Wolski Paul W, Dana Craig M, Clark Douglas S, Blanch Harvey W
Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Graduate Group of Comparative Biochemistry at University of California, Berkeley, CA, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.
Protein Eng Des Sel. 2016 Apr;29(4):117-22. doi: 10.1093/protein/gzv066. Epub 2016 Jan 26.
Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes.
在某些离子液体（ILs）中溶解木质纤维素生物质可以在纤维素酶解糖化用于生物燃料生产之前提供有效的预处理。为了实现预处理和酶促水解相结合的目标，我们对艾默生篮状菌Cel7A的酶变体进行了改造，使其在水-离子液体溶液（高达43%（w/w）的1,3-二甲基咪唑鎓二甲基磷酸盐和20%（w/w）的1-乙基-3-甲基咪唑鎓乙酸盐）中比野生型艾默生篮状菌Cel7A或里氏木霉Cel7A更具活性和稳定性。一般来说，在高温缓冲液中酶的稳定性越高，在水-离子液体中的稳定性也越高。通过谷氨酰胺环化酶将N端谷氨酰胺残基翻译后修饰为焦谷氨酸增强了艾默生篮状菌Cel7A及其变体的稳定性。差示扫描量热法显示，在水性缓冲液和离子液体-水混合物中，变体1M10的熔点比野生型艾默生篮状菌Cel7A提高了1.9-3.9°C。无论酶是否经过谷氨酰胺环化酶处理，我们都观察到了这种升高。
