2020 ; Vol.30-1: 146~154
|Author||Wei Jiang, Yali Wang|
|Place of duty||Department of Bioengineering and Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen, Fujian, 361021, P.R. China|
|Title||Improving Catalytic Efficiency and Changing Substrate Spectrum for Asymmetric Biocatalytic Reductive Amination|
J. Microbiol. Biotechnol.2020 ;
|Abstract||With the advantages of biocatalytic method, enzymes have been excavated for the synthesis of
chiral amino acids by the reductive amination of ketones, offering a promising way of
producing pharmaceutical intermediates. In this work, a robust phenylalanine dehydrogenase
(PheDH) with wide substrate spectrum and high catalytic efficiency was constructed through
rational design and active-site-targeted, site-specific mutagenesis by using the parent enzyme
from Bacillus halodurans. Active sites with bonding substrate and amino acid residues
surrounding the substrate binding pocket, 49L-50G-51G, 74M,77K, 122G-123T-124D-125M,
275N, 305L and 308V of the PheDH, were identified. Noticeably, the new mutant PheDH
(E113D-N276L) showed approximately 6.06-fold increment of kcat/Km in the oxidative
deamination and more than 1.58-fold in the reductive amination compared to that of the wide
type. Meanwhile, the PheDHs exhibit high capacity of accepting benzylic and aliphatic ketone
substrates. The broad specificity, high catalytic efficiency and selectivity, along with excellent
thermal stability, render these broad-spectrum enzymes ideal targets for further development
with potential diagnostic reagent and pharmaceutical compounds applications.|
|Key_word||Chiral compounds, asymmetric catalysis, substrate spectrum, biocatalysis, rational design, substrate binding pocket|
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