| 绿色木霉AS3.3711的葡聚糖内切酶Ⅲ基因的克隆与表达 |
| 摘要点击 1392 全文点击 1802 投稿时间：2003-11-07 修订日期：2004-02-11 |
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| 中文关键词 绿色木霉 葡聚糖内切酶Ⅲ 酿酒酵母 表达 |
| 英文关键词 Trichoderma viride endo β-glucanase Ⅲ Saccharomyces cerevisiae expression |
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| 中文摘要 |
| 为研究构建可降解纤维类固体废弃物的工程菌,采用RT-PCR方法克隆到绿色木霉(Trichoderma viride)AS3.3711的葡聚糖内切酶Ⅲ(EGⅢ)的cDNA 基因,测序后构建到酿酒酵母(Saccharomyces cerevisiae)诱导型表达载体pYES2 上,用正交实验对超声波辅助酵母转化系统进行了优化.转化获得的EGⅢ转化子用2%的β-D-半乳糖诱导,用Northern 杂交、刚果红染色法和CMC糖化力法分别对目的基因的转录和表达产物的葡聚糖内切酶活性进行检测.结果表明, EGⅢ的cDNA 基因开放阅读框长度为1254bp,编码418个氨基酸,推测蛋白质分子量为44.1×103.正交实验中较优组合为第5组(超声波处理60s,温育40min,单链DNA 150μg,热激5min); 刚果红染色显示,转化子可产生明显的水解圈;CMC酶活检测显示该基因能在酿酒酵母中表达有生物活性的EGⅢ并分泌到胞外,发酵液中的酶活在培养60h达到最高0.041 U/mL;最适酶解温度为50℃;最适pH 值为5.8.
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| 英文摘要 |
| To study the construction of yeast bioengineering strain which can degrade cellulosic waste, an endo β glucanase Ⅲ(EGⅢ) cDNA gene of Trichoderma viride AS3.3711 was isolated with RT-PCR protocol. After sequencing it was constructed on S. cerevisiae induceable expression vector pYES2. A L9(34) orthogonal design was used to optimize yeast sonication assistant transformation. The expression of EGⅢ gene was induced by 2% β-D-glactose,the transcription and expression of it was detected by Northern blotting and Congo Red method respectively. The endo β-glucanase activity was assayed as CMCase activity with CMC-Na as a substrate. The results show that the ORF of EGⅢ was 1254bp ,encoding 418aa,deducing molecular weight 44.1×103, group 5 (sonication treat time 60 s, incubate 40 min, SS DNA 150μg, heat shock 5 min) was the optimum one of the orthogonal experiment, and EGⅢ transformants can produced clear hydrolysis halos on the Congo Red CMC plate. The measure of the enzyme activity show that the expression product can be expressed in active forms and secreted to the medium.The enzyme activity was approached the highest level (0.041U/mL)when the culture time was 60h .The optimized enzyme reaction temperature was 50℃ and the optimized pH was 5 8. |
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