易错PCR技术提高嗜热酸性Ⅲ型普鲁兰水解酶TK-PUL催化活性的研究  

Research on Improving the Catalytic Activity of Thermoacidophilic Type Ⅲ Pullulan Hydrolase TK-PUL by Error-prone PCR

作　　者：曾静[1] 郭建军[1] 袁林[1] ZENG Jing;GUO Jianjun;YUAN Lin(Institute of Microbiology,Jiangxi Academy of Sciences,Nanchang 330096,China)

机构地区：[1]江西省科学院微生物研究所,江西南昌330096

出　　处：《食品工业科技》2022年第18期130-136,共7页Science and Technology of Food Industry

基　　金：国家自然科学基金地区科学基金项目(32160579);江西省杰出青年科学基金项目(20202ACBL215001);江西省主要学科学术和技术带头人培养计划(20212BCJ23033)。

摘　　要：嗜热酸性Ⅲ型普鲁兰水解酶TK-PUL在液化条件下可完全水解淀粉为淀粉糖,在“液化糖化一步法”的淀粉制糖工艺中具有巨大的应用潜力。本研究拟采用易错聚合酶链式反应(polymerase chain reaction,PCR)技术提高TK-PUL的催化活性。经过两轮易错PCR及高通量筛选,获得了催化活性提高的突变体L538D。与TK-PUL相比,L538D以可溶性淀粉为底物的比酶活力提高了50%,以普鲁兰多糖为底物的比酶活力提高了21%;L538D以可溶性淀粉、普鲁兰多糖为底物的k/K值分别提高了44%、27%。即L538D的α-1,4-糖苷键水解活性和α-1,6-糖苷键水解活性均明显提高,并且其α-1,4-糖苷键水解活性的提高幅度更大。同源模建得到的蛋白质分子结构显示,将TK-PUL中Leu538替换为Asp,可能通过缩短氨基酸残基侧链的长度和减弱氨基酸残基的疏水性,提高了催化活性位点Glu538所在loop结构的柔性,从而提高酶的催化活性。本研究结果表明,TK-PUL中Leu538是影响其催化活性的重要氨基酸残基。Thermoacidophilic type Ⅲ pullulan hydrolase TK-PUL can completely hydrolyze starch to starch sugar under liquefaction condition. It has great application potential in the ’’one step liquefaction-saccharification’’ starch syrup production process. In this study, error-prone PCR technology was used to improve the catalytic activity of TK-PUL. After two sequential error-prone PCR and high-throughput screening, the mutant L538D with improved catalytic activity was obtained. Compared with TK-PUL, the specific activity of L538D with soluble starch as substrate increased by 50%, the specific activity of L538D toward pullulan increased by 21%. And the kcat/Km value of L538D for soluble starch, pullulan increased by 44% and 27%, respectively. In other words, the hydrolytic activity of L538D toward α-1,4-glycosidic bond andα-1,6-glycosidic bond were significantly improved, and the hydrolytic activity of L538D toward α-1,4-glycosidic bond increased to a greater degree. Homology modeling showed that replacing Leu538 with Asp in TK-PUL may improve the flexibility of the loop structure including the catalytic site Glu538 by shortening the length of the side chain of amino acid residues and reducing the hydrophobicity of amino acid residues, thus improving the catalytic activity of the enzyme. The results demonstrated that Leu538 in TK-PUL is vital for its catalytic activity.

关 键 词：Ⅲ型普鲁兰多糖水解酶 定向进化 易错聚合酶链式反应(易错PCR) 高通量筛选技术 催化活性本 

分 类 号：Q814[生物学—生物工程]