1(天津科技大学 化工与材料学院, 天津, 300457) 2(天津科技大学 生物工程学院, 天津, 300457) 3(Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, 4001, South Africa)
Enzymatic characterization of the pullulanase mutant N467G
1(College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China) 2(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China) 3(Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, 4001, South Africa
Abstract: Pullulanase is extensively used in preparation of glucose syrup from starch, combined with glucoamylase.However, its catalytic activity at low pH and high temperature is the main factor affecting its efficiency during starch debranching.In this study, the difference of the protein unfolding free energy (ΔΔG) of Bacillus naganoensis pullulanase (PulA) mutants was calculated and the stability of each mutation site was analyzed.A mutant, N467G, was obtained through site-directed mutagenesis of the Asn467 residue ofPulA and its biochemical properties were examined.Compared to the wild-type PulA, the optimal temperature of mutant N467G was increased by 5 ℃ to 60 ℃ and the optimal pH was reduced by 0.5 pH value to 4.5.Its relative activity was over 80% after incubating the enzyme at 60 ℃ for 2.5 h or at pH 4.0-5.0 for 1 h, which represented a significantly higher stability.The result lays a foundation for the subsequent high-efficiency protein production and industrial application of this mutant.
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