| β葡聚糖酶杂合基因bglHAM的克隆及在P.pastoris中的表达 |
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| 引用本文： | 苏华波,韦宇拓,黄鲲,赵颖怡,黄日波.β葡聚糖酶杂合基因bglHAM的克隆及在P.pastoris中的表达[J].广西大学学报(自然科学版),2002,27(1):10-13. |
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| 作者姓名： | 苏华波 韦宇拓 黄鲲 赵颖怡 黄日波 |
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| 作者单位： | 广西大学,生物技术实验中心,广西,南宁,530005 |
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| 基金项目： | 国家"八六三计划"(国科生字[1997]192号) |
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| 摘 要： | 采用PCR的方法，以Bacillus macerans总DNA为模板，构建出β-1，3-1，4葡聚糖酶杂合基因bglHAM，与巴斯德毕赤酵母表达载体pPIC9K重组，得到重组质粒pPIC9K-HAM，电脉冲转化法转化酵母菌GS115，KM71，在MM，MD平板上筛选表型，YPD-G418平板上筛选多拷贝重组子，重组菌株经甲醇诱导后，刚果红平板染色法检测到β-葡聚糖酶活性，SDS-PAGE证明表达产物的分子量约为24kD，摇瓶诱导培养筛选到的重组菌株，胞外每mL发酵液最高酶活达240U。
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| 关 键 词： | bglHAM P.pastoris β－1 3－1 4葡聚糖酶 巴斯德毕赤酵母 杂合基因 基因克降 基因表达 PCR扩增 |
| 文章编号： | 1001-7445(2002)01-0010-04 |
| 修稿时间： | 2001年12月20 |
Cloning of a hybrid betaglucanase gene bglHAM and its expression in Pichia pastoris |
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| SU Hua bo,WEI Yu tuo,HUANG Kun,ZHAO Ying yi,HUANG Ri bo.Cloning of a hybrid betaglucanase gene bglHAM and its expression in Pichia pastoris[J].Journal of Guangxi University(Natural Science Edition),2002,27(1):10-13. |
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| Authors: | SU Hua bo WEI Yu tuo HUANG Kun ZHAO Ying yi HUANG Ri bo |
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| Abstract: | A hybrid beta1,31,4 glucanase gene bgl HAM was constructed by PCR on Bacillus macerans genomic DNA. A recombinant plasmid, pPIC9KHAM was constructed by inserting bgl HAM into Pichia expression vector pPIC9K . The recombinant plasmid was transformed into Pichia pastoris strains GS115 and KM71 with electroporation method. Phenotypes of transformants and multicopy transformants were screened on MM, MD plates and YPDG418 plates. The recombinant strains were induced to express betaglucanase with methanol. SDSPAGE showed that the relative molecular weight of the expression product was about 24kD.The activity of the hybrid enzyme was detected by YPML plate stained by congored. The highest extrcellular activity was more than 240U/mL when recombinant strains were induced in flask. |
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| Keywords: | beta1 31 4 glucanase Pichia pastoris hybrid gene cloning induced expression |
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