Cloning Of A Laccase Gene Form Coprinopsis Cinerea And Study On Its Expression In Pichia Pastoris

Laccase (EC1.10.3.2) is a type of copper-containing polyphenol oxidase that was discovered in the exudates of the Japanese lacquer tree in1883, and then finded it in fungi, bacteria and higher plants, but it mainly exists in plants and fungi. Due to laccase could react to various substrate, and it could catalytic oxidation many kinds of phenolic compounds and many kinds of aromatic compounds, laccase became a very active research field of biology and environmental science. Because of its strong ability of oxidation, laccase is widely used in paper pulp industry, textile Industry, food industry, biological detection and biosynthesis.Laccase was widely found in fungi, especially in white-rot fungus. In this stuy the material was coprinopsis cinerea. It belongs to the basidiomycota, hymenomycetes, agaricales, coprinus section, coprinus genus, and it was one kind of white-rot fungus. It contained a large laccase family, and it had17different laccases. In this study, we select one of laccase gene to builded a Inducible and secretion expression vector of pichia pastoris, at last we got engineering strain that express laccase effectively.In this study, results were summarized as follow:1. Selection and cloning of the laccase geneCoprinopsis cinerea okayama7#130contains17different laccase genes, in the pre-experimental, five laccase gene probe primers were designed and four cDNA sequences of laccase were amplified, the laccase gene one was choose finaliy which the length of laccase amino acid sequences was moderate and containing conserved sequence named copper ion binding site. The DNA and cDNA sequence was obtained by PCR and RT-PCR respectively. After the analysis of signal peptide sequence, the the laccase gene clacl which excluding the signal peptide sequence was obtained.2. Construction of Pichia pastoris induced expression vectorIn this study, the induced secretion expression vector pPIC9K-clacl of the Pichia pastoris was constructed, the laccase gene one can be expressed under secreting in pichia pastoris because it does not contain the signal peptide.3. The secreted expression of Laccase gene in Pichia pastoris GS115and the study on the properties of the recombinant enzymeAfter transformed the constructed expression vector pPIC9K-clacl into competent cells pichia pastoris, transformants GS115-pPIC9K-clacl were gained, the initial activity of laccase reach to1.108U/mL. Analyse the enzymatic properties of laccase:the optimum reaction temperature of recombinase was45℃and the optimum pH value was4.3, the temperature stability and pH stability were both good.4. The preliminary optimization of the conditions of induced expression on the engineering bacteriaAfter chose the best conditions in the progress of the induced expression of the engineered bacteria GS115-pPIC9K-clacl,such as the amount of methanol, speed, medium volume, copper ions, initial pH value, cultivate temperature and the amount of inoculation, the activity of laccase in GS115-pPIC9K-clacl catch up to1.805U/mL which increased by62.91%.