AUTHOR=Yang Lijuan , Zhang Xian , Chen Jing , Zhang Yao , Feng Zhiping
TITLE=Expanding the pH range of glutamate decarboxylase from L. pltarum LC84 by site-directed mutagenesis
JOURNAL=Frontiers in Bioengineering and Biotechnology
VOLUME=11
YEAR=2023
URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1160818
DOI=10.3389/fbioe.2023.1160818
ISSN=2296-4185
ABSTRACT=Introduction: Glutamate decarboxylase is a class Ⅱ amino acid decarboxylase dependent onpyridoxal-5′-phosphate (PLP), which catalyzes the decarboxylation of substrateL-glutamate (L-Glu) to synthesize γ-aminobutyric acid (GABA). The low activity ofglutamic acid decarboxylase (GAD) and its ability to catalyze only under acidicconditions limit its application in biosynthesis of GABA.
Methods: Taking glutamic acid decarboxylase from Lactobacillus plantarum, which produces GABA, as the research object, the mutation site was determined by amino acid sequence analysis of GAD, the mutation was introduced by primers, and the mutant was constructed by whole plasmid PCR and expressed in Escherichia coli. Then, the enzymatic properties of the mutant were analyzed. Finally, the three-dimensional structure of the mutant was simulated to support the experimental results.
Results and Discussion: In this case, mutants E313S and Q347H of glutamate decarboxylase from L. pltarum LC84 (LpGAD) were constructed by targeted mutagenesis. Compared with the wild-type, their enzyme activity increased by 62.4% and 12.0% at the optimum pH 4.8, respectively. In the range of pH 4.0–7.0, their enzyme activity was higher than that of the wild-type, and enzyme activity of mutant E313S was 5 times that of the wild-type at pH 6.2. Visualization software PyMOL analyzed the 3D structure of the mutant predicted by homologous modeling, and the results showed that mutant E313S may broadened the reaction pH of LpGAD through the influence of surface charge, while mutant Q347H may broadened the reaction pH of LpGAD through the stacking effect of aromatic rings. In a word, mutants E313S and Q347H were improved the enzyme activity and were broadened the reaction pH of the enzyme, which made it possible for it to be applied in food industry and laid the foundation for the industrial production of GABA.