摘要
酿酒酵母水解菊糖的能力与蔗糖转化酶SUC2的分泌表达水平直接相关,而绝大多数酿酒酵母中SUC2的低水平分泌表达限制了菊糖基乙醇工业化生产的进程。通过全基因合成获得α交配因子分泌信号肽编码基因片段αFSP,并利用RF克隆手段将SUC2自身的天然分泌信号肽替换为αFSP。重组菌株BY-mS发酵48h后的酶活力测定结果表明,全细胞和发酵液上清菊糖酶活力分别为2.6和3.4U/mL,蔗糖酶活力分别为34.5和9.7U/mL均高于对照菌株BY-BS。说明相对于SUC2自身的天然分泌信号肽,α交配因子分泌信号肽的融合能够有效地提高SUC2在酿酒酵母中的分泌水平,且从不同分泌场所中的酶活差异可以推测出,SUC2分泌定位于细胞壁表面时的构象更加利于蔗糖酶酶活的展示,而当SUC2分泌至发酵液上清中时,结构的变化导致该酶的菊糖酶活力更高。
The inulin-degrading ability of Saccharomyces cerevisiae is directly related to the secretory level of invertase SUC2.The low secretory level of SUC2 in most Saccharomyces cerevisiae strains limited the process of industrial production of inulin ethanol.The gene fragmentαFSP encodingαmating factor secretory signal peptide was obtained by whole gene synthesis,and the natural secretory signal peptide of SUC2 was replaced byαFSP through RF cloning.The enzyme activity of the recombinant strain BY-mS after 48 h fermentation showed that the inulinase activities of the whole cell and the supernatant were 2.6 and 3.4 U/mL,respectively,while the sucrase activities were 34.5 and 9.7 U/mL,respectively,which were higher than those of the control strain BY-BS.The results indicated that compared to the natural secretion signal peptide of SUC2,the fusion ofα-mating factor secretory signal peptide could effectively improve the secretory level of SUC2 in Saccharomyces cerevisiae.The variation in enzyme activities from different locations suggested that the conformation of SUC2 anchored in cell surface was benefit to higher sucrase activity,while SUC2 was secreted into supernatant,the changed conformation lead to higher inulinase activity.
作者
郑聪聪
周海龙
赵晶晶
李宪臻
杨帆
ZHENG Congcong;ZHOU Hailong;ZHAO Jingjing;LI Xianzhen;YANG Fan(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China;China Biotech Fermentation Industry Association,Beijing 100833,China)
出处
《大连工业大学学报》
CAS
北大核心
2021年第1期1-5,共5页
Journal of Dalian Polytechnic University
基金
国家自然科学基金项目(31801469)
辽宁省高校杰出青年学者成长计划(LJQ2015009)。
关键词
蔗糖转化酶
菊糖
分泌表达
invertase
inulin
secretory expression
