Mutagenesis on the surface of a β-agarase from Vibrio sp. ZC-1 increased its thermo-stability

• Published in: Enzyme and microbial technology 2019-08, Vol.127, p.22-31
• Main Authors: Su, Bing-Mei, Xu, Xin-Qi, Yan, Ren-Xiang, Xie, Yong, Lin, Juan
• Format: Article
• Language: English
• Subjects: Hydrolysis kinetics; Mutagenesis; Thermal deactivation kinetics; Thermo-stability; β-Agarase
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2019.04.006

•The engineering bacteria for expressing rAgaZC-1 and its mutant D622G were constructed.•The thermo-stability and thermal deactivation kinetics were compared between rAgaZC-1 and D622G.•Hydrolysis kinetics of agarose by rAgaZC-1 and D622G was compared.•Reasons for the improved thermo-stability of D622G were analyzed. The recombinant rAgaZC-1 was a family GH50 β-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.