Jul 2026· Journal of Agricultural and Food Chemistry· Vol 74, pp. 23000-23015· 0 citations· 81 references
Medicine
TL;DR
This work offers the first molecular insight into P. eryngii xylanases, highlighting PeXyn1 for agro-waste valorization and multiivalent interactions at the CBM1-catalytic interface restrict structural fluctuations, promoting a stable conformation.
Abstract
The widely cultivated mushroom Pleurotus eryngii utilizes lignocellulosic biomass as a growth substrate, yet its xylanases remain poorly understood. Here, we identified PeXyn1, a novel cellulose-upregulated GH11 xylanase featuring a C-terminal carbohydrate-binding module (CBM1) domain, using integrated multiomics. Recombinant PeXyn1 exhibited an optimal pH of 4.5 and temperature of 50 °C, with remarkable stability across pH 4.5-7.5. Kinetic characterization yielded a Km of 27.51 mg mL-1 and a kcat of 20.95 s-1. Purified PeXyn1 efficiently saccharified corncob, releasing 4.258 mg mL-1 reducing sugars after 2 h. Notably, CBM1 truncation increased specific activity from 10.04 ± 0.29 to 28.79 ± 2.23 U mg-1, confirming the core's hydrolytic function while CBM1 mediates substrate anchoring. Simulations revealed an induced-fit mechanism with a high xylohexaose binding affinity (-11.7 kJ mol-1). Multivalent interactions at the CBM1-catalytic interface restrict structural fluctuations, promoting a stable conformation. This work offers the first molecular insight into P. eryngii xylanases, highlighting PeXyn1 for agro-waste valorization.
Xylanases play important roles in lignocellulosic biomass degradation and have broad industrial applications. The discovery of new xylanases expands our understanding of these enzymes and provides new opportunities for industrial applications. This study performed biochemical and structural characterization of the xylanase Xyn11D from Hypocrea virens (HviXyn11D) and evaluated its potential for XOS production from agro-food by-products. HviXyn11D exhibited optimal hydrolytic activity against beechwood xylan at 50 °C and pH 5. Its enzymatic activity was enhanced in the presence of the biomass-derived compounds furfural, 5-hydroxymethylfurfural, and vanillin. The crystal structure of HviXyn11D exhibited a typical β-jelly roll fold and showed a unique substrate-binding cleft compared with homologous xylanases, adopting a closed conformation of the substrate-binding cleft with rigid thumb and finger domains. Molecular dynamics simulations demonstrated that higher temperatures increased the flexibility of the thumb and finger domains, leading to an open conformation of the substrate-binding cleft. Recombinant HviXyn11D enabled the hydrolysis of agro-biomass by-products derived from rice straw, corn cob, and sugarcane bagasse, resulting in successful XOS production. Together, these findings highlight the potential industrial applications of HviXyn11D in biomass degradation and provide new insights into the molecular and structural functions of HviXyn11D and the GH11 xylanase family.
Yena Kim, Bomin Jung, Kyeong-Min Kim et al.· International Journal of Bio...· 0 citations
Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
Kaijuan Wu, Ke Guo, Zheng Yu et al.· Applied Biochemistry and Bio...· 0 citations
BACKGROUND
The discovery of novel biocatalysts for the sustainable valorization of complex biomass feedstocks remains a significant challenge. Domain-centric exploration of characterized CAZyme families offers a promising but underexplored strategy for identifying enzymes with unusual architectures and potentially expanded substrate specificities.
RESULTS
Systematic analysis of archaeal glycoside hydrolase family 18 (GH18) chitinases using the CANDy domain annotation pipeline led to the identification of TcChi from Thermococcus chitonophagus, a multidomain enzyme combining a GH12 and a GH18 catalytic domain alongside two carbohydrate-binding modules. Given that T. chitonophagus also encodes dedicated standalone cellulases and chitinases, we hypothesized that this multidomain assembly may have evolved a broader functional range than either composing domain alone. Biochemical assays of truncated constructs confirmed this hypothesis: the GH18 domain hydrolyzed chitin, chitosan, and β-1,3-glucan, marking the first report of β-1,3-glucanase activity (EC 3.2.1.58) in a GH18 chitinase, while the GH12 domain exhibited strong cellulase activity alongside unexpected chitosanase activity (EC 3.2.1.132), extending the known functional range of this family. Both domains demonstrated high thermostability consistent with the hyperthermophilic origin of T. chitonophagus.
CONCLUSIONS
TcChi is a thermostable, multifunctional biocatalyst capable of degrading chitin, chitosan, cellulose, and β-1,3-glucan from a single protein scaffold, making it a promising candidate for consolidated biomass deconstruction and waste valorization. These findings also demonstrate that domain-centric analysis of CAZyme families is an effective strategy for uncovering hidden functional diversity in well-characterized enzyme families.
Alex Windels, S. Dhaene, Tom Desmet· Biotechnology for Biofuels a...· 0 citations
MDBE, a novel multifunctional isoamylase derived from Myxococcus sp. V11, was heterologously expressed in the Escherichia coli BL21 (DE3). Phylogenetic analysis indicated that MDBE is a multifunctional starch debranching enzyme belonging to the glycoside hydrolase GH13_11 family. Functional identification revealed that MDBE exhibits bifunctional hydrolytic activity, enabling it to hydrolyze both α-1,4 and α-1,6 glycosidic bonds, thereby allowing the degradation of both amylose and amylopectin. Moreover, MDBE can transfer α-1,4-glucan oligosaccharides between molecules through a newly discovered 4-α-D-glucanotransferase activity, resulting in the production of serial oligosaccharides. Under conditions devoid of any added ions, the specific enzyme activity of MDBE was determined to be 250.5 μmol·min⁻1·mg⁻1 and 209. μmol·min⁻1·mg⁻1 when using corn starch and amylose as substrates, respectively. Application of the Michaelis-Menten equation allowed the calculation of the Vmax, Km, and kcat of MDBE for amylose, yielding values of 3171.8 μmol·min⁻1·mg⁻1, 17.7 mM, and 18.9 min⁻1, respectively. The hydrolysis rates of MDBE towards corn starch, amylose, and amylopectin were 50%, 48%, and 44%, respectively. In combined enzyme hydrolysis of amylopectin, the yields of G1 and G2 increased by 1.1-fold and 1.8-fold, respectively, compared to the application of maltogenic amylase (BMAL) alone. Our results suggest that MDBE has potential for future applications in starch hydrolysis and bioconversion, pending further optimization of its catalytic efficiency and stability under industrial conditions.
Siting Feng, Peipei Peng, Jinsong Ouyang et al.· Applied Microbiology and Bio...· 0 citations
β-Mannanases are involved in the hydrolysis of mannan, a major component of hemicellulose. The genome of Glutamicibacter halophytocola sp., an endophytic bacterium isolated from Crithmum maritimum, a halophilic plant from the Island of Crete, encodes a putatively secreted β-mannanase (Gh_GH26) belonging to glycoside hydrolase 26 family (GH26). This sequence occupies a poorly explored region of the GH26 sequence space. Gh_GH26 consists of three domains, including a C-terminal carbohydrate-binding module 23 (CBM23), which is uncommon among GH26s and has a peculiar amino acidic composition at the -3/-4 substrate subsites. Gh_GH26 displays halophilic behavior, retaining activity up to 2.5 M NaCl, and exhibits endo-mannanase activity. Functional analyses combined with structural predictions suggest that Gh_GH26 coordinates Ca2+ ions through a single binding site located within the CBM23 domain. The residues involved in Ca2+ binding are conserved in the CBMs of other characterized GH26s, despite their low similarity with Gh_GH26. Site-directed mutagenesis shows that Ca2+ ions contribute to both enzyme activity and stability. These results suggest that Ca2+ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain. Overall, this work expands our understanding of the functional and structural diversity of the GH26 family, highlighting its potential for application in the degradation of mannan-containing biomass under saline conditions.
M. Orlando, Mattia Salvadori, P. Sarris et al.· International Journal of Bio...· 0 citations