Improving Catalytic Efficiency of Bacillus Cereus Amine Dehydrogenase for Acetophenone Reduction by Iterative Saturation Mutagenesis †

doi:10.7503/cjcu20190621

Abstract

Abstract:

Based on the 3D structure of Bacillus cereus amine dehydrogenase established by homology modeling, 8 amino acid residues(L42, G43, M67, A115, E116, T136, V293 and V296) around the substrate binding pocket were selected to site-directed saturation mutagenesis. Positive mutants were screened on 3 sites(116, 136, 293) by color-rendering method. The best triple-sites mutant(V293A/E116V/T136S) was obtained by iterative saturation mutation strategy and its catalytic efficiency for acetophenone reached 2.54 L·min ^-1·mmol^-1, which was 719% higher than BcAmDH. Compared with BcAmDH, the substrate concentrations and conversions of asymmetric reduction reaction of acetophenone were increased from 100 mmol/L to 300 mmol/L and from 42.1% to 80.2% at the optimal reaction conditions, respectively. It was suggested that the decrease of steric hindrance in the substrate binding pocket of the mutant and the expansion of the substrate entrance channel were the main reasons for improving catalytic efficiency by molecular docking results.

Key words: Iterative saturation mutagenesis, Amine dehydrogenase, Catalytic efficiency, Acetophenone, (R)-α-Phenethylamine

CLC Number:

O621.2

TrendMD:

WU Tao,MU Xiaoqing,NIE Yao,XU Yan. Improving Catalytic Efficiency of Bacillus Cereus Amine Dehydrogenase for Acetophenone Reduction by Iterative Saturation Mutagenesis ^†[J]. Chem. J. Chinese Universities, 2020, 41(5): 1018.

Figures/Tables 7

References 34

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Mutant	Acetophenone			NADH
Mutant	K_m/ (mmol·L^-1)	k_cat/ min^-1	(k_cat/K_m)/ (L·min^-1·mmol^-1)	K_m/ (mmol·L^-1)	k_cat/ min^-1	(k_cat/K_m)/ (L·min^-1·mmol^-1)
BcAmDH	31.11±1.74	9.58±0.51	0.31	0.026±0.0025	4.13±0.32	158.85
V293A	36.18±1.65	26.02±1.48	0.72	0.031±0.0023	8.77±0.76	282.90
V293A/E116V	31.52±1.92	44.89±3.37	1.42	0.028±0.0034	16.09±0.97	574.64
V293A/E116V/T136S	28.87±1.22	73.25±6.21	2.54	0.027±0.0031	21.14±1.31	782.96

Mutant	Acetophenone			NADH
Mutant	K_m/ (mmol·L^-1)	k_cat/ min^-1	(k_cat/K_m)/ (L·min^-1·mmol^-1)	K_m/ (mmol·L^-1)	k_cat/ min^-1	(k_cat/K_m)/ (L·min^-1·mmol^-1)
BcAmDH	31.11±1.74	9.58±0.51	0.31	0.026±0.0025	4.13±0.32	158.85
V293A	36.18±1.65	26.02±1.48	0.72	0.031±0.0023	8.77±0.76	282.90
V293A/E116V	31.52±1.92	44.89±3.37	1.42	0.028±0.0034	16.09±0.97	574.64
V293A/E116V/T136S	28.87±1.22	73.25±6.21	2.54	0.027±0.0031	21.14±1.31	782.96