Research On Genetic Mining And Catalytic Mechanism Of Novel Carbohydrate-Active Enzyme

Enzymolysis of xylan is one of the main bioway to produce xylo-oligosaccharide which is a kind of prebiotics.However,effective enzymolysis of xylan has to be carried out in the multi-enzymatic system,because the conformation of xylan is complex due to the interaction between the main chain and side chain of xylan.Hence,bifunctional enzymes capable of simultaneous hydrolysis of main and side chains are effective for enzymolysis of xylan.In the present work,a bifunctional acetyl ester-xyloside hydrolase(CLH10)was obtained by genetic mining.The primary sequence of CLH10 contains the fragments of the conserved sequence of esterase and glycosidase,which distribute in a mixed type.While the crystal structure revealed that the primary sequence folded into two independent structural regions to undertake both acetyl esterase and ?-1,4-xylanase hydrolase functions.CLH10 is capable of cleaving both the?-1,4-xylosidic bond-linked main chain and the ester bond-linked acetylated side chain of xylan,which renders it valuable because it can degrade acetylated xylan within one enzyme.Significantly,the ?-1,4-xylanase activity of CLH10 appears to have been fortuitously obtained due to the variable Asp10 and Glul39 located in its loop region,which suggested that the exposed loop region might act as a potential hot-spot for the design and generation of promising enzyme function in both directed evolution and rational protein design.