Study On Laccase Gene Cloning And Enhancing The Expression Of Laccase Gene

Laccase is a copper-containing polyphenol oxidase which has a broad spectrum of substrates,capable of oxidizing a variety of organic substances and has potential industrial value in the degradation of organic pesticide residues and lignin.In this study,the laccase gene lac1 was isolated from Humicola insolens Y1 and its total length was 1 803 bp,encoding 600 amino acids.The deduced amino acid sequence was aligned with available protein sequences held in the Gen Bank,the results showed that the highest similarity was 71% which is the polyphenol oxidase sequence from Chaetomium globosum CBS 148.51?XP₀01228806?,indicating a new laccase gene.When the lac1 gene fragment was cloned into the E.coli expression vector p ET30 a,the recombinant expression vector p ET30a-lac1 was constructed and then transferred into E.coli BL21?DE3?.The recombinant enzyme did not show any enzymatic activity and was insoluble.Then,the lac1 gene fragment was cloned into Pichia pastoris expression vector p PIC9 to construct the recombinant expression vector p PIC9-lac1.The results of enzyme activity assay and SDS-PAGE confirmed that the gene was incorporated into the yeast genome and the recombinant laccase Lac1 was secreted expression.After fermented in 3 L fermentor,Lac1 protein expression reached 3.79 mg/m L fermentation broth,and the enzyme activity reached 1.3 U/m L fermentation broth,which was 10 times higher than the result of shake flask fermentation enzyme activity.The analysis of the enzymatic properties showed that the optimum temperature of laccase was 65?,the optimum p H was 4.5,and the relative activity of enzyme was above 70% in the range of p H 6-11.It laid the foundation for the discovery of new laccases with excellent enzyme properties from fungi.In order to improve the expression level of laccase Cot A derived from Bacilluslicheniformis,cot A was optimized by the synonymous mutation of rare codons.By improving the codon usage of arginine in the sequence of cot A,the arginine in cot A gene was homologously mutated using a rare arginine codon,and CGT/CGA was mutated to CGA/CGT.The T of positions 534/747/1317/1533 and the A mutation of 738/918 were mutated to A and T respectively.The mutants were inserted into p ET30 a and the recombinated vectors were constrcted and transferred into E.coli BL21?DE3?.The assay of laccase activity showed that the 534-site mutant decreased,the 738-site mutant did not change and other mutants increased compare with the wild-type.As a result,the closer the mutant site was to the C-terminal,the higher the enzyme activity was.The enzyme activity of the 1533-site was the highest and 2 times that of the wild type.To improve the expression of laccase provides a new idea.In addition,it has been reported that molecular chaperones can improve the secretion and expression of foreign proteins.In order to improve the enzyme activity of laccase in Pichia pastoris,co-expression of chaperone genes and the lacccase gene eueo from Ecoli was studied.Eight functional chaperone genes?GCN4/SSE1/SEC53/KIN2-1/KIN2-2/BMH2/HAC1/PDI?were screened out from previously reported chaperones and cloned into the vector p GAPZA or p PICZA.Ten recombinant vectors?p GAPZA-GCN4/SSE1/SEC53/KIN2-1/KIN2-2/BMH2/HAC1 and p PICZA-GCN4/BMH2/PDI?were constructed,respectively.The recombinant vectors were transferred into the yeast strain GS115/p PIC9-cueo which contained the laccase gene cueo respectively.The strains harboring p PICZA-GCN4/BMH2/PDI had many transformants and had enzymatic activity.The other strains had few transformants and no enzyme activity was detected.The strains harboring p PICZA-GCN4/BMH2/PDI were fermented,and the enzyme activity was measured.The results showed that the enzyme activities of the chaperones BMH2 and PDI were increased by 80% and 20%,respectively.To improve the expression of laccase provides a new idea.In this study,a new laccase gene was screened,and the optimal temperature,p H,and enzyme properties of the enzyme were discovered.In addition,two ideas for improving the expression of laccase were obtained.The aspect can be through mutating rare arginine codons in laccase,and on the other hand through co-expression with molecular chaperones.Lay the foundation for the application of laccase.