ABSTRACT
Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.
Subject(s)
Glutamate Decarboxylase , Metabolism , Half-Life , Industrial Microbiology , Levilactobacillus brevis , Mutagenesis, Site-Directed , Mutation , TemperatureABSTRACT
Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (k cat) of V445A was increased by 7.5 times, while its K m value decreased by 9.2 %. Consequently, the catalytic efficiency (k cat/K m) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Directed Molecular Evolution , Indoles/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Bacterial Proteins/chemistry , Cytochrome P-450 Enzyme System/chemistry , Electron Transport , Gene Library , Hydroxylation , Indoles/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Mutation , NADPH-Ferrihemoprotein Reductase/chemistry , Protein Conformation , Stereoisomerism , Substrate SpecificityABSTRACT
Objective: To study a new purification method of Polysaccharide Peptide of Coriolus versicolor in affinity chromatography. Methods: Isotherm and kinetics in absorption and the optimal conditions of absorption and elution were studied through static experiments. The static absorption capacity q m and absorption constant K d were calculated according to Chase model. Results: q m =55.57mg/g wet resin, K d =5.312g/L, the dynamic absorption capacity is 43.1mg, polysaccharide/g wet resin and 10.3mg protein/g wet resin.Conclusion: Affinity chromatography can be used to purify PSP preliminarily.
