Improving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp. S38: Structural comparison and mutational analysis

Abstract

Endo‐β‐1,4‐xylanases of the family 11 glycosyl‐hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M‐1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three‐dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt‐bridges and the presence of more charged residues in the catalytic cleft as well as an eight‐residue‐longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site‐directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt‐bridge in the N92D/E139K mutant.