Semi-rational Design Of ?-transaminase For High-efficient Preparation Of L-2-aminobutyric Acid

L-2-aminobutyric acid(L-ABA)is a kind of unnatural chiral amino acid.As an important chemical raw material and drug intermediate,L-2-aminobutyric acid is widely used in the synthesis of chemicals and drugs,such as antiepileptic drugs levetiracetam and buxactam,antituberculous drugs ethambutol,etc.The methods of L-2-aminobutyric acid production include chemical synthesis and biological synthesis.Chemical synthesis method is expensive and easy to cause environmental pollution,which is not conducive to industrial application.In contrast,biological synthesis is green and pollution-free,which is more suitable for the green synthesis of L-2-aminobutyric acid.L-threonine deaminase(TD,EC 4.2.1.6)and ?-transaminase is one of the most commonly used biosynthesis methods for the preparation of L-2-aminobutyric acid.The cheap substrates of the reaction are L-thr and isopropylamine.The by-product acetone is volatile,which is conducive to the forward reaction and the purification of L-2-aminobutyric acid.It is a popular solution to improve the activity of ammonia enzyme by molecular modification.Using ?-ketobutyric acid as a ligand to dock with ?-transaminase,six amino acid sites were selected for saturation mutation within 5 (?) in the range of ?-ketobutyric acid and active site,which were Y20,L57,W58,G229,A230 and M419,respectively.A total of 114 single point mutations were constructed.The activity of L57 A,L57C,A230 S and M419 I was increased by 1.6,2.7,1.7 and 2.7 times,respectively.The activity of double point mutation L57C/M419 I was increased by 3.2 times.In comparison with the docking pose of ?-ketobutyric acid in the wild-type enzyme,the altered binding of ?-ketobutyric acid in the active site of the mutant led to a significant decrease in the distance between carbonyl carbon of the substrate and the PMP from 4.420 (?) to 3.455 (?),which might contribute to the increased catalytic activity.Molecular dynamics(MD)simulations have been very useful for investigating of effects of mutations on protein structure and the impact of protein motions on catalysis.Molecular dynamics simulation results showed that the hydrophobic interaction between I419 and substrate was destroyed,and hydrogen bond was formed between C57 and ?-ketobutyric acid,which led to the transfer of carbonyl carbon to PMP.The increase of the accessibility and stability between the substrate and the active site of the mutant may lead to the increase of kcat.We then combined the most active double point mutation with L-threonine deaminase to prepare L-2-aminobutyric acid.In 50 m L reaction system,the conversion rate of 300 m M threonine substrate was 94.09% and the purity was 75.16% after 10 h,while the conversion rate of wild type was 70%.L5C/M419 I,a double mutant of ?-transaminase,has higher catalytic efficiency than wild type without changing the configuration of the product,In the process of one pot preparation of L-2-aminobutyric acid,the improvement of catalytic efficiency and conversion rate can reduce the purification cost of L-2-aminobutyric acid,which is efficient for industrial application.