生物技术进展 ›› 2013, Vol. 3 ›› Issue (3): 211-217.DOI: 10.3969/j.issn.2095-2341.2013.03.11

• 研究论文 • 上一篇    下一篇

阴沟肠杆菌过氧化氢酶基因的重组表达及酶学性质研究

周利伟1§,张艳丽1,2§,左冬阳1,张伟1,张宇宏1*   

  1. 1.中国农业科学院生物技术研究所， 北京 100081;
    2.西南大学园艺园林学院， 重庆 400715
  • 收稿日期:2013-01-21 出版日期:2013-05-25 发布日期:2013-04-20
  • 通讯作者: 张宇宏，助理研究员，博士，从事微生物基因工程研究。E-mail：zhangyuhong@caas.cn
  • 作者简介:周利伟与张艳丽为本文共同第一作者。周利伟，硕士研究生，主要从事生物化学与分子生物学方向的研究。E-mail: zlwei_815@163.com；张艳丽，硕士研究生，主要从事微生物酶工程的研究。E-mail: zyl_18651@126.com
  • 基金资助:

    国家自然科学基金青年基金项目(31201313)资助。

Recombinant Expression and Characterization of a Catalase from Enterobacter cloacae

ZHOU Li-wei, ZHANG Yan-Li,, ZUO Dong-yang, ZHANG Wei, ZHANG Yu-hong   

  1. 1.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
    2.College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China
  • Received:2013-01-21 Online:2013-05-25 Published:2013-04-20

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摘要： 过氧化氢酶又称触酶，在食品、纺织和造纸等领域应用广泛。本研究从阴沟肠杆菌Enterobacter cloacae XTL13中克隆得到一个过氧化氢酶基因cat1，该基因全长2 250 bp，编码749个氨基酸和一个终止密码子。将cat1基因连接pPIC9载体并转化毕赤酵母GS115，得到高效表达CAT1的重组酵母菌株。在摇床水平过氧化氢酶活性可达300 U/mL。重组CAT1最适温度为37℃，最适pH为6.5，比活力为1 667 U/mg；50℃处理2 h或pH 5~8处理1 h后，仍然能保留80%以上的酶活力。当CAT1与葡萄糖氧化酶(GOD)联合使用（酶活力比例为1∶30）时，能够解除过氧化氢对GOD活力的抑制，使得GOD能够更好地发挥其作用。另外，本研究还建立了一种快速筛选过氧化氢酶重组菌株的方法。

关键词: 过氧化氢酶, 阴沟肠杆菌, 毕赤酵母, 基因表达, 葡萄糖氧化酶

Abstract: Hydrogen peroxidase, commonly named catalase, has been widely used in food, textile and paper industries. A catalase gene cat1 was cloned from Enterobacter cloacae by using touch-down PCR and TAIL-PCR. Length of cat1 is 2 250 bp, which encode 749 amino acids and a termination codon. This gene was cloned into an expression vector pPIC9, and overexpressed in Pichia pastoris GS115 with the maximum catalase activity of 300 U/mL at shaker level. The recombinant CAT1 exhibited optimal activity at pH 6.5 and 37℃. Specific activity of CAT1 was 1667 U/mg, towards hydrogen peroxide as substrate. CAT1 remains above 80% enzyme activity after treated at 50 C for 2 h or stay at pH 5~8 for 1 h. The effects of CAT1 on glucose oxidase (GOD) activity was investigated. When the activity ratio of CAT1 to GOD was 1∶30, the GOD activity was no longer inhibited by hydrogen peroxide. This study also introduced a rapid screening method for P. pastoris with recombinant catalase.

Key words: catalase, Enterobacter cloacae, Pichia pastoris, gene expression, glucose oxidase

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