The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans.

Talaromyces emersonii, a thermophilic aerobic fungus, produces a complete xylan-degrading enzyme system when grown on appropriate substrates. In this paper we present the physicochemical and catalytic properties of three enzymes, xylosidase (Xyl) I (M(r) 181,000; pI 8.9), II (M(r) 131,000; pI 5.3) and III (M(r) 54,200; pI 4.2). Xyl I and II appear to be dimeric and Xyl III is a single-subunit protein. All three enzymes catalyse the hydrolysis of aryl beta-D-xylosides and xylo-oligosaccharides. Xyl I is a classic beta-xylosidase (1,4-beta-D-xylan xylohydrolase; EC 3.2.1.37), and Xyl II and III are novel xylanases (endo-1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) which we believe have not hitherto been reported. In addition to the above substrates, they also catalyse the extensive hydrolysis of unsubstituted xylans, and may have considerable biotechnological potential. The hydrolysis product profiles and bond-cleavage frequencies with various substrates are presented

Structural Insights into the Specificity of Xyn10B from Paenibacillus barcinonensis and Its Improved Stability by Forced Protein Evolution*

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)8 barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His249–Glu250, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme