Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity

Abstract

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear β-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA2Xyln. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan

“…In detail, numbered from the nonreducing end of modeled X4, the 2nd xylose (Xyl2) forms hydrogen bond interactions with the carbonyl oxygen of I428 and the guanidium group of R357, via its C2 hydroxyl group, while its C3 hydroxyl group also interacts with the nitrogen atom of the pentane ring of W420. Sequence alignment of biochemically characterized eukaryotic GH30_7s (Šuchová et al, 2022) indicates a conservation of a positively charged amino acid (either R or K) at the position corresponding to R357, while all of them feature either a W or a Y at the position corresponding to W420. Xyl3 is accommodated by pi‐pi interactions with W350, an amino acid conserved in all GH30_7s, while its C3 hydroxyl group of Xyl3 interacts with atom ND2 of N58.…”

“…Here, the distorted conformation of xylose at subsite −1 observed in Tt Xyn30AEE‐UXX complex, combined with the fact that part of the catalytic activity is retained when both hypothesized catalytic glutamates were mutated to alanine, provides further evidence for our previous hypothesis that E233 participates in the catalytic mechanism, and potentially in the endo‐glucuronoxylan function. Interestingly, one of the two other bifunctional GH30_7 xylanases, Sl Xyn30A from Sugiyamaella lignohabitans also features a glutamate at the corresponding position (Šuchová et al, 2022). The fact that in the case of neutral XOs we did not observe any distortion of −1 xylose could be an indication that E188 is engaged in “exo” activity whereas E233 is the major player in “endo” activity.…”

“…Even though xylanases have already been applied in the food and feed industries as well as in textile and paper pulp processes, the supplementation of enzymatic systems with thermophilic GH30 xylanases enhances the overall efficiency of the process, especially when glucuronoxylan‐containing raw material is used (Li, Dilokpimol, et al, 2022; Puchart et al, 2021). Tt Xyn30A is one of the three characterized GH30_7 members that catalyze glucuronoxylan hydrolysis in a dual way: endo‐activity, releasing aldouronic xylooligosaccharides (UXOs) substituted at the penultimate xylose from the reducing end, and exo‐activity, releasing xylobiose from the nonreducing end (Figure 1a) (Katsimpouras et al, 2019; Nakamichi, Fouquet, Ito, Watanabe, et al, 2019; Šuchová et al, 2022). Thus, compared to other biochemically characterized GH30_7 enzymes, Tt Xyn30A is functionally positioned somewhere between strict endo‐xylanases and strict nonreducing end xylobiohydrolases (Šuchová et al, 2020).…”

“…TtXyn30A is one of the three characterized GH30_7 members that catalyze glucuronoxylan hydrolysis in a dual way: endo-activity, releasing aldouronic xylooligosaccharides (UXOs) substituted at the penultimate xylose from the reducing end, and exo-activity, releasing xylobiose from the nonreducing end (Figure 1a) (Katsimpouras et al, 2019;Nakamichi, Fouquet, Ito, Watanabe, et al, 2019;Šuchová et al, 2022). Thus, compared to other biochemically characterized GH30_7 enzymes, TtXyn30A is functionally positioned somewhere between strict endo-xylanases and strict nonreducing end xylobiohydrolases (Šuchová et al, 2020).…”

Valorization of lignocellulosic wastes for sustainable xylanase production from locally isolated Bacillus subtilis exploited for xylooligosaccharides’ production with potential antimicrobial activity

“…Among others, microbial xylanases fulfill the biotechnological requirements for large-scale production in terms of scalability and production titer (Basit et al 2020 ). Different microbial sources were reported for xylanase production including yeasts (Miao et al 2021 ; Šuchová et al 2022 ), fungi (Cekmecelioglu and Demirci 2020 ; Patel and Dudhagara 2020a ), and actinomycetes (Danso et al 2022 ). Currently, eubacteria are largely applied at commercial-scale xylanases’ production (Chakdar et al 2016 ; Ghosh et al 2021 ).…”