Heterologous Expression Of The GC.u01 Laccase Gene In Monot And The Application

According to previous reports,most laccase-related reactions are mild,harmless and do not produce toxic byproducts.These characteristics also lead to more and more research on laccase.Because the fermentation cycle of fungus laccase is too long,it greatly increases the time cost and economic cost of industrial production of laccase.Therefore,it is of great value and significance to use genetic engineering technology to integrate laccase genes into other hosts for expression in order to shorten the production cycle of laccase.In this study,Trichodontoides GC.u01 was selected as the research object and its whole genome was sequenced.After analysis,laccase gene A2820 was selected as the research object and transferred into GS115 for exogenous expression.The effect of inorganic ions on the activity of laccase in recombinant bacteria GS115-A2820*,protein purification and enzymatic properties of recombinant enzyme Lac-2 and its application were studied.The main results were as follows:（1）The whole genome of Trichodontodon GC.u01 was sequenced,and 11 laccase sequences were predicted by GO,KEGG,KOG,NR,Pfam and Swiss-Prot database annotation.They are AA1-1,AA1-2,A2726,A2820,A6023,A6049,A6061,A6378,A6400,A1620 and A3567,respectively.The laccase sequence was screened,and finally a laccase A2820 which could be expressed exogenously was obtained.（2）Laccase A2820 was synthesized according to the codon bias of Pichia Pastoris GS115,and A2820*was obtained.The CAI of A2820*increased from 0.71 to 0.94,and the square variance between the actual codon frequency and the preferred codon frequency changed from 2.59 to 6.46.GC content of the whole sequence was homogenized,and GC content of A2820*was reduced from 52%to 39%.Hairpin structure in A2820*reduced from 1399 to 1292.The optimized gene A2820*was connected to plasmid p PIC9K to obtain recombinant plasmid p PIC9K-A2820*,and p PIC9K-A2820*was transferred into Pichia Pastoris to construct recombinant yeast GS115-A2820*.（3）The effects of inorganic ions on the activity of laccase in recombinant yeast GS115-A2820*were studied using BMMY as the base medium.The results showed that adding Cu²⁺in the medium could improve the activity of laccase,especially when the content of Cu²⁺was 0.05 mmol/L,the activity of laccase was increased from135.93 U/L to 245.73 U/L,which was 40.43%higher than that of the control group.Both Mn²⁺and Ca²⁺can inhibit the activity of laccase,and the inhibitory effect of Mn²⁺is particularly significant.When Mn²⁺is added in the medium,the activity of laccase is below 40 U/L,and when the concentration of Mn²⁺reaches 0.3 mmol/L,there is no activity of laccase.When Ca²⁺was added in the medium,there was little difference between the enzyme activity and that of the control group in the first 5 days of culture.With the increase of culture time,Ca²⁺began to inhibit the activity of laccase,and low concentration of Ca²⁺had a stronger inhibitory effect on the activity of laccase.When the concentration of Ca²⁺reached 0.6 mmol/L,the activity of laccase only decreased by about 50%.In this experiment,the initial OD value of the medium and the amount of the inducer methanol were also optimized.When the initial OD value of the medium was 1,the activity of laccase was the highest,reaching 98.68 U/L.Under the same other conditions,only the amount of methanol was changed.When the amount of methanol was 0.5%,the laccase activity on the 10th day reached the maximum,which was 269.09 U/L.（4）In this study,laccase was isolated and purified by Ni affinity chromatography,and was verified by SDS-PAGE as a single bright band with a molecular weight of about 70 k Da,which was named Lac-2.After purification,the specific activity of laccase was 8.29 U/mg,the purification ratio was 4.21,and the recovery was 29.21%.The enzymatic properties of the purified laccase Lac-2 were studied.The results show that the optimum reaction temperature of Lac-2 is 50℃and the optimum p H is 3.6.Lac-2 is stable at 0-30℃and p H 3-6.5,which indicates that Lac-2 has greater adaptability to temperature and p H value,and its tolerance to acidic environment is stronger than alkaline environment.When the concentration of metal ions was 1mmol/L,Lac-2 showed relatively strong ion tolerance to Na⁺,Mg²⁺,K⁺,Cu²⁺,Mn²⁺,Zn²⁺,Ca²⁺,Co²⁺,Al³⁺,NH₄⁺,and Cu²⁺could enhance the activity of Lac-2.However,when the concentration of metal ions increased to 50 mmol/L,most of the metal ions had a great effect on the activity of Lac-2.（5）For Lac-2 degradation of pyrimethanil and phoxim,the optimal degradation temperature of both was 30℃,but the difference between the two was that Lac-2concentration and degradation time were 100%and 60 minutes when the degradation rate of pyrimethanil was the maximum.When the degradation rate of phoxiphos was the highest,Lac-2 concentration and degradation time were 40%and 90 minutes.