Induction Of Laccase Production From Coprinus Comatus And CDNA Clone

Laccase is a kind of copper–contained polyphenol oxidase produced mainly by white rot fungi and excreted into media in form of glycoprotein. It can degrade lignin and catalyze the oxidation of a broad number of phenolic compounds and aromatic amines. Laccase is applied in many areas. Coprinus comatus,a sort of white rot fungi produces laccase efficiently.In this dissertation, laccase production of Coprinus comatus in four different media was studied under liquid culture at the temperature of 25℃. It was shown that the best medium was medium A which contained the glucose and yeast extract as carbon and nitrogen source respectively. The relation between the laccase production of Coprinus comatus and different metal ions and aromatic compounds was also discussed. It was shown the production of the laccase can be mostly induced by the addition of Mn2+ at concentration of 0.8 mmol/L and caffeic acid at the concentration of 3.0mmol/L. Other chemical agents contributed to the increase of the enzyme production most at different concentration. 4-methoxycinnamic acid, anisic acid, 3,4,5-trimethoxy-cinnamic acid, guaiacol, 3,5-dimethoxy-4-hydoxycinnamic acid, vanillin and 3-(4-methoxyphenyl)propionic acid induced the enzyme production most at low concentration, however, Cu2+, 3,4-dimethoxybenzaldehyde, cinnamic acid,4-hydroxycinnamic acid, and 3-(4-hydroxyphenyl)propionic acid did at high concentration. The result also showed that glucose was the best carbon source and peptone was the best nitrogen source. From the discontinuous polyacrylamide gel electrophoresis, we found different compounds at diverse concentration induced many laccase isoenzymes of Coprinus comatus displayed different bands with various pI. The optimum pH value and temperature of reaction of laccase was 6.0 and 60℃.In this paper, extraction of total RNA from Coprinus comatus and RT technology was used to gain the cDNA sequences. A series of degenerate oligonucleotide primers were designed , coding for conservative amino acid regions in fungi laccase protein family. A PCR amplification was performed on Coprinus comatus cDNA template and a specific fragment of 143 bp was obtained. By using the program of BLAST on NCBI GenBank database, the sequence presented a high match with the laccase gene from other fungi. The cDNA sequences of 5′ends were isolated by using RACE(rapid amplification of cDNA ends) technique from Coprinus comatus.The length of sequence was 626bp.