Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution |
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| Affiliation: | 1. Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;2. School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;3. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China;1. College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;2. School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;3. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China;1. Department of Biotechnology, University of Verona, Strada Le Grazie 15, Verona (VR), Italy;2. Paul Scherrer Institut, Villigen 5232, Switzerland |
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| Abstract: | Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of l-glutamate to form γ-aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity 55.4 mmol·L? 1·min? 1·(mg protein)? 1, 120% higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD. |
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