A homodimeric bacterial exo-ß-1,3-glucanase derived from moose rumen microbiome shows a structural framework similar to yeast exo-ß-1,3-glucanases.

The impact of various ß-glucans on the gut microbiome and immune system of vertebrates is becoming increasingly recognized. Besides the fundamental interest in understanding how ß-glucans support human and animal health, enzymes that metabolize ß-glucans are of interest for hemicellulose bioprocessing. Our earlier metagenomic analysis of the moose rumen microbiome identified a gene coding for a bacterial enzyme with a possible role in ß-glucan metabolization. Here, we report that the enzyme, mrbExg5, has exo-ß-1,3-glucanase activity on ß-1,3-linked glucooligosaccharides and laminarin, but not on ß-1,6- or ß-1,4-glycosidic bonds. Longer oligosaccharides are good substrates, while shorter substrates are readily transglycosylated into longer products. The enzyme belongs to glycoside hydrolase subfamily GH5_44, which is a close phylogenetic neighbor of the subfamily GH5_9 exo-ß-1,3-glucanases of the yeasts Saccharomyces cerevisiae and Candida albicans. The crystal structure shows that unlike the eukaryotic relatives, mrbExg5 is a functional homodimer with a binding region characterized by: (i) subsite +1 can accommodate a branched sugar on the ß-1,3-glucan backbone; (ii) subsite +2 is restricted to exclude backbone substituents; and (iii) a fourth subsite (+3) formed by a unique loop. mrbExg5 is the first GH5_44 enzyme to be structurally characterized, and the first bacterial GH5 with exo-ß-1,3-glucanase activity.

Comparative study of ß-glucan-degrading enzymes from Coprinopsis cinerea for their capacities to induce stipe cell wall extension.

We previously reported endo-ß-1,3-glucanase ENG in combination with ß-glucosidase BGL2 at low concentration induced stipe cell wall extension. This study further explored ENG could be replaced by endo-ß-1,3(4)-glucanase ENG16A in combination with BGL2 to induce stipe cell wall extension; similarly, BGL2 could be replaced by ß-glucosidase BGL1 to cooperate with ENG to induce stipe cell wall extension. However, ENG could not be replaced by exo-ß-1,3-glucanase EXG in combination with BGL2 to induce stipe cell wall extension, although EXG alone released higher level of soluble sugars from the stipe cell walls during the reconstituted wall extension than that released from the stipe cell walls by a combination of ENG16A or ENG and BGL2 or BGL1, which was different from chitinase-mediated stipe cell wall extension. These results indicate endo-ß-1,3-glucanases loosen the stipe cell wall, whereas exo-ß-1,3-glucanases and ß-glucosidases play a synergistic role to maintain a low and efficient concentration of endo-ß-1,3-glucanases for stipe cell wall extension. Furthermore, ENG was expressed at a very high level in the matured pilei, in contrast, ENG16A was expressed at a very high level in the elongating apical stipe. Therefore, ENG16A might be involved in stipe elongation growth, while ENG might participate in autolysis of pilei.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Structure Prediction of a Novel Exo-ß-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12.

We report a detailed structural analysis of the psychrophilic exo-ß-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-ß-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-ß-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-ß-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.

Stratum corneum lipid liposome-encapsulated panomycocin: preparation, characterization, and the determination of antimycotic efficacy against Candida spp. isolated from patients with vulvovaginitis in an in vitro human vaginal epithelium tissue model.

In this study, a liposomal lyophilized powder formulation of panomycocin was developed for therapeutic purposes against vulvovaginal candidiasis which affects 80% of women worldwide. Panomycocin is a potent antimycotic protein secreted by the yeast Wickerhamomyces anomalus NCYC 434. This study involved the preparation of panomycocin-loaded stratum corneum lipid liposomes (SCLLs), characterization of the SCLLs, and determination of antimycotic efficacy of the formulation against Candida albicans and Candida glabrata clinical vaginal isolates in a human vaginal epithelium tissue model. The encapsulation and loading efficiencies of SCLLs were 73% and 76.8%, respectively. In transmission electron microscopy images, the SCLLs appeared in the submicron size range. Dynamic light scattering analyses showed that the SCLLs had uniform size distribution. Zeta potential measurements revealed stable and positively charged SCLLs. In Fourier transform infrared spectroscopy analyses, no irreversible interactions between the encapsulated panomycocin and the SCLLs were detected. The SCLLs retained >98% of encapsulated panomycocin in aqueous solution up to 12 hours. The formulation was fungicidal at the same minimum fungicidal concentration values for non-formulated pure panomycocin when tested on an in vitro model of vaginal candidiasis. This is the first study in which SCLLs and a protein as an active ingredient have been utilized together in a formulation. The results obtained in this study led us to conduct further preclinical trials of this formulation for the development of an effective topical anti-candidal drug with improved safety.

Expression and Characterization of a Novel Antifungal Exo-ß-1,3-glucanase from Chaetomium cupreum.

A novel ß-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 ß-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein's molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 °C. Heavy metal ions Fe2+, Mn2+, Cu2+, Co2+, Ag+, and Hg2+ had inhibitory effects on Ccglu17A, but Ba2+ promoted the enzyme's activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing ß-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 µmol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-ß-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.

ß-1,3-glucan modifying enzymes in Aspergillus fumigatus.

In Aspergillus fumigatus like in other filamentous ascomycetes, ß-1,3-glucan constitutes a prominent cell wall component being responsible for rigidity of the cell wall structure. In filamentous fungi, softening of the cell wall is absolutely required during conidial germination and hyphal branching. Because of the central structure of ß-1,3-glucans, it is expected that ß-1,3-glucanases play a major role in cell wall softening. Based on in silico and experimental data, this review gives an overview of ß-1,3-glucan modifying enzymes in A. fumigatus genome and their putative role during morphogenesis.