GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

Abstract

Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (XnU4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing XnU4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system. IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

Crystal structure of GH30-7 endoxylanase C from the filamentous fungusTalaromyces cellulolyticus

“…TcXyn30C (GenBank/DDBJ/EMBL accession No. GAM40414) and TcXyn30C-ÁCBM1, in which the CBM1 domain and linker region (residues Gly456-Ala524) at the C-terminus were deleted, were purified from the culture supernatant of the T. cellulolyticus Y251 and Y283 strains, respectively, using anion-exchange chromatography, hydrophobic interaction chromatography and cation-exchange chromatography as described previously (Nakamichi, Fujii et al, 2019).…”

“…The plasmid pANC287 expressing TcXyn30C F47R, in which Phe47 was substituted with arginine, was constructed by site-directed mutagenesis of the plasmid pANC251 expressing TcXyn30C (Nakamichi, Fujii et al, 2019). Escherichia coli DH5 (Takara Bio, Shiga, Japan) was used for DNA procedures.…”

“…In contrast, the GH30-7 xylanases, which are primarily found in fungi and certain actinobacteria, comprise several types of xylanases, such as reducing-end xylose-releasing exoxylanases (ReXs), endoxylanases, glucuronoxylanases and xylobiohydrolases (Nakamichi, Fujii et al, 2019). For instance, XYN IV from Trichoderma reesei and Xyn30A from Talaromyces cellulolyticus (TcXyn30A) both have ReX activity, which releases xylose from the reducing ends of glucuronoxylan, arabinoxylan and XOSs (Biely et al, 2014;.…”

“…Xyn30C from T. cellulolyticus (TcXyn30C) is an endoxylanase that degrades both glucuronoxylan and arabinoxylan with a similar catalytic efficiency. The enzyme has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal carbohydrate-binding module family 1 (CBM1) connected by a serine/threonine-rich linker region (Nakamichi, Fujii et al, 2019). The hydrolysis of glucuronoxylan by TcXyn30C results in the specific accumulation of 2 2 -MeGlcAxylobiose (U 4m2 X) through a two-step reaction ( Fig.…”

“…1, iii). In contrast, the hydrolysis of arabinoxylan by Xyn30C produces various arabino-XOSs (Nakamichi, Fujii et al, 2019). In this study, the crystal structure of TcXyn30C in which the CBM1 domain and linker region had been deleted (TcXyn30C-ÁCBM1) was determined at 1.65 Å resolution to elucidate the structural factors that are responsible for the endoxylanase activity towards glucuronoxylan and arabinoxylan.…”