摘要
目的克隆、表达宇佐美曲霉(Aspergillus usami)羧肽酶成熟肽基因,并检测其酶学性质。方法根据前期克隆的宇佐美曲霉羧肽酶基因序列设计引物,克隆羧肽酶成熟肽基因AucpA,构建重组表达质粒pPIC9k-AucpA,电击转化毕赤酵母GS115,甲醇诱导重组蛋白表达。表达的重组蛋白经Sephadex-50层析纯化后,进行酶活性及其影响因素的检测。结果重组表达质粒pPIC9K-AucpA经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约81 000,纯化的重组羧肽酶最高比酶活为57.15 nkat/mg,最适反应温度为45℃,最适反应pH为3.5,金属离子、EDTA对重组羧肽酶的酶活性无影响,而PMSF对重组羧肽酶酶活性的抑制率达50%以上。结论已成功在毕赤酵母中表达了具有较高酶活的宇佐美曲霉羧肽酶,为进一步研究该重组酶的应用奠定了基础。
Objective To clone and express the carboxypeptidase mature peptide gene of Aspergillus usamii and analyze its enzymatic property. Methods Primers were designed based on the previously cloned carboxypeptidase sequence of A spergillus usamii, with which carboxypeptidase mature peptide gene A ucpA was cloned and inserted into vector pPIC9K. The constructed recombinant plasmid pPIC9K-AucpA was transformed to Pichia pastori GS115 by electroporation for expression under induction of methanol. The expressed protein was purified by Sephadex-50 chromatography, and determined for activity and its influencing factors. Results Restriction analysis and sequencing proved that recombinant plasmid pPICgK-A ucpA was constructed correctly. The expressed recombinant carboxypeptidase, with a relative molecular mass of 81 000, reached a specific activity of 57. 15 nkat/mg at most, of which the optimal temperature and pH value were 45℃ and 3.5 respectively. Metal ions and EDTA showed no effect on the activity of recombinant carboxypeptidase. However, the inhibiting rate of PMSF to the activity was more than 50%. Conclusion The carboxypeptidase gene of Aspergillus usamii, with a high activity, was successfully expressed in P. pastoris, which laid a foundation of further study on its application.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期176-180,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(311012299)
关键词
宇佐美曲霉
羧肽酶
基因表达
酶学
Aspergillus usamii
Carboxypeptidase
Gene expression
Enzymology
