基于宏基因组学方法挖掘新型α-L-鼠李糖苷酶资源

Discovering Novel α-L-Rhamnosidases Based on the Metagenomic Approach

α-L-鼠李糖苷酶是特异性切割末端含α-L-鼠李糖的天然化合物的一类糖苷水解酶,该酶在食品、医药、化学等行业都有广泛的应用。本研究旨在利用保守氨基酸基序结合PCR驱动的宏基因组学方法,从提取的健康人体粪便宏基因组DNA中获得新型细菌源α-L-鼠李糖苷酶基因。通过对CAZy数据库GH78家族中193条细菌源α-L-鼠李糖苷酶氨基酸序列进行多重序列比对,首次将α-L-鼠李糖苷酶家族分为3个亚家族,并确定了其中两个亚家族的两对保守氨基酸基序。基于保守基序氨基酸序列设计简并引物,PCR扩增保守基序间基因片段,对PCR产物克隆测序,结果获得12条α-L-鼠李糖苷酶基因片段,对其编码的氨基酸序列在Gen Bank数据库进行Blast序列比对,其中两条基因片段的氨基酸序列一致性仅为52%,一条为73%,其余9条在94%以上。根据Gen Bank数据库中序列一致性94%以上片段对应全长基因序列设计上下游引物,以人体粪便宏基因组DNA为模板,扩增获得3条α-L-鼠李糖苷酶全长基因,并将其克隆于载体p ET-28a,在E.coli BL21(DE3)内进行异源表达,目的蛋白多以包涵体形式存在于沉淀中,少量以可溶性形式存在于上清中。人体肠道细菌宏基因组为新型α-L-鼠李糖苷酶基因发现提供了潜在的基因资源库,基于保守氨基酸基序驱动的宏基因组学方法,从人体肠道以及环境宏基因组中直接获取新酶基因是可行的。

α-L-rhamnosidase is a kind of glycoside hydrolase which cleaves terminal α-L-rhamnose specifically from a large number of natural products,it was used widely in the food,medicine,chemical and other industries. We want to obtain the novel bacterial α-L-rhamnosidase genes with the approach based on the conserved amino acid motifs of α-L-rhamnosidase family and degenerate primers PCR from the metagenomic DNA which was extracted from healthy human feces. 193 amino acid sequences of bacterial α-L-rhamnosidases belonging to the GH78 family from the CAZy database were analyzed by multiple sequences alignment. The α-L-rhamnosidase family was firstly divided into three subfamilies and two pairs of conserved amino acid motifs from two subfamilies were defined. Degenerate primers weredesigned on the basis of the conserved amino acid motifs. The gene fragments between conserved motifs were amplified by degenerate primers PCR which used the total DNA from feces metagenome as the template. The PCR products were cloned and sequenced. And 12 α-L-rhamnosidase gene fragments were obtained. Amino acid sequences encoded by these gene fragments were aligned via blasting in the Gen Bank. The results showed that amino acid sequence identities of two fragments were only 52%,one fragment was 73%,and the remaining nine fragments were more than 94%. The PCR primers for amplifying full-length genes which were high identities（ 94%） were designed according to the corresponding sequences from Gen Bank,and three full genes were amplified via metagenomic DNA as the template. Finally,the three α-L-rhamnosidase genes were cloned into the vector p ET-28 a and were heterogeneously overexpressed with the precipitate as inclusion bodies in E. coli BL21（ DE3）. The human intestinal bacterial metagenome provides a potential source for discovering novel α-Lrhamnosidases. It is possible that finding novel enzymes from intestinal and environmental microbiome directly by the metagenomic method based on the conserved amino acid motifs and PCR.