Celińska Ewelina, Borkowska Monika, Białas Wojciech
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland.
Appl Microbiol Biotechnol. 2016 Mar;100(6):2693-707. doi: 10.1007/s00253-015-7098-8. Epub 2015 Nov 7.
Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain's secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different.
淀粉是生物乙醇生产中消耗的主要原料，占全球生物乙醇产量的60%。鉴于生物乙醇对全球燃料市场的重大贡献，其主要操作技术的任何改进在经济上都非常有吸引力。据估计，最终乙醇单价的高达40%来自底物预处理所需的能量输入。应用生淀粉水解酶（RSHE），并结合同时糖化发酵（SSF）策略进行工艺操作，是解决当前技术局限性最有前景的方案。在本研究中，我们在专用于蛋白质过表达的酿酒酵母菌株中表达了一种源自昆虫的新型RSHE。随后，在由工业产乙醇酵母或耐热酵母菌株进行的SSF过程中评估了该酶的性能。将昆虫源RSHE制剂与市售淀粉分解性RSH制剂进行了比较。我们的结果表明，来自米象的重组α-淀粉酶可以在酿酒酵母INVSc-pYES2-Amy1表达系统中通过其天然信号肽高效表达和分泌（占菌株分泌蛋白质组的近72%）。基于重组酶的制剂在SSF过程中的应用确保了足够的淀粉分解活性，以促进酵母细胞增殖和从生淀粉形成乙醇。比较制剂产生的（寡）糖谱在糖混合物的均匀性方面有所不同。同时，正如本研究开发的动力学模型所表明的，描述比较制剂活性的动力学参数是不同的。
