克隆来自Thermobifida fusca(嗜热放线菌)的编码角质酶基因tfu,并在N端人为添加了α-factor信号肽,以引导重组角质酶的分泌表达。在成功构建了重组菌株后,深入分析了半乳糖浓度对重组菌株表达角质酶的影响,并对诱导方式进行了优化分析。此外,还分析了混合碳源对重组菌株产角质酶的影响。依靠α-factor信号肽的引导,成功实现了重组酵母菌株角质酶的活性分泌表达。混合碳源实验表明,棉子糖和半乳糖的组合能够更好地促进角质酶的分泌表达,这为角质酶发酵生产及应用于食品医药及保健品行业提供了参考和借鉴。

In the present study,we cloned the tfu gene encoding cutinase from Thermobifida fusca.To accomplish the secretion of cutinase,α-factor signal peptide was added at the N terminal of cutinase.After successfully construction of the recombinant strain,the effects of concentration of galactose and induction mode were deeply analyzed.Moreover,we investigated the effects of different combinations of mixture of carbon source on the production of cutinase.As a result,raffinose was proved to be much more active than other sugars applied for overexpression of cutinase.The results achieved herein would provide an alternative for cutinase production and its potential application.This would be more attractive in the food and pharmaceutical industry.