转氨酶新酶的挖掘、表征及在2-氨基丁酸生物转化中的应用

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

摘要/Abstract

摘要：

目的通过挖掘高性能转氨酶和引入丙酮酸代谢酶形成催化级联体系抑制逆反应活性，提升产物L-2-氨基丁酸转化水平。方法利用基因挖掘技术在数据库中对转氨酶进行大规模挖掘，以2-酮丁酸作为底物，筛选高效转氨酶，并对新酶进行生化分析，表征酶学性质，通过建立全细胞生物转化和催化级联体系调控反应平衡，减缓逆反应水平，提升产物转化率。结果从数据库中发现了源自大肠杆菌的Ec4a转氨酶，以2-酮丁酸为底物，Ec4a的最适温度为45 ℃，最适pH值为9.0，比酶活1.25 U/mg，酶蛋白的熔融温度（T_m值）为68.2 ℃。酶蛋白在55 ℃和70 ℃下的酶活半衰期分别为321 min和150 min。在pH 8.5的条件下孵育6 h相对酶活剩余59%。在全细胞催化体系中两种底物最佳的浓度比为1∶1。分别以30 mmol/L的2-酮丁酸和30 mmol/L的L-Ala为底物，在菌体为OD₆₀₀=10的条件下，2-氨基丁酸的转化率为37.5%。引入枯草芽胞杆菌乙酰乳酸合成酶（Bsalss）可以消耗副产物丙酮酸抑制逆反应，形成体外级联后相同全细胞催化体系下，转化率提高至61.4%。结论挖掘到稳定性好的转氨酶Ec4a，建立并优化2-氨基丁酸的全细胞生物转化体系，并通过引入Bsalss构建级联体系提高转化率至61.4%。

关键词: 转氨酶, L-2-氨基丁酸, 酶学性质, 生物催化, 逆反应消除

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

叶妍, 吴雨萱, 周哲敏, 崔文璟. 转氨酶新酶的挖掘、表征及在2-氨基丁酸生物转化中的应用[J]. 生物技术通报, 2025, 41(11): 134-142.

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.

图/表 6

表1 本研究中使用的主要菌株和质粒

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

图1 转氨酶的结构聚类和序列同源性分析以及重组表达SDS-PAGEA：结构聚类； B：同源性分析； C：重组表达SDS-PAGE （b代表菌液，s代表破碎菌液上清）

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)

图2 不同转氨酶菌株全细胞催化转化率测定

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

图3 Ec4a的最适温度，最适pH，温度稳定性和pH稳定性A：最适温度；B：最适pH；C：温度稳定性；D：pH稳定性

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

图4 Ec4a的熔融温度

Fig. 4 The melting temperature of Ec4a

图5 全细胞催化体系优化和生物催化级联体系A：不同底物配比转化率； B：不同底物浓度转化率； C：级联体系示意图； D：对照组催化转化率

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

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