›› 2010, Vol. 12 ›› Issue (5): 80-85.DOI: 10.3969/j.issn.1008-0864.2010.05.14

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High-efficiency Expression of Xylanase XYN-W with High Specific Activity in Pichia pastoris

LUO Jian-jie1, WANG Ya-ru1, YUAN Tie-zheng1, BAI Ying-guo1, HUANG Huo-qing1, LUO Hui-ying1, YAO Bin1, FAN Yun-liu2   

  1. (1.Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Feed Research Institute, Chinese Academy of |Agricultural Sciences, Beijing 100081|2.Biotechnology Research Institute, Chinese Academy of |Agricultural Sciences, Beijing 100081, China)
  • Received:2010-04-06 Revised:2010-06-23 Online:2010-10-15 Published:2010-09-15

高比活木聚糖酶XYN-W及其突变体在酵母中的高效表达

罗建杰1,王亚茹1,袁铁铮1,柏映国1,黄火清1,罗会颖1,姚斌1,范云六2   

  1. (1.中国农业科学院饲料研究所, 农业部饲料生物技术重点开放实验室, 北京 100081|2.中国农业科学院生物技术研究所, 北京 100081)
  • 通讯作者: 姚斌,研究员,博士,博士生导师,从事饲料生物技术研究。Tel:010-82106053;E-mail: yaobin@mail.caas.net.cn
  • 作者简介:罗建杰,研究方向为生物技术。E-mail: luojianjie521@163.com。
  • 基金资助:

    国家863计划项目(2007AA100601);国家肉鸡产业技术体系(nycytx-42-G2-05)资助。

Abstract:

xyn-w gene encoding a xylanase with high specific activity from ruminal fungus Neocallimastix frontalis was inserted into pPIC9 with the yeast α-mating factor and transformed into Pichia pastoris by electroporation to obtain the recombinants. It was shown that the recombinant xylanase xyn-w was high efficiently expressed and secreted into the supernatant medium by SDS-PAGE and expression products analysis. In 5 liter fermentor, xylanase protein  was expressed by the level of 1 mg/mL and its activity was more than 13 000 IU /mL. Using PCR method, a truncated xyn-m was obtained by deleting the 57 amino acids at the C-terminus of the xylanase XYN-W, which were binding domains and anchered domains. Similarly, the Pichia pastoris strains with high expression of xylanase XYN-M were constructed and the enzyme character of purified product was analyzed. The result showed that the specific activity and Kcat of XYN-M was 19 856.6 IU/mg and 4 433.8 s-1 respectively, increased 43.9% and 63.2% compared to those of XYN-W (13 795.3 IU/mg and 2 717.1 s-1), respectively.

Key words: xylanase, Pichia pastoris, XYN-W, high-efficient expression, improvement of specific activity

摘要:

将来源于瘤胃真菌Neocallimastix frontalis的高比活木聚糖酶基因xyn-w整合到毕赤酵母表达载体pPIC9上,通过电击转化得到重组转化子。SDS-PAGE分析和表达产物的研究表明,木聚糖酶基因xyn-w得到了高效分泌表达,在5 L发酵罐中木聚糖酶蛋白表达量达到1 mg/mL,酶活性达到13 000 IU以上。XYN-W 的C端57个氨基酸序列为连接序列和锚定区域,利用PCR方法将此段序列去除,得到截短的木聚糖酶序列xyn-m。用相同方法构建高效表达XYN-M蛋白的毕赤酵母工程菌株,表达产物经纯化后进行酶学性质测定, 结果表明, 其比活为19 856.6 IU/mg, Kcat值为4 433.8 s-1,与纯化的XYN-W蛋白(比活性13 795.3 IU/mg、Kcat值2 717.1 s-1)相比,分别提高了43.9%和63.2%。

关键词: 木聚糖酶;毕赤酵母;XYN-W;高效表达;比活性改良

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