Fungal glycoside hydrolase family 44 xyloglucanases are restricted to the phylum Basidiomycota and show a distinct xyloglucan cleavage pattern

Abstract

Summary Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cell walls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several glycoside hydrolase (GH) families. So far, family GH44 has been shown to contain bacterial XEGs only. Detailed genome analysis revealed GH44 members in fungal species from the phylum Basidiomycota, but not in other fungi, which we hypothesized to also be XEGs. Two GH44 enzymes from Dichomitus squalens and Pleurotus ostreatus were heterologously produced and characterized. They exhibited XEG activity and displayed a hydrolytic cleavage pattern different from that observed in fungal XEGs from other GH families. Specifically, the fungal GH44 XEGs were not hindered by substitution of neighboring glucosyl units and generated various “XXXG-type,” “GXXX(G)-type,” and “XXX-type” oligosaccharides. Overall, these fungal GH44 XEGs represent a novel class of enzymes for plant biomass conversion and valorization.

Unraveling the diversity within CAZy families related to hemicellulose degradation

“…Using methyl ferulate as a substrate for FAE activity, Po. anserina FaeD also showed optimum pH at 7.0 and optimum temperature at 50°C, these properties are within the range of other reported fungal FAEs, mainly with activity at pH 4-8 and temperature 30-65°C [37,38,47,48] (Table 1). It also showed a high pH stability over a broad pH range from pH 3.0 to 10.0, which is more superior than most reported CE1 enzymes (Table 1).…”

“…PsExlA showed a similar hydrolytic product pattern to T. reesei XYN VI [38], both of which resembled the bacterial endoxylanases from GH30_8 [28,[46][47][48]. TtExlA predominantly released xylobiose, but also different (MeGlcA substituted) XOS (Figure 5A), similar to the earlier report [4], which shows that it is an endoxylanase with xylobiohydrolase activity.…”

“…The functional characterization showed pH optima for PsXLNs ranged from acidic to neutral (pH 4-7), with most PsXLNs having temperature optima of 40°C (Table 1). They are similar to the properties of characterized enzymes clustered with PsXLNs in the phylogenetic tree [45][46][47][48][49][50] (Figure 1A). Surprisingly, four PsXLNs (PsXlnB and PsXlnC of GH10, and PsXlnI and PsXlnF of GH11) displayed the highest activity at 70°C (Table 1).…”

“…GH10 XLNs generally have a broader substrate specificity than GH11 XLNs, as GH10 XLNs tolerate the presence of decorations on the xylan backbone more than GH11 XLNs do [47]. As a result, GH10 XLNs can cleave the glycosidic linkage next to a D-xylosyl residue decorated with one or two residues (e.g., at the non-reducing side), whereas GH11 XLNs only cleave the xylan backbone that has at least three consecutive undecorated D-xylosyl residues, and cannot cleave at the glycosidic linkage next to a branch [38,48]. BXL is characterized generally using an artificial substrate, i.e., p-nitrophenyl-β-D-xylopyranoside (pNPXyl).…”

“…For example, for one bacterial GH44 XEG (PpXG44), it has been shown that the compound XXXGXXXG could be hydrolyzed into XXX and GXXXG [23]. Similarly, for the bacterial Cel44O, identified as an endocellulase, xyloglucan degrading activity and cleavage between XXX and GXXX have been suggested [48].…”