学术
摘 要:
拟南芥谷胱甘肽S-转移酶Zeta类（AtGSTZ）是一种与细胞代谢和环境净化密切相关的多功能酶．应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库，再利用pH指示剂颜色改变法对突变文库进行筛选，获得了9个二氯乙酸脱氯活性提高的突变子．其中，NN23含25个氨基酸突变，比活力提高120％，NN20含24个氨基酸突变，比活力提高102％，ECl含2个氨基酸突变，比活力提高47％，其他6个为单点突变，比活力分别提高9％～60％．酶学分析显示，所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高，但热稳定性均没有明显改善．同时，对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论．[著者文摘]
文章出处:
《生物化学与生物物理进展》-2008年35卷2期 -208-216页
栏目信息:
Obtainment and Characterization of The Evolved Enzymes From Arabidopsis thaliana Glutathione S-Transferase Zeta Class
TAO Su-Dan, CHEN Xi-Wen, LIU Jia, JIA Xiang-Dong, CHEN De-Fu （College of Life Sciences, Nankai University, Tianjin 300071, China）
Abstract:
The A rabidopsis thaliana glutathione S-transferases zeta class （AtGSTZ） is a multi-functional enzyme, which plays important role in cellular metabolism and environmental purification. Error-prone PCR and cycles of DNA shuffling were used to construct a mutagenesis library of AtGSTZ. The screening of the resultant libraries was carried out by a pH indicator dye-based colorimetric assay. Nine mutants which enhanced the dichloroacetic acid dechlorination activity were obtained. Among them, NN23 contained 25 amino acid substitutions with the activity improving 120%, whereas NN20 contained 24 amino acid substitutions with the activity improving 102%. ECl contained 2 amino acid substitutions with the activity improving 47%. The rest 6 mutants contained one amino acid substitution with their activity increasing from 9% to 60%. The enzymatic characterization showed that all the evolved enzymes increased their catalytic efficiencies towards dichloroacetic acid and binding affinity towards glutathione whereas some of them increased the renaturability. However there is no obvious change in their thermostability. Based on these data, functional residues related to catalysis and refolding of AtGSTZ were discussed.[著者文摘]
Key words:
evolved mutants, dichloroacetic acid dechlorination （DCA-DC） activity, thermostability, renaturability, A rabidopsis thaliana glutathione S-transferases zeta class （AtGSTZ）
基金资助:
国家自然科学基金资助项目（30671183）. 致谢 感谢南开大学生命科学学院2003级明铭同学在本工作上给予的协助.
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