Discovering Novel ?-L-Rhamnosidases Based On The Metagenomic Approach

?-L-Rhamnosidase(E.C.3.2.1.40)belong to glycoside hydrolase,which specifically cleaves terminal ?-L-rhamnose from a number of natural products,such as hesperidin,rutin,poncirin and quercitrin.It was reported that ?-L-rhamnosidase has wide occurrence in bacteria,yeasts,fungi,plants and animal tissues.Biotechnological applications of the enzyme include debittering and removing turbidity of fruit juices,enhancing the aroma of wine,derhamnosylating of many natural products containing terminal ?-L-rhamnose and improving drug properties used as many drug precursors.193 bacterial ?-L-rhamnosidases belonging to GH78 family are obtained from the CAZy database.These bacterial ?-L-rhamnosidase were divided into three subfamilies based on the difference of conserved amino acid motif at the general acid and general base after multiple sequence alignment.Two pair of conserved amino acid motifs of two subfamilies at the general acid and base were obtained by sequence alignment,respectively.Degenerate primers were designed on the basis of the conserved amino acid motifs.The gene fragments between conserved motifs were amplified using total DNA from feces metagenome as the template by degenerate primers PCR.The PCR products were cloned and sequenced.And 12 ?-L-rhamnosidase gene fragments were obtained.Amino acid sequences encoded by these gene fragments were aligned by 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 primers for amplifying three full length genes which possessing high sequence identities(>94%)were designed according to the corresponding gene from GenBank.The full length gene which displaying the lowest sequence identity(52%)was obtained by de novo sequencing on the human feces metagenome.Finally,the four genes were cloned into the vector pET-28 a and were heterologously expressed in E.coli BL21(DE3).SDS-PAGE showed that the target proteins were expressed in supernatant and precipitation.Catalytic activity of four ?-L-rhamnosidases was detected using the synthetic substrate p-nitrophenyl-?-L-rhamnopyranoside(pNPR).The activity was detected only in the supernatant of RhaC.Subsequently,Rha C was purified by Ni-NTA affinity chromatography.In summary,the human intestinal bacterial metagenome provides a potential source for discovering novel ?-L-rhamnosidases.It is possible that finding novel enzymes from intestinal and environmental microbiome by the metagenomic method based on the conserved amino acid motifs and PCR.