黑木耳漆酶基因在毕赤酵母中的表达及其酶学性质研究

Expression of laccase gene from Auricularia auricula in Pichia pastoris and characterization of recombinant enzyme

应用SignalP 3.0 Server软件对黑木耳漆酶基因lac1(GenBank No.AY450405)编码的蛋白质序列进行分析,发现在其N端存在18个氨基酸的信号肽序列。通过RT-PCR克隆了lac1基因,分别构建带有自身信号肽的lac1基因毕赤酵母表达载体pPICN-lac1和以α因子信号肽替换自身信号肽的lac1基因毕赤酵母表达载体pPIC9-lac。将这两个载体转化巴斯德毕赤酵母(Pichia pastoris)GS115细胞,获得基因工程菌GS115-pPICN-lac1与GS115-pPIC9-lac。SDS-PAGE分析和酶活性测定结果表明,在转化后者中漆酶基因得到了有效分泌表达,而在转化前者中漆酶基因未能得到有效分泌表达。进一步研究确定GS115-pPIC9-lac菌株液体发酵的最适pH为4.0,在此发酵条件下,其分泌表达的漆酶最高酶活为0.149U·mL-1。酶学性质研究表明,该酶的最适反应温度为40℃,最适反应pH为5.0,在最适反应条件下其pH稳定性和热稳定性均较好。

Through SignalP 3.0 Sewer analysis, a signal peptide sequence of 18 amino acids was found at N-terminal of deduced amino acid encoded by lacl from Auricularia auricula （GenBank No. AY450405）. The lacl gene had been cloned by RT-PCR, construction of Pichia pastoris expression plasmid pPICN-lacl which contained signal peptide itself and Pichia pastoris expression plasmid pPIC9＇lac which contained a -factor prepropeptide instead of signal peptide itself. Two plasmids were tranformed into the Pichia pastoris GSl15, and the genetically engineered bacterium known as GS115-pPICN-lac1 and GS115-pPIC9-1ac. The ineffective secreting expression of pPICN-lacl and effective secreting expression of pPIC9-1ac in the recombinant were confirmed by SDS polyacrylamide gel electrophoresis （SDS-PAGE） and the determined result of enzyme activity. The further resesrch determined the optimal fermentative pH of GSl15-pPIC9-1ac was 4.0, under optimum fermentative condition, the activity of the laccase secreting expression was 0.149 U .mL-1. The properties of enzyme were determined. The optimum temperature and pH value were 40 ℃ and oH 5.0, respectively. Thermostability and pH stability were very well under the optimal conditions.