信号肽对木聚糖酶在马克斯克鲁维酵母中分泌表达的影响

Effect of Signal Peptide on Secretory Expression of Xylanase in Kluyveromyces marxianus

马克斯克鲁维酵母(Kluyveromyces marxianus)是一种新型的非常规酵母,具有生长快、分泌能力强、安全性高等优势.在酵母表达系统中,分泌信号肽不仅介导了外源蛋白沿着正确途径分泌到胞外,也在蛋白质翻译后加工等方面发挥了重要作用.本文分别研究了酿酒酵母α-Factor信号肽和克鲁维酵母菊粉酶信号肽对耐热木聚糖酶在马克斯克鲁维酵母中的分泌表达影响.工程菌摇瓶发酵72h后,在含有菊粉酶信号肽和α-Factor信号肽的工程菌株的发酵上清中,木聚糖酶酶活分别为318.91U/mL和117.90U/mL,表明马克斯克鲁维酵母表达木聚糖酶时,菊粉酶信号肽介导的蛋白分泌表达效果优于α-Factor信号肽.重组表达木聚糖酶最适反应条件是pH5.5和65℃,在75℃处理1h后,该酶的剩余酶活保留73%以上.在5L发酵罐中,重组菌高密度发酵72h,木聚糖酶酶活高达157 757U/mL,结果表明,马克斯克鲁维酵母表达系统在工业酶领域中应用具有非常广阔的前景.

As a new type of non-conventional yeast,Kluyveromyces marxianus is the fastest growing eukaryote with strong secretion and high safety.In yeast,secretory signal peptide plays an important role in mediating the heterologous proteins into the post-translational modifications,such as glycosylation,which is important for the synthesis of correctly folded and active form,and also to secrete them.In this study,we employed two secretory signal peptides,includingα-Factor signal peptide fromSaccharomyces cerevisiae and inulinase signal peptide from K.marxianus,to compare the secretion efficiency of the thermostable xylanase.The xylanase activity in the supernatant produced by the recombinant strains Fim-1/pUKD-Inu-xyn and Fim-1/pUKD-α-Factor-xyn was318.91U/mL and 117.90U/mL,respectively,indicating that the secretion efficiency of inulinase signal peptide was higher than that ofα-factor signal peptide.The optimum pH and temperature of xylanase was pH 5.5 and 65℃,respectively,and incubated at 75 ℃ for 1h,the residual activity was more than 73%.In a 5 Lfermentor,the expression level of xylanase reached 157 757U/mL.Our results showed that the K.marxianus expression system has a very broad application prospect in industrial enzyme field.