Properties, Gene Cloning And Structure Analysis Of Laccase Produced By Pycnoporus Sanguineus SYBC-L1

Laccase （EC1.10.3.2） can catalyze various substrates, such as phenols, aromatic diamines amine, and biological pigment, etc, to form quinones, carbonyl compound, and water. It has very broad application prospects and potential application of the value, including environmental remediation, drug production, food processing and other areas. Therefore, to study the isolation and purification, molecular biological characteristics and enzymology of laccase has the important theoretical and practical significance.The thesis uses Pycnoporus sanguineus SYBC-L1 as the material to study some research on the main methods of the isolation and purification of laccase, its enzyme property, gene cloning, structure analysis, etc. Finally we got some data as the following.1) By the combination of ammonium sulfate precipitation, DEAE-cellulose anion exchange, and Sephadex G-100 gel filtration, the laccase （Lac II） with electrophoretic homogeneity was isolated and purified from liquid culture of Pycnoporus sanguineus SYBC-L1. The purity of purified Lac II reached 12.61-folds and total yield was obtained as 42.70%.2) The properties of the laccase produced by Pycnoporus sanguineus SYBC-L1 were characterized. The results showed that the optimum pH and temperature of Lac II was 3.0 and 75℃respectively. Lac II was quite stable at the pH from neutral to basic and temperature from 40℃to 70℃. The activity of Lac II was obviously inhibited by SDS、K+、Fe3+and Fe²⁺ and weakly activated by Na+、Mg²⁺、EDTA.3) Effects of several different methods on the DNA extraction from Pycnoporus sanguineus were compared. According to the fungal laccase conserve sequences registered on NCBI, primers for Lac II gene cloning were designed. A 1,084bp laccase gene fragment was amplified by using genomic DNA from Pycnoporus sanguineus as template.4) According to the BLAST results, we found that the sequence had the homology more than 99% to Pycnoporus coccineus. The alignment of amino acids shared the homology of 88% to Pycnoporus coccineus. Therefore, the genomic DNA should be Pycnoporus. The secondary structure of purified enzyme was determined. And by simulating the three-dimensional space structure of the enzyme, the relationship between the structure and function of the enzyme was analyzed.