The Polymerization Of Diphenols Catalyzed By Trametes Sp.SQ01 Yellow Laccase And The Expression Of Yellow Laccase In Trichoderma Reesei

The biosynthesis of phenolic polymers has great significance in dye synthesis,cosmetics manufacturing,pharmaceutical industry,and plastic industry production.Yellow laccase catalyzes the synthesis of organic compounds with many advantages: a wide range of substrates,simple processes,mild reaction conditions,and basically non-toxic products.In the synthesis of high molecular polymer dyes,a very promising new type of dye,the yellow laccase produced by the white-rot fungus Trametes sp.SQ01 can polymerize diphenols in the absence of mediator,and during the reaction,no intermediate is formed and the polymer have different colors.However,there are two major problems in the reaction: first,the low utilization rate of yellow laccase and the low efficiency of product synthesis during the reaction;second,the reaction has shortcomings such as a low production of yellow laccase,a long acquisition cycle,and a unstable production.In order to solve the first problem in this paper,we optimized the reaction conditions of the polymerization of diphenols to catalyzed by the yellow laccase,We analyze and study the catalytic kinetics,investigate the effects of reaction time,enzyme concentration,p H,temperature,rotation speed,and substrate concentration on the reaction,and grasp the differences in biological oxidation characteristics of different diphenols.The results showed that the optimal reaction conditions for the three substrates weredifferent under the several conditions examined.The optimal conditions for catechol were: reaction time of 80 min,enzyme concentration of 0.8 U/ml,p H of 5,and temperature of 50?,the rotation speed is 300 r/min,and the substrate concentration is 2 m M.The optimum conditions of resorcinol are:reaction time is 80 min,enzyme concentration is 1.0 U/ml,p H is 5,temperature is 50?,and rotation speed is 300 r/min,substrate concentration is 5 m M.The optimal conditions for hydroquinone are: reaction time is 80 min,enzyme concentration is 1.0 U/ml,p H is 4,temperature is 50?,rotation speed is 300 r/min,substrate concentration is 5 m M.The p H,enzyme concentration,and substrate concentration have a greater impact on the reaction;redundant analysis confirm that p H has the greatest effect on the polymerization of diphenols,followed by enzyme concentration,temperature,and substrate concentration;kinetic analysis shows that Catechol is the most suitable substrate catalyzed by yellow laccase.In order to solve the shortage of yellow laccase produced by white rot fungus SQ01,we use yellow laccase expression in Trichoderma reesei to solve this problem.First,we obtained the yellow laccase gene sequence by purifying the yellow laccase protein,sequencing the peptide map,sequencing the transcriptome of Trametes sp.SQ01,constructing the local protein database of Trametes sp.SQ01,blast alignment,and bioinformatics analysis.The results showed that the gene sequence of the white rot fungus Trametes sp.SQ01 yellow laccase is c13470_g1.Then we construct engineering strainthat expresses yellow laccase efficiently by the recombinant expression vector p SKPss T,the recombinant expression vectors p SKPss LT,Trichoderma Tu6 strain protoplasts,transformation and selection.The results show that we successfully constructed an engineered strain that expresses yellow laccase,but the recombinant protein may not be successfully expressed and secreted into the culture medium,or the recombinant protein has been degraded,resulting the engineered strain has not secreted an active laccase.