The Study Of Functional Expression And Activity Enhancement Of CotA-laccase Mutant WLF Through Site-directed Mutagenesis

The CotA laccase of Bacillus is a green enzyme with the prospect of industrial application,CotA laccase has important application potential for basic industrial waste-water treatment,such as printing and dyeing waste-water,due to their remarkable advantages,including broad substrate spectrum,high thermostability,wide pH scope,and especial tolerance to alkaline environments.However,the low catalytic activity of wild CotA laccase to dyes is the bottleneck of industrial application.L386W/G417L/G57F?WLF?,a mutant of CotA-laccase from Bacillus pumilus W3,has been constructed and reported by our laboratory with four times improved catalytic efficiency.However,the low expression level of mutant WLF in Escherichia coli was a shortcoming.Fivemutants,M502L/WLF,M502F/WLF,K317N/WLF,D501G/WLFand K317N/D501G/WLF,were constructed through rational analysis of sequence alignment to further improve its catalytic activity and the expression of WLF in this study.According to the results,CotA of M502L/WLF and M502F/WLF both are inactive,it showed that the site of M502 is a key site for Cot A active.The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E.coli enhanced 4.48-fold and 3.63-fold level,respectively.The K317N/WLF failed to increase the soluble expression level,but slightly improved the stability of CotA-laccase.Results showed that mutants D501G/WLF and K317N/D501G/WLF still remained its high thermostability,resistance of acid,alkaline and salt solution,and conspicuous decolorizing efficiency.Different metal ions have different effects on CotA laccase mutants;Cu²⁺can significantly enhance the activity of CotA laccase,Fe²⁺has a strong inhibitory effect on laccase,Na⁺,K⁺and Ca²⁺have a weak inhibitory effect,while Zn²⁺,Mn²⁺and Mg²⁺have a weaker promoting effect.Through bioinformatics analysis combined with CD to determine the secondary structure,mutation of the site will affect the number of hydrogen bonds between amino acid residues,the secondary structure will change,The potential mechanism of increased soluble expression is that the increase of?-fold in the central structure of T1Cu promotes the post-translational folding efficiency of CotA mutants.This work is the first to improve the soluble expression of CotA-laccase of B.pumilus genus in E.coli by site-directed mutagenesis.The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.