[Directed evolution of cellobiose 2-epimerase and its application in the synthesis of lactulose].

内容摘要

We had engineered Caldicellulosiruptor morganii CE (CmCE) to construct the enzyme CmCE/D226G for catalyzing the isomerization of lactose to lactulose. However, the poor thermostability and catalytic efficiency of CmCE/D226G limits its application under high substrate concentrations. To address these issues, we employed directed evolution to improve the catalysis performance of CmCE/D226G. First, we used error-prone PCR to construct a variant library and identified the positive variants through cysteine-tryptophan chromogenic high-throughput screening. Then, aiming the crucial residue positions, we conducted saturation mutagenesis to obtain the variants with improved thermostability and catalysis. Accordingly, we successfully obtained a variant named CmCE/D226G/S180T. Its half-life and catalysis efficiency (kcat/Km) at 70 ℃ were 184.2 min and 36.5 L/(mmol·s), respectively, which were higher than those (117.7 min and 14.96 L/(mmol·s), respectively) of CmCE/D226G. The improvement of isomerization efficiency resulted from the shortening distance between His377 and the O1 of substrate enol intermediate. The improvement of thermostability was attributed to the rigidness enhancement of a flexible loop. With 350 g/L lactose as the substrate, CmCE/D226G/S180T achieved a lactulose yield of 54.8% and an epilactose yield of 9.2%, while CmCE/D226G only reached a lactulose yield of 50% and an epilactose yield of 10.3%. Taken together, this work provides an elite enzyme for catalyzing the transformation of lactose to lactulose, which especially facilitates the innovation of eco-friendly synthesis of lactulose. 来自热解纤维素菌(Caldicellulosiruptor morganii)的纤维二糖差向异构酶(CmCE)的突变体CmCE/D226G可以催化乳糖异构化为乳果糖，但热稳定性和催化活性制约了其在高底物浓度下的生物转化。为解决上述问题，本研究针对CmCE/D226G开展定向进化，采用易错PCR建立突变体文库，结合盐酸半胱氨酸-色氨酸显色高通量筛选正向突变体，再针对关键位点开展饱和突变，获得了热稳定性和催化活性提高的突变体CmCE/D226G/S180T。该酶在70 ℃下的半衰期从117.7 min提高到184.2 min，催化效率从14.96 L/(mmol·s)提高到36.5 L/(mmol·s)。机理分析表明，突变酶的His377和底物烯醇式中间体O1的距离更近，从而提升了异构化效率，同时突变增强了柔性环刚性，提高了酶热稳定性。以350 g/L乳糖进行生物转化，CmCE/D226G/S180T的乳果糖得率达54.8%，依匹乳糖得率为9.2%;相比之下，CmCE/D226G的乳果糖和依匹乳糖得率仅分别为50%和10.3%。本研究为生物法制备乳果糖提供了优势酶，有利于促进乳果糖的绿色合成技术创新。.