Mutation Breeding And Gene Analysis Of Laccase From Myrothecium Verrucaria

In this paper,the mutagenesis was carried out by chemical mutagenesis,which was treated with nitrosoguanidine(NTG).Two strains of laccase with good heat-resistance were screened by high-throughput screening method of 96-well plate,named MF-01 and MF-02.They made a physiological indicator of the laccase produced by them.The results showed that the enzyme activity,pH and the growth of the two strains were not changed in the fermentation broth compared with the non-mutant strains.The pH value of the fermentation broth was decreased and the cell growth rate decreased after the line rose.The laccase of both strains and nonmutant strains was purified to give a single pure protein.MF-01 reaction of the optimum pH value of 3.0,pH stability of the best time in the pH3.0-3.5.The optimum pH for the MF-02 reaction is 4.0,the pH stability is preferably between 3.5 and 4.0,and the two values of the non-mutant are between pH 4.5 and pH 4-5,respectively.Indicating that the two mutants are more resistant to acid and have better stability.In the experiment of thermal stability and optimum temperature,the mutant strain MF-01 had relatively high enzyme activity at 30-50?,but the difference between the optimum temperature of MF-02 and non-mutant was not very obvious.The results showed that the laccase produced by the two strains had good stability,but the performance of one of the MF-01 was more prominent and could be stably inherited,which was a mutant to change the gene sequence of laccase.In order to verify this conjecture,the luciferase gene of the mutant strain was cloned by RACE method,and the whole sequence of the laccase gene was obtained.The primers were designed and the laccase Gene coding region of the cloning,the two gene comparison found that in the coding area of the 1312 bp position,the base has changed,from GCT into GAT,resulting in the amino acid encoded by alanine into the day Aspartic acid,mutations did not cause structural changes in the copper center of the laccase,the copper center was a relatively conserved region,and the active copper ion sites were located in this group consisting of one cysteine and 10 histidine residues Base structure of the ligand structure.Alanine is a neutral amino acid,and transformed into aspartic acid,it becomes a negatively charged acidic amino acid,histidine belongs to a positively charged alkaline amino acid;analysis of these two amino acids may produce intermolecular forces,The laccase protein molecules are more stable,but the laccase protein not only by the amino acid composition,as well as the participation of glycosylation,analysis of the stability of the changes may also be induced mutagenesis of the glycosylation process has undergone some changes,Laccase gene level to do a basic study.