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Rational engineering of cofactor specificity of glutamate dehydrogenase for poly-γ-glutamic acid synthesis in Bacillus licheniformis.
- Source :
-
Enzyme & Microbial Technology . Apr2022, Vol. 155, pN.PAG-N.PAG. 1p. - Publication Year :
- 2022
-
Abstract
- Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis. [Display omitted] • Three-dimension structure of B. licheniformis RocG was attained by homology modeling. • Potential mutation sites of RocG were scoped by cMD and RAMD simulations. • The specificity of RocG was altered to NADH-dependent by rational enzyme design. • Cofactor engineering was proven as an effective strategy for increasing γ-PGA production. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01410229
- Volume :
- 155
- Database :
- Academic Search Index
- Journal :
- Enzyme & Microbial Technology
- Publication Type :
- Academic Journal
- Accession number :
- 155122129
- Full Text :
- https://doi.org/10.1016/j.enzmictec.2021.109979