Redesign of ( R )-Omega-Transaminase and Its Application for Synthesizing Amino Acids with Bulky Side Chain
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY(2021)
Abstract
ω-Transaminase (ω-TA) is an attractive biocatalyst for stereospecific preparation of amino acids and derivatives, but low catalytic efficiency and unfavorable substrate specificity hamper their industrial application. In this work, to obtain applicable ( R )-ω-TA responsible for amination of α-keto acids substrates, the reactivities of eight previously synthesized ω-TAs toward pyruvate using ( R )-α-methylbenzylamine (( R )-α-MBA) as amine donor were investigated, and Gibberella zeae TA (GzTA) with the highest ( R )-TA activity and stereoselectivity was selected as starting scaffold for engineering. Site-directed mutagenesis around enzymatic active pocket and access tunnel identified three positive mutation sites, S214A, F113L, and V60A. Kinetic analysis synchronously with molecular docking revealed that these mutations afforded desirable alleviation of steric hindrance for pyruvate and α-MBA. Furthermore, the constructed single-, double-, and triple-mutant exhibited varying degrees of improved specificities toward bulkier α-keto acids. Using 2-oxo-2-phenylacetic acid ( 1d ) as substrate, the conversion rate of triple-mutant F113L/V60A/S214A increased by 3.8-fold relative to that of wide-type GzTA. This study provided a practical engineering strategy for improving catalytic efficiency and substrate specificity of ( R )-ω-TA. The obtained experience shed light on creating more industrial ω-TAs mutants that can accommodate structurally diverse substrates. Graphical abstract
MoreTranslated text
Key words
ω-Transaminase,Site-directed mutagenesis,Catalytic efficiency,Substrate specificity
AI Read Science
Must-Reading Tree
Example
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined