|Table of Contents|

Directional modification of heat resistance and enzyme activity of polygalacturonase AnPGA from Aspergillus niger(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2021 05
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Directional modification of heat resistance and enzyme activity of polygalacturonase AnPGA from Aspergillus niger
YAN Jiachao1 CAO Yu2 YANG Danni1 WU Yong1 XU Hui1 QIAO Dairong1 & CAO Yi1?
1 Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610065, China 2 Laboratory and Equipment Management Office, Sichuan University, Chengdu 610065, China
polygalacturonase directional modification site-directed mutation thermal stability enzyme activity

Pectin is a rich source of biomass. Polygalacturonase is the key enzyme for degrading pectin and is widely used in the food industry. However, its enzyme activity and heat resistance are key factors that restrict its utilization as well as that of pectin. In this study, polygalacturonase AnPGA from Aspergillus niger SC323 was modified to improve either its heat resistance or enzyme activity. PoPMuSiC was used to predict the amino acid sites that significantly affected the unfolding free energy, and a simulation of molecular dynamics was used to predict flexible amino acid sites. These strategies were combined to design the mutation sites for AnPGA. Site-directed mutagenesis was carried out by overlapping PCR, expression vectors were constructed for mutants, and the mutant Pichia pastoris GS115 expression strains were constructed. After all mutants were expressed, the mutant D73P which had improved enzyme activity and thermal stability was successfully screened. The Vmax of D73P was 2.72 times higher than that of the wild type, and the half-life (t1/2) of D73P was extended by 7 min at 65 ℃. Furthermore, the D73P showed more residual activity than the wild type (1.86 times greater) after 30 min of treatment at 65 ℃. This study was the first to use a PoPMuSiC design strategy combined with a simulation of molecular dynamics to improve the activity and heat resistance of AnPGA. The results provided new enzyme resources for industrial applications and a reference for the directional modification of other industrial enzymes.


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Last Update: 2021-10-25