Dehydrogenase Reveals a "Polar Gate" in Stereorecognition of Diaryl Ketones[J]. J Am Chem Soc, 2018,140(39):12645-12654. doi: 10.1021/jacs.8b08640
18. [18]
  Tang C D, Shi H L, Jiao Z J. Exploitation of Cold-Active Cephalosporin C Acylase by Computer-Aided Directed Evolution and Its Potential Application in Low-Temperature Biosynthesis of 7-Aminocephalosporanic Acid[J]. J Chem Technol Biotechnol, 2018,93(10):2925-2930. doi: 10.1002/jctb.5647
1. [1]
  Yerong Chen , Bingbin Yang , Xinglei He , Yuqi Lin , Keyin Ye . Enzyme-Directed Evolution Enables Bioconversion of Organosilicon Compounds. University Chemistry, 2025, 40(10): 121-129. doi: 10.12461/PKU.DXHX202411054
2. [2]
  Zhaoxin LI , Ruibo WEI , Min ZHANG , Zefeng WANG , Jing ZHENG , Jianbo LIU . Advancements in the construction of inorganic protocells and their cell mimic and bio-catalytical applications. Chinese Journal of Inorganic Chemistry, 2024, 40(12): 2286-2302. doi: 10.11862/CJIC.20240235
3. [3]
  Shaohua Wu , Jianfeng Yan , Chunfa Xu , Yuanming Li , Kai Yang , Caixia Lin . Construction of the synthetic chemistry experiment course under the Chemistry “101 Plan”: instructional design and implementation of the L-proline-catalyzed asymmetric aldol condensation reaction experiment. University Chemistry, 2026, 41(6): 152-159. doi: 10.12461/PKU.DXHX202511100
4. [4]
  Yingying Wang , Yang Yu . 基于统计能量分析的酶催化机理探究：以丝氨酸水解酶为例. University Chemistry, 2026, 41(9): 373-381. doi: 10.12461/PKU.DXHX202508066
5. [5]
  Hong Lu , Yidie Zhai , Xingxing Cheng , Yujia Gao , Qing Wei , Hao Wei . Advancements and Expansions in the Proline-Catalyzed Asymmetric Aldol Reaction. University Chemistry, 2024, 39(5): 154-162. doi: 10.3866/PKU.DXHX202310074
6. [6]
  Heng Zhang . Determination of All Rate Constants in the Enzyme Catalyzed Reactions Based on Michaelis-Menten Mechanism. University Chemistry, 2024, 39(4): 395-400. doi: 10.3866/PKU.DXHX202310047
7. [7]
  Li'na ZHONG , Jingling CHEN , Qinghua ZHAO . Synthesis of multi-responsive carbon quantum dots from green carbon sources for detection of iron ions and L-ascorbic acid. Chinese Journal of Inorganic Chemistry, 2025, 41(4): 709-718. doi: 10.11862/CJIC.20240280
8. [8]
  Yu Dai , Xueting Sun , Haoyu Wu , Naizhu Li , Guoe Cheng , Xiaojin Zhang , Fan Xia . Determination of the Michaelis Constant for Gold Nanozyme-Catalyzed Decomposition of Hydrogen Peroxide. University Chemistry, 2025, 40(5): 351-356. doi: 10.12461/PKU.DXHX202407052
9. [9]
  Jian Li , Yu Wu , Wenling Gu , Chengzhou Zhu . 纳米酶生物医学应用研究进展. University Chemistry, 2026, 41(9): 173-181. doi: 10.12461/PKU.DXHX202509068
10. [10]
  Meiyu Lin , Yuxin Fang , Songzhang Shen , Yaqian Duan , Wenyi Liang , Chi Zhang , Juan Su . Exploration and Implementation of a Dual-Pathway Blended Teaching Model in General Chemistry Experiment Course: A Case Study of Copper Glycine Synthesis and Its Thermal Analysis. University Chemistry, 2024, 39(8): 48-53. doi: 10.3866/PKU.DXHX202312042
11. [11]
  Yuai Duan , Xuanyu Gan , Yao Fu , Yingjie Cao , Hongliang Han , Zhanfang Ma . Application and Innovative Design of Digital Technology in the Preparation Experiment of Cis(Trans)-Diglycine Copper Complexes. University Chemistry, 2026, 41(1): 373-381. doi: 10.12461/PKU.DXHX202504048
12. [12]
  Xuejie Wang , Guoqing Cui , Congkai Wang , Yang Yang , Guiyuan Jiang , Chunming Xu . Research Progress on Carbon-based Catalysts for Catalytic Dehydrogenation of Liquid Organic Hydrogen Carriers. Acta Physico-Chimica Sinica, 2025, 41(5): 100044-0. doi: 10.1016/j.actphy.2024.100044
13. [13]
  Zhi Zheng , Feiyang Liu , Junlong Zhao . D-Amino Acids and Mirror-Image Proteins. University Chemistry, 2026, 41(2): 353-359. doi: 10.12461/PKU.DXHX202505017
14. [14]
  Lele Feng , Xueying Bai , Jifeng Pang , Hongchen Cao , Xiaoyan Liu , Wenhao Luo , Xiaofeng Yang , Pengfei Wu , Mingyuan Zheng . Single-atom Pd boosted Cu catalysts for ethanol dehydrogenation. Acta Physico-Chimica Sinica, 2025, 41(9): 100100-0. doi: 10.1016/j.actphy.2025.100100
15. [15]
  Feiyang Liu , Liuhong Song , Miaoyu Fu , Zhi Zheng , Gang Xie , Junlong Zhao . Tryptophan’s Employment Journey. University Chemistry, 2024, 39(9): 16-21. doi: 10.12461/PKU.DXHX202404037
16. [16]
  Hanxue LIU , Shijie LI , Meng REN , Xuling XUE , Hongke LIU . Design and antitumor properties of dehydroabietic acid functionalized cyclometalated iridium(Ⅲ) complex. Chinese Journal of Inorganic Chemistry, 2025, 41(8): 1483-1494. doi: 10.11862/CJIC.20250031
17. [17]
  Yingchun ZHANG , Yiwei SHI , Ruijie YANG , Xin WANG , Zhiguo SONG , Min WANG . Dual ligands manganese complexes based on benzene sulfonic acid and 2, 2′-bipyridine: Structure and catalytic properties and mechanism in Mannich reaction. Chinese Journal of Inorganic Chemistry, 2024, 40(8): 1501-1510. doi: 10.11862/CJIC.20240078
18. [18]
  Yihui Song , Shangshang Qin , Kai Wu , Chengyun Jin , Bin Yu . 生物化学在高水平创新型药学人才培养中的交叉融合应用——以去甲基化酶LSD1抑制剂的活性评价为例. University Chemistry, 2025, 40(6): 341-352. doi: 10.12461/PKU.DXHX202406018
19. [19]
  Wenwen Ma , Lian Kong , Jinyang Chu , Li Ma , Ziqing Ma , Heyu Cheng , Xinyuan Li , Zhan Yu , Zhen Zhao . Digitalization-Driven Olefin Production: Digital Design of Catalysts for CO₂-Assisted Oxidation Dehydrogenation of Ethane to Ethylene. University Chemistry, 2026, 41(1): 363-372. doi: 10.12461/PKU.DXHX202506055
20. [20]
  Bingnan Wang , Duanyang Kong . 对“脱氢乙酸钠”译名的异议. University Chemistry, 2026, 41(10): 413-415. doi: 10.12461/PKU.DXHX202510070

Get Citation

PDF

XML

Metrics

PDF Downloads(9)
Abstract views(1538)
HTML views(272)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142