Improving Hydrolysis Characteristics of Xylanases by Site-Directed Mutagenesis in Binding-Site Subsites from Streptomyces L10608

The preparation of oligosaccharides via xylan hydrolysis is an effective way to add value to hemicellulosic material of agricultural waste. The bacterial strain Streptomyces L10608, isolated from soil, contains genes encoding xylanases of glucoside hydrolase family 10/11 (GH10/11), and these have been cloned to catalyze the production of xylooligosaccharide (XOS). To improve the XOS proportion of hydrolysates produced by xylanase, four amino acid residues were substituted by site-directed mutagenesis, and the mutant genes were overexpressed in Escherichia coli. Mutations replaced the codons encoding Asn214 (+2) and Asn86 (−2) by Ala and removed the Ricin B-lectin domain in GH10-xyn, and mutants Y115A (−2) and Y123A (−2) were produced for GH11-xyn. Interestingly, GH10-N86Q had significantly increased hydrolysis of XOS and almost eliminated xylose (X1) to <2.5%, indicating that the −2 binding site of GH10-xyn of L10608 is required for binding with xylotriose (X3). The hydrolytic activity of GH10-N86Q was increased approximately 1.25-fold using beechwood xylan as a substrate and had high affinity for the substrate with a low K(m) of about 1.85 mg·mL(−1). Otherwise, there were no significant differences in enzymatic properties between GH10-N86Q and GH10-xyn. These mutants offer great potential for modification of xylanase with desired XOS hydrolysis.