Improving The Catalytic Efficiency Of Bacillus Pumilus CotA-laccase By Protein Engineering

The fungal laccases have been commercialized.However,the applications of fungal laccases in industrial printing and dyeing wastewater treatment are greatly limited as a result of the low activity or inactivation in alkaline environment and poor thermal stability.Besides,the glycosylation of fungal laccases is not conducive to molecular modification.In contrast,bacterial laccases(such as Cot A)display relatively high enzyme activity and stability in alkaline pH and high temperature although natural activity of bacterial laccases is less than fungal laccases.Therefore,bacterial laccases are more suitable for popularization and application in printing and dyeing wastewater treatment.The critical point is that bacterial laccases have no glycosylation and are suitable for enzyme engineering.In this study,Cot A,a bacterial laccase from Bacillus pumilus,was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability.Main research results are as follows:(1)L386W/G417 L,a Cot A double-mutant,was constructed through site-directed mutagenesis.The catalytic efficiency of L386W/G417 L for ABTS was 4.3 fold higher than that of wild-type Cot A-laccase.(2)The thermostability of L386W/G417 L was decreased than that of wild-type and other mutants.The half-life(t1/2)of wild-type and G417 L were 1.14 h and 1.47 h,but the half-life of L386W/G417 L was only 0.37 h when incubating the enzyme at 80 °C.In order to improve the catalytic efficiency of L386W/G417 L,we constructed L386W/G417L/G57 F by bioinformatics prediction and experimental verification.Results showed that the half-life of L386W/G417L/G57 F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity,but the residual activity of L386W/G417 L was less than 40 % when incubating the enzyme at 90 °C for 5 min.(3)The catalytic efficiency and specificity of four mutants using SGZ and DMP as substrates were decreased than that of wild-type Cot A-laccase,which showed that the preference of mutants for ABTS was increased compared to the decreased preference for SGZ and DMP.(4)L386W/G417 L and L386W/G417L/G57 F exhibited an efficient decolorization ability in decolorizing various industrial dyes at pH 10.In conclusion,L386W/G417L/G57 F is appropriate for biotechnological applications because of its high activity and thermostability in enhanced biotreatment of printing and dyeing wastewater.