| 内切葡聚糖酶基因在毕赤酵母中高效表达及表达条件研究 |
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| 引用本文： | 吴振芳,陈惠,曾民,吴琦.内切葡聚糖酶基因在毕赤酵母中高效表达及表达条件研究[J].农业生物技术学报,2009,17(3):529-535. |
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| 作者姓名： | 吴振芳 陈惠 曾民 吴琦 |
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| 作者单位： | 四川农业大学8-20号信箱 |
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| 摘 要： | 根据枯草芽孢杆菌内切葡聚糖酶基因序列设计引物，采用PCR扩增到获得去除信号肽后约1.4Kb的内切葡聚糖酶表达片段。以此片段成功构建了pPIC-End载体，并转化至巴斯德毕赤酵母GS115。经过MD、MM平板筛选和酶活性测定，获得了高效表达的转化子GS115-pPIC-EndⅠ、GS115-pPIC-EndⅦ、GS115-pPIC-EndⅧ。在摇瓶培养条件下，对酵母工程菌表达条件进行了优化研究：在pH4-8条件下均能稳定表达，诱导起始OD600=5表达水平最高，甲醇诱导最佳浓度为0.5%-1%，加大培养通气量对表达有显著的促进作用。三种工程菌在优化条件后诱导培养，酶活性可达860.7U、760.3U、786.2U，分别为原始菌株酶活（63.78U）的13.5倍、11.9倍和12.3倍。SDS-PAGE分析表明表达产物分子量约为79.82KDa，热稳定性分析表明该酶在65℃保温30min可保持最高酶活的80%以上。
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| 关 键 词： | 枯草芽孢杆菌 中性内切葡聚糖酶 毕赤酵母 表达条件 高效表达 |
| 收稿时间： | 2000-8-8 |
| 修稿时间： | 2008-9-23 |
Over-Expression of Endoglucanase Gene in Pichia Pastories and Study of Expression Condition |
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| Abstract: | Endoglucanase is one of the most important cellulose enzyme, which cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose completely. Our objectives were to express an endoglucanase gene in Pichia pastoris and find out expression conditions for high-level expression of neutral endoglucanase. According to the sequence of endoglucanase gene (GenBank Accession No.DQ782954) from Bacillus subtilis, a pair of primers was designed and synthesized, and a high-fidelity polymerase (Pfu DNA polymerase) was used for amplifying the mature endoglucanase expression gene. A 1.4kb fragment, which does not contain signal peptide sequence obtained, and was ligated to expression vector pPIC9k. pPIC-End was constructed and transformed to Pichia pastoris GS115. Three transformants named GS115-pPIC-EndⅠ, GS115-pPIC-EndⅦ, GS115-pPIC-EndⅧ were obtained through MD,MM plate screening and enzymatic activity test. The expression conditions containing the initial cell density, pH and methanol concentration were studied in shaking culture. Optimal endoglucanase production was observed when the initial cell density OD600was 5, and the optimal methanol concentration was 0.5%-1%. The endoglucanase production increased obviously when provided adequate aeration, whereas it did not seem to vary when cells were induced at pH4-8. Under the above expression condition, the endoglucanase activity of the three transformants was 860.7U, 760.3U, 786.2U, 13.5 times, 11.9 times and 12.3 times compared with the endoglucanase activity of the original strain (63.78U) respectively. The enzyme maintained over 80% of the original enzyme activity after incubated at 65℃ for 30 min, and the SDS-PAGE showed that The molecular weight is about 79.82KDa. |
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