Functional Improvement of Xylanase by Introducing Mutated Xylan-binding Domain

Abstract

Alkaliphilic Bacillus sp. strain 41M-1 secretes a xylanase (termed xylanase J) that has an alkaline pH optimum. Xylanase J is a multidomain enzyme and consists of two functional domains: a glycoside hydrolase family 11 catalytic domain and an additional domain of unknown function. Protein engineering study of xylanase J indicated that the functionally unknown domain should be a xylan-binding domain (XBD) belonging to carbohydrate binding module family 36. The XBD bound to insoluble xylan and enhanced hydrolyzing activity of the adjacent catalytic domain. The XBD was successfully displayed on the surface of filamentous phage. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on phage. Sequencing analysis of the xylan-binding activity-deficient mutants revealed that Phe284, Asp286, Asp313, Trp317 and Asp318 might contribute to the xylan-binding activity of XBD. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of xylanase J toward insoluble xylan was improved by introducing mutation T316I.