该研究成功构建4种杂合纤维小体支架蛋白,并将纤维素酶与其在胞外组装成空间结构不同的嵌合纤维小体,分析了嵌合纤维小体的酶的热稳定性和酶解动力学特征。并基于酿酒酵母EBY100细胞表面展示系统锚定优化前后的支架蛋白以组装嵌合纤维小体进行纤维素同步糖化发酵产乙醇性能分析。结果表明嵌合纤维小体的酶活性在50 ℃下维持相对稳定状态达到120 h。其中ScafI-IV型支架蛋白嵌合纤维小体的Vmax=0.147 mg/（mL·min）,Km=6.085 mg/mL及水解纤维素的还原糖产量均高于其他3种结构嵌合纤维小体。CBP酿酒酵母菌群以磷酸膨胀纤维素为底物进行同步糖化发酵产乙醇,ScafI-IV型结构嵌合纤维小体在96 h时乙醇产量达到1.12 g/L,产量为0.263 g/g,相当于乙醇产率理论值的51.50%。该结果证明通过优化支架蛋白结构可改善嵌合纤维小体的酶解性能,并对于人工设计纤维小体的研究具有一定的理论意义。
Abstract
In this study, four different heterozygous fibrin scaffold proteins were successfully constructed. Cellulase and its extracellular assembly into a different spatial structure of chimeric designer cellulosome. The thermal stability and enzymatic hydrolysis kinetics of chimeric designer cellulosome were analyzed. The influence of different structures on the hydrolysis activity of designer cellosome and its optimal scaffold proteins structure was explored. Based on the Saccharomyces cerevisiae eby100 surface display system, the optimized scaffold protein was anchored and the performance of simultaneous saccharification and ethanol fermentation were assayed on PSAC. The results show that the enzyme activity of the designer cellosome remains relatively stable at 50 ℃ for 120 hours, the Vmax=0.147 mg/（mL·min）, Km=6.085 mg/mL and the reducing sugar production of ScafI-IV designer cellosome are all higher than those of the other cellosomes structural, which indicates the ScafI-IV type has a excellence substrate affinity and hydrolysis property. The maximum ethanol production is 1.12 g/L after 96 h, and the output is 0.263 g/g, which is equivalent to 51.50% of the theoretical value of ethanol yield. This result proves that the enzymolysis performance of the designer cellosome can be improved by structure optimizing of the scaffold proteins, which has reference significance for the research of artificially designing the cellosome.
关键词
酿酒酵母 /
纤维素乙醇 /
空间结构 /
表面展示 /
纤维小体
Key words
Saccharomyces cerevisiae /
cellulosic ethanol /
spatial structural /
surface display /
fibrous corpuscle
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基金
国家自然科学基金（31971202）; 国家科技支撑计划（2019YFB1503802）; 科技创新服务能力建设资助项目（025185305000/198）
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