Mutagenesis Of A Laccase Gene From Pleurotus Eryngii In Vitro And Expression In Pichia Pastoris

Laccases, belonging to the blue copper oxidase, are the most important components of the ligninolytic enzymes 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 result, they play a more and more important role in environmental protection, delignification of paper pulp, food industry, biosensor and immunological detection. The majority of laccases are produced by white-rot-fungi, which can 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 the mutagenesis of its laccase gene (lac, GenBank gi: FJ231091 ) in vitro and expression in Pichia pastoris were studied. There are three parts in this study.1. Mutagenesis of lac in vitro by the means of error-prone PCR. Using this method after one round of mutagenesis, the establishment of mutant library and screening were done and the expression system was constructed, but no activity mutant of lac was obtained. Nucleotide sequence analysis showed that two mutated bases were changed in one of the mutant gene: the first one located on aβ-strand is T209C (Valâ†'Ala), the other belonging to synonymous mutation is T237C, which located on a coil. They both located in Domain 1, which participated in connection of T2/T3 trinuclear cluster.2. Mutagenesis of lac in vitro by the means of overlap extension PCR (OE-PCR). Since GTC is superior codon of high expression, there was the reverse mutation from GCC. Then the recombinant plasmid pMD19-lacM was connected between the gene (lacM) with the pMD19T-Simple Vector .3. The laccase was expressed in Pichia pastoris: The laccase gene lacM with theα-factor signal sequence was inserted into the secreted expression vector of pHBM905A and transformed into Pichia pastoris strain GS115. After the 7 days of induction with 0.5 % methanol at 28℃, the lacM was expressed successfully, but its enzyme activity was low.