Molecular Cloning Of A Laccase Gene From Pleurotus Eryngii And Study On Its Heterogenous Expression

Laccases are the most important components of the lignolytic complex of wood-destroying white-rot-fungi responsible for decomposition of lignin.They can also break down lignin structure-like materials that are harmful to environment.As a results,They play a more and more important role in environment protection,delignification of paper pulp,food and biosensor industry.the majority of laccases are produced by white-rot-fungi,which are easily join together in the process of culture.The production of laccase from white-rot-fungi is rather low,so It can hardly meet the need of industry. After sceening,we find the optimum temperature and thermo stability of P.eryngii laccase is excellent.Therefore we clone its laccase gene and studied its heteroexpression,There are five parts in this study.1. Clone of laccase gene. We obtained cDNA and gDNA of P.eryngii by the means of RT-PCR and PCR technologis. The sequences are aubmitted to BeneBank and the accession number are FJ231091, FJ231092 respectively. P.eryngii laccase contains 14 introns, the length of which rank from 50bp to 70bp. And most of them subject to GT-AG rule.2. The perdiction of laccase senior structure. We predicted the senior structure of laccase with the help of Bioinformatics. The secondary and tertiary structure are respectively displayed by the sofes of CLC protein work bench and pymol.3. The laccase was expressed in Escherichia coli:The laccase was inserted into the expression vector of pET21b(+) and transformed into E.coli BL21(DE3) which was expressed successfully in Escherichia coli by inducing with IPTG. But wasn’t secreted. After breaking up cells,we either detected the enzyme activity.4. The xynlll was expressed in Pichia pastoris:The laccase gene with the a-factor signal sequence was inserted into the expression vector of pPIC9K and transformed into Picha pastoris GS115.At the 6 days of induction with 0.5% methanol at 28-30℃,the produced crude enzyme was detected to reach the highest enzyme activity of 122U/L.5. The recombinant xylanases were characterized:Both of the wild enzyme and the recombinant laccase was optimally active at 55℃and pH3.2. These enzyme properties suggested that this acidophilic enzyme encoded by laccase could be used in the industry.