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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
首发时间：2025-03-14
摘要：【目的】转氨酶（transaminase,ta）是一类催化氨基转移反应的酶，可用于合成众多手性氨类化合物以及非天然氨基酸，广泛用于生物医药、生物化工等领域。然而，大多数转氨酶活性较低、反应平衡不适宜等问题，导致合成和转化率不高，迫切需要高性能转氨酶来提升产物转化水平。【方法】本研究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（tm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ph8.5的条件下孵育6h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 mm的2-酮丁酸和30 mm的l-ala为底物，在菌体为od600的条件下，2-氨基丁酸的转化率为37.5%。引入枯草芽孢杆菌乙酰乳酸合成酶（bsalss）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】本研究为挖掘高效转氨酶以及建立合成2-氨基丁酸的全细胞生物转化体系提供了重要的技术依据。
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exploration, characterization, and application of transaminase new enzymes in the biocatalytic conversion of 2-aminobutyric acid
abstract：【objective】transaminases (tas) are a class of enzymes that catalyze the transfer of amino groups, and can be used for the synthesis of numerous chiral amines and unnatural amino acids. they are widely applied in the fields of biomedicine and biochemical engineering. however, most transaminases have low activity and an unfavorable reaction equilibrium, leading to low synthesis and conversion rates. there is an urgent need for high-performance transaminases to enhance product conversion levels. 【methods】in this study, 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 was increased.【results】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 (tm 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 hours, 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 mm of 2-ketobutyric acid and 30 mm of l-ala were used as substrates under the condition of a bacterial od600, the conversion rate of 2-aminobutyric acid was 37.5%. after introducing the bacillus subtilis acetolactate synthase (bsalss) into the in vitro cascade, the conversion rate increased to 61.4% under the same whole-cell catalytic system.【conclusion】this study provides important technical basis for the mining of efficient transaminases and the establishment of a whole-cell biotransformation system for the synthesis of 2-aminobutyric acid.
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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
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