棘孢木霉几丁质酶tachi2基因的原核表达及酶学性质研究  

Prokaryotic Expression of tachi2 Gene from Trichoderma asperellum and Characterization of Recombinant Enzyme

作　　者：张军霞[1] 丛大鹏[1] 李雅华[1] 咸洪泉[1] 

机构地区：[1]青岛农业大学生命科学学院,青岛266109

出　　处：《中国生物工程杂志》2013年第6期45-51,共7页China Biotechnology

基　　金：山东省自然科学基金资助项目(ZR2010CM040)

摘　　要：目的:实现棘孢木霉(Trichoderma asperellum)几丁质酶基因tachi2的原核高效表达,研究几丁质酶Tachi2的酶学性质。方法:利用PCR技术扩增得到几丁质酶基因tachi2,将其克隆到原核表达载体pEHISTEV中,测序后,转化大肠杆菌BL21感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后进行Tachi2蛋白的纯化和复性。用纯化的目的蛋白Tachi2进行几丁质酶酶学性质的研究。结果:tachi2基因在重组大肠杆菌中正确表达,其主要以包涵体形式存在;重组蛋白Tachi2分子量约为44kDa,经过纯化和复性后得到的Tachi2有较高的几丁质酶活性。该酶的最适温度为40℃,最适pH值为7.0,几丁质酶在40℃以下比较稳定、pH 6～9时酶有较高活性,受Cu2+和Zn2+的强烈抑制。结论:成功实现了棘孢木霉几丁质酶基因tachi2的原核高效表达,表达纯化了重组蛋白,明确了几丁质酶Tachi2的酶学性质,为该几丁质酶的进一步开发利用和深入研究奠定了基础。Objective： To realize high level prokaryotic expression of the tachi2 gene from Trichoderma asperellum and characterize the recombinant enzyme. Methods： The tachi2 gene was amplified by PCR, and recombinant plasmid pEHISTEV /tachi2 was constructed successfully by inserting the tachi2 gene into pEHISTEV. The recombinant plasmid pEHISTEV/tachi2 was transformed into Escherichia coli BI21, which was induced with isopropyl-β-D-thiogalactoside （IFFG）. The recombinant protein Tachi2 was purified and renatured to analyze enzymatic properties. Results：The recombinant protein Tachi2 was expressed successfully in the form of inclusion body in the recombinant E. coll. The molecular weight of Tachi2 was about 44 kDa, Taehi2 was purified and renatured by a serial of treatments, the renatured protein had high chitinase activity. The optimum temperature and pH for the enzyme activity were 40℃ and 7.0 respectively. The enzyme activity was stable under 40 ℃ and in the pH range of 6 - 9, its activity was significantly reduced by 0.05 mol/L of Cu2 ＋ and Zn2＋ Conclusion：The recombinant strain Escherichia coli BL21 showed high level prokaryotic expression of the tachi2 gene. And the enzymatic properties were further defined. It will provide theoretical basis for application and further research in this chitinase.

关 键 词：棘孢木霉 几丁质酶 原核表达 酶学性质 

分 类 号：Q93[生物学—微生物学]