College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China.
Mol Biol Rep. 2012 Apr;39(4):3871-7. doi: 10.1007/s11033-011-1166-7. Epub 2011 Jul 9.
A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V (max) values are 243.9 and 10.55 Um min(-1) mg(-1), respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.
根据酵母偏性密码子，化学合成了来自扩展枝孢霉的新型漆酶基因，并通过电穿孔将其整合到毕赤酵母 GS115 的基因组中。从培养上清液中回收表达的酶并进行纯化。酶活性测定和 SDS-PAGE 的结果表明，重组漆酶在毕赤酵母中被诱导并分泌到细胞外。对该漆酶的主要生化性质，如热依赖性和热稳定性、最适 pH 和 pH 稳定性，以及金属离子和抑制剂的影响进行了表征。以 2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）为底物时，MfLcc 的最适 pH 为 3.5，最适温度为 45°C。ABTS 和愈创木酚的 Km 值分别为 0.012 和 0.016 mM，相应的 V（max）值分别为 243.9 和 10.55 Um min(-1) mg(-1)。在最佳条件下，重组漆酶在 8 小时内可降解 80%的 2,4,6-三氯苯酚。重组菌株及其漆酶可被视为处理三氯苯酚污染废水的候选物。
