Production, Purification and Some Properties of Extracellular Laccase from Thermobifida fusca

• Main Authors: Wei, Chienmei, 魏千媚
• Other Authors: Yang, Chaohsun
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/72045339295902128958

碩士 === 靜宜大學 === 化粧品科學系 === 101 === Bagasse was the best substrate for production of extracellular laccase from the newly isolated thermophilic actinomycetes, Thermobifida fusca BCRC 19214. After 42 h of fermentation in the 5-liter fermentor, the culture broth accumulated 2.3 U/ml laccase activities. The laccase was purified 4.64 fold through Q-Sepharose FF column and SephacrylTM S-200 column chromatography. The overall yield of the partial purified laccase was 7.49%. The molecular mass of purified laccase as estimated by gel filtration and by SDS-PAGE on Sephacryl S-200 was found to be 73.3 kDa and 24.7 kDa, respectively, indicated that the laccase from T. fusca BCRC 19214 is a trimer. The internal amino acid sequences of the purified laccase determined by LC-MALDI-TOF/MS had high homology with superoxide dismutase product from T. fusca YX. The laccase also had superoxide dismutase activity. The optimal pH and temperature of the purified enzyme were 8.0 and 60℃, respectively. About 95% of the original activity still remained after treatment at 50℃ for 4 h. The laccase can effective oxidize the L-3,4-dihydroxyphenylalanine (L-Dopa), 2,6-dimethoxyphenol (2,6-DMP) and 4-dimethoxybenzylalcohol (veratryl alcohol). Using 2,6-DMP as a substrate, the partial purified laccase were determined Km and Vmax of 1.82 mM and 15.11 U/mL, respectively. The enzyme was slight inhibited by 1mM Hg2+. This laccase was strongly inhibited by 1 mM -mercaptoethanol, 10 mM EDTA and 0.1 mM DTT. The laccase activity was not reduced in the organic solvents like acetonitrile and dimethylformamide (DMF). The laccase could oxidize dye intermediate, especially for 2,6-dimethylphenylalanine (DMP), p-aminophenol (PAP), o-aminophenol (OAP) and p-phenylenediamine (PPD), to produce coloring. These properties demonstrated that the newly isolated laccase had potentials for the specific industrial applications. In this study, extracellular laccase gene from T. fusca BCRC 19214 was cloned, according to the genomic information of T. fusca YX, to pETDuet-1 vector, and the recombinant protein was expressed in E.coli AD494 (DE3). The cloned laccase gene encodes the gene consists of 612 base pairs and encodes a protein of 204 amino acids. The laccase of E.coli transformant (pED-Tfu 0957) was cultured in LB (including ampicillin and kanamycin) broth and purified by Ni2+ - NTA column chromatography. The purified laccase could be detected as a major band by SDS-polyacrylamide gel electrophoresis and western blotting.