Exploration， Characterization， and Application of Transaminase New Enzymes in the Biocatalytic Conversion of 2-aminobutyric Acid

doi:10.13560/j.cnki.biotech.bull.1985.2025-0223

Abstract

Abstract:

Objective To enhance the conversion level of the product L-2-aminobutyric acid by exploring high-performance transaminases and introducing pyruvate metabolic enzymes to form a catalytic cascade system to inhibit the reverse reaction activity. Method Gene mining technology was used to conduct large-scale mining of transaminases in the database, with 2-ketobutyric acid as the substrate to screen for efficient transaminases. The new enzymes were subjected to biochemical analysis to characterize their enzymatic properties. By establishing a whole-cell biotransformation and catalytic cascade system to regulate reaction equilibrium and reduce the level of reverse reactions, the product conversion rate increased. Result A transaminase named Ec4a from Escherichia coli was discovered in the database, with 2-ketobutyric acid as the substrate. The optimal temperature for Ec4a was 45 ℃, the optimal pH value was 9.0, and the specific enzyme activity was 1.25 U/mg. The melting temperature (T_m value) of the enzyme protein was 68.2 ℃. The half-life of the enzyme protein at 55 ℃ and 70 ℃ was 321 min and 150 min respectively. Incubated under pH 8.5 conditions for 6 h, the relative enzyme activity remained at 59%. In the whole-cell catalytic system, the optimal concentration ratio of the two substrates was 1∶1. When 30 mmol/L of 2-ketobutyric acid and 30 mmol/L of L-Ala were used as substrates under the condition of a bacterial OD₆₀₀, the conversion rate of 2-aminobutyric acid was 37.5%. The introduction of Bacillus subtilis acetolactate synthase (Bsalss) consumed the byproduct pyruvate to inhibit the reverse reaction. After forming an in vitro cascade, the conversion rate increased to 61.4% under the same whole-cell catalytic system. Conclusion This study identified the stable transaminase Ec4a, established and optimized the whole-cell biotransformation system of 2-aminobutyric acid, and improved the conversion rate to 61.4% by introducing Bsalss to construct a cascade system.

Key words: transaminase, L-2-aminobutyric acid, enzymatic properties, biocatalysis, reverse reaction elimination

YE Yan, WU Yu-xuan, ZHOU Zhe-min, CUI Wen-jing. Exploration， Characterization， and Application of Transaminase New Enzymes in the Biocatalytic Conversion of 2-aminobutyric Acid[J]. Biotechnology Bulletin, 2025, 41(11): 134-142.

Figures/Tables 6

Table 1 Main strains and plasmids used in this study

菌株与质粒 Strain and plasmid	性质 Properties	来源 Source
E.coli BL21（DE3）	表达宿主	实验室保存
E.coli JM109	克隆宿主	实验室保存
pET-28a-EsRTA	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-Ec4a	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-Bs	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-CbRTA	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-AtRTA	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-TsRTA	His 标签、Kan 抗性、T7 启动子	本实验构建
pET-28a-TsRTA	His 标签、Kan 抗性、T7 启动子	本实验构建
Pbad-Bsalss	AmpR 抗性、araBAD 启动子	本实验构建

Fig. 1 Structural clustering and sequence homology analysis of transaminases and recombinant expression of SDS-PAGEA: Structural clustering; B: sequence homology analysis; C: recombinant expression of SDS-PAGE (b indicates bacterial liquid, s indicates supernatant of lysed bacterial liquid)

Fig. 2 Determination of whole-cell catalytic conversion rates of different transaminase strains

Fig. 3 Optimal temperature, optimal pH, temperature stability and pH stability of Ec4aA: Optimal temperature for Ec4a. B: Optimal pH for Ec4a. C: Temperature stability of Ec4a. D: pH stability of Ec4a

Fig. 4 The melting temperature of Ec4a

Fig. 5 Optimization of whole-cell catalytic systems and biocatalysis cascade systemsA: Conversion rates under different substrate ratios. B: Conversion rates under different substrate concentrations. C: Schematic diagram of the cascading system. D: Catalytic conversion rate of the control group

References 33

[1]	Kelly SA, Pohle S, Wharry S, et al. Application of ω-transaminases in the pharmaceutical industry [J]. Chem Rev, 2018, 118(1): 349-367.
[2]	蔡雪, 孙晨阳, 翟增春, 等. 磷酸吡哆醛依赖型酶的研究进展及其应用[J]. 生物工程学报, 2024, 40(9): 2771-2785.
	Cai X, Sun CY, Zhai ZC, et al. Research progress and applications of pyridoxal phosphate-dependent enzymes [J]. Chinese Journal of Biotechnology, 2024, 40(9): 2771-2785.
[3]	Slabu I, Galman JL, Lloyd RC, et al. Discovery, engineering, and synthetic application of transaminase biocatalysts [J]. ACS Catalysis, 2017, 7(12): 8263-8284.
[4]	Farkas E, Sátorhelyi P, Szakács Z, et al. Transaminase-catalysis to produce trans-4-substituted cyclohexane-1-amines including a key intermediate towards cariprazine [J]. Commun Chem, 2024, 7(1): 86.
[5]	夏温娜, 孙雨, 闵聪, 等. 转氨酶催化不对称合成芳香族L-氨基酸 [J]. 生物工程学报, 2012, 28(11): 1346-1358.
	Xia WN, Sun Y, Min C, et al. Asymmetric synthesis of aromatic L-amino acids catalyzed by transaminase [J]. Chin J Biotechnol, 2012, 28(11): 1346-1358.
[6]	Park ES, Dong JY, Shin JS. ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor [J]. Org Biomol Chem, 2013, 11(40): 6929-6933.
[7]	Babu KC, Reddy RB, Mukkanti K, et al. Enantioselective synthesis of antiepileptic agent, (-)-levetiracetam, through Evans Asymmetric strategy [J]. Journal of Chemistry, 2013, 72(64): 13846-13855.
[8]	Chen JJ, Zhu RS, Zhou JP, et al. Efficient single whole-cell biotransformation for L-2-aminobutyric acid production through engineering of leucine dehydrogenase combined with expression regulation [J]. Bioresour Technol, 2021, 326: 124665.
[9]	张利坤, 肖延铭, 杨卫华, 等. 亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸 [J]. 生物工程学报, 2020, 36(5): 992-1001.
	Zhang LK, Xiao YM, Yang WH, et al. Synthesis of L-2-aminobutyric acid by leucine dehydrogenase coupling with an NADH regeneration system [J]. Chin J Biotechnol, 2020, 36(5): 992-1001.
[10]	奚强, 丁友友, 林丫丫, 等. 2-氨基丁酸的酶拆分[J]. 武汉工程大学学报, 2009, 31(3): 26-29.
	Xi Q, Ding YY, Lin YY, et al. Enzymatic resolution of 2-aminobutyric acid [J]. Journal of Wuhan Institute of Technology, 2009, 31(3): 26-29.
[11]	Liu YF, Han LC, Cheng ZY, et al. Enzymatic biosynthesis of L-2-aminobutyric acid by glutamate mutase coupled with L-Aspartate-β-decarboxylase using L-glutamate as the sole substrate [J]. ACS Catalysis, 2020, 10(23): 13913-13917.
[12]	Shin JS, Kim BG. Transaminase-catalyzed asymmetric synthesis of L-2-aminobutyric acid from achiral reactants [J]. Biotechnol Lett, 2009, 31(10): 1595-1599.
[13]	Tao RS, Jiang Y, Zhu FY, et al. A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH-regeneration system based on L-leucine dehydrogenase and formate dehydrogenase [J]. Biotechnol Lett, 2014, 36(4): 835-841.
[14]	周俊平. L-2-氨基丁酸的微生物高效制备及关键酶的理性改造 [D]. 无锡:江南大学, 2019.
	Zhou JP, Microbial high-efficiency preparation of L-2-aminobutyric acid and rational modification of key enzymes [D]. Wuxi: Jiangnan University, 2019.
[15]	Zhang ZW, Liu Y, Zhao J, et al. Active-site engineering of ω- transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid [J]. BMC Biotechnol, 2021, 21(1): 55.
[16]	Luo W, Hu JG, Lu JP, et al. One pot cascade synthesis of L-2-aminobutyric acid employing ω-transaminase from Paracoccus pantotrophus [J]. Mol Catal, 2021, 515: 111890.
[17]	Cui YM, Gao YD, Yang LC. Transaminase catalyzed asymmetric synthesis of active pharmaceutical ingredients [J]. Green Synth Catal, 2024.
[18]	Gomm A, O'Reilly E. Transaminases for chiral amine synthesis [J]. Curr Opin Chem Biol, 2018, 43: 106-112.
[19]	Pinto A, Contente ML, Tamborini L. Advances on whole-cell biocatalysis in flow [J]. Curr Opin Green Sustain Chem, 2020, 25: 100343.
[20]	Lin BX, Tao Y. Whole-cell biocatalysts by design [J]. Microb Cell Fact, 2017, 16(1): 106.
[21]	胡佳桂. ω-转氨酶基因挖掘、级联反应体系构建及其应用 [D]. 无锡: 江南大学, 2021.
	Hu JG. Gene mining of ω-aminotransferase, construction of cascade reaction system and its application [D]. Wuxi: Jiangnan University, 2021.
[22]	Jiang JJ, Chen X, Zhang DL, et al. Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast [J]. Appl Microbiol Biotechnol, 2015, 99(6): 2613-2621.
[23]	Kelly SA, Mix S, Moody TS, et al. Transaminases for industrial biocatalysis: novel enzyme discovery [J]. Appl Microbiol Biotechnol, 2020, 104(11): 4781-4794.
[24]	蒙丽钧. 转氨酶在大肠杆菌中的克隆表达及应用 [D]. 杭州: 浙江大学, 2019.
	Meng LJ. Cloning and expression of transaminase in Escherichia coli and its application [D]. Hangzhou: Zhejiang University, 2019.
[25]	蔡婷婷, 曹佳仁, 邱帅, 等. 半理性设计进化土曲霉来源的ω-转氨酶AtTA热稳定性 [J]. 生物工程学报, 2023, 39(6): 2126-2140.
	Cai TT, Cao JR, Qiu S, et al. Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability [J]. Chin J Biotechnol, 2023, 39(6): 2126-2140.
[26]	Meng QL, Ramírez-Palacios C, Wijma HJ, et al. Protein engineering of amine transaminases [J]. Frontiers in Catalysis, 2022, 2. DOI: 10.3389/fctls.2022.1049179
[27]	Khatik AG, Muley AB, More PR, et al. Transaminase-mediated chiral selective synthesis of (1R)-(3-methylphenyl)ethan-1-amine from 1-(3-methylphenyl)ethan-1-one: process minutiae, optimization, characterization and ‘What If studies’ [J]. Bioprocess Biosyst Eng, 2023, 46(2): 207-225.
[28]	郑裕国, 程峰, 金利群, 等. 一种转氨酶突变体及其生产L-草铵膦的应用:中国,CN201810540686.1 [P]. 2018-05-30.
	Zheng YG, Cheng F, Jin LQ, et al. A transaminase mutant and its application in the production of L-phosphinothricin: China, CN201810540686.1 [P]. 2018-05-30.
[29]	李仲霞, 刘妍, 罗泉, 等. 从专利角度分析ω-转氨酶在我国手性胺生物合成应用中的研究进展 [J]. 生物工程学报, 2023, 39(8): 3169-3187.
	Li ZX, Liu Y, Luo Q, et al. The advance of ω-transaminase in chiral amine biosynthesis in China from the perspective of patents [J]. Chin J Biotechnol, 2023, 39(8): 3169-3187.
[30]	Cárdenas-Fernández M, Sinclair O, Ward JM. Novel transaminases from thermophiles: from discovery to application [J]. Microb Biotechnol, 2022, 15(1): 305-317.
[31]	Wang ZC, Xu ML, Xie YY, et al. One-pot two-stage biocatalytic cascade to produce l-phosphinothricin by two enantioselective complementary aminotransferases at high substrate loading via a deracemization process [J]. J Agric Food Chem, 2024.
[32]	Höhne M, Kühl S, Robins K, et al. Efficient asymmetric synthesis of chiral amines by combining transaminase and pyruvate decarboxylase [J]. Chembiochem, 2008, 9(3): 363-365.
[33]	Guo F, Berglund P. Transaminase biocatalysis: optimization and application [J]. Green Chem, 2017, 19(2): 333-360.