Glycoside Hydrolase Activities Research Articles

Molecular characterization revealed the role of thaumatin-like proteins of Rhizoctonia solani AG4-JY in inducing maize disease resistance

The gene family of thaumatin-like proteins (TLPs) plays a crucial role in the adaptation of organisms to environmental stresses. In recent years, fungal secreted proteins (SP) with inducing disease resistance activity in plants have emerged as important elicitors in the control of fungal diseases. Identifying SPs with inducing disease resistance activity and studying their mechanisms are crucial for controlling sheath blight. In the present study, 10 proteins containing the thaumatin-like domain were identified in strain AG4-JY of Rhizoctonia solani and eight of the 10 proteins had signal peptides. Analysis of the TLP genes of the 10 different anastomosis groups (AGs) showed that the evolutionary relationship of the TLP gene was consistent with that between different AGs of R. solani. Furthermore, it was found that RsTLP3, RsTLP9 and RsTLP10 were regarded as secreted proteins for their signaling peptides exhibited secretory activity. Prokaryotic expression and enzyme activity analysis revealed that the three secreted proteins possess glycoside hydrolase activity, suggesting they belong to the TLP family. Additionally, spraying the crude enzyme solution of the three TLP proteins could enhance maize resistance to sheath blight. Further analysis showed that genes associated with the salicylic acid and ethylene pathways were up-regulated following RsTLP3 application. The results indicated that RsTLP3 had a good application prospect in biological control.

Rice false smut virulence protein subverts host chitin perception and signaling at lemma and palea for floral infection.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the 2 bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens-rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host-pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

Simulated digestions of free oligosaccharides and mucin-type O-glycans reveal a potential role for Clostridium perfringens.

The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

Induction and Characterisation of Lignocellulolytic Activities from Novel Deep-Sea Fungal Secretomes

Fungi are increasingly recognised as being able to inhabit extreme environments. The deep sea is considered an extreme environment because of its low temperatures, high hydrostatic and lithostatic pressures, 3.5% salinity, and low oxygen, nutrient and light availability. Fungi inhabiting the deep sea may have evolved to produce proteins that allow them to survive these conditions. Investigation and characterisation of fungal lignocellulolytic enzymes from extreme environments like the deep sea is needed, as they may have unusual adaptations that would be useful in industry. This work, therefore, aimed to profile in detail the lignocellulolytic capabilities of fungi isolated from deep-sea sediments in the Atlantic Ocean, and a comparative lignocellulolytic terrestrial isolate. The isolates were strains of Emericellopsis maritima, Penicillium chrysogenum, P. antarcticum and Talaromyces stollii. Lignocellulolytic enzyme induction was achieved using liquid-state fermentation (LSF) with wheat bran as the main carbon source, while enzyme characteristics were evaluated using biochemical assays and gel-based proteomics. This study revealed that the isolates were halotolerant, produced xylanase over wide pH and temperature ranges, and produced a variety of glycoside hydrolase and feruloyl esterase activities. The T. stollii secretome demonstrated remarkable levels of exo-glycoside hydrolase activity, with xylanase activity optimum between pH 1.5–6.0 and temperatures between 1–60 °C, making this isolate an ideal candidate for biotechnological applications. This study is the first to quantitatively characterise xylanase activities and exo-glycoside hydrolase activities secreted by E. maritima, P. antarcticum and a marine T. stollii strain. This study is also the first to quantitatively characterise xylanase activities by a marine strain of P. chrysogenum during LSF.

Genome mining reveals chitin degradation potential of \(\textit{Streptomyces parvulus}\) VCCM 22513

The genus Streptomyces is not only known as a natural producer of antibiotics but also a prolific source of chitinolytic enzymes that digest recalcitrant chitin to chitooligosaccharides. However, only a few reports have used whole-genome sequencing to study chitin degradation of Streptomyces to date. In the present study, out of 22 Streptomyces strains, Streptomyces parvulus VCCM 22513 produced the highest chitinase activity. Time courses of incubation revealed that the maximum chitinase (0.91 ± 0.04 U/mL) of this strain was observed after 96 hours in the yeast extract salts medium supplemented with 10.0 g/L colloidal chitin. Additional genomic analysis of VCCM 22513 was also conducted to discover the genomic information related to chitin degradation. The VCCM 22513 genome consists of 341 CAZy genes divided into 6 families including glycoside hydrolase (134 genes), carbohydrate-binding module (88 genes), glycosyl transferase (87 genes), carbohydrate esterase (18 genes), polysaccharide lyase (7 genes), and auxiliary activity (7 genes). Further genome mining revealed the presence of 10 chitinases, 4 lytic polysaccharide monooxygenases, and 14 β-N-acetylhexosaminidases, which mainly contribute to the degradation of chitin polymers. This is the first report revealing the mechanism underlying the chitin degradation of S. parvulus. Further investigations are required to characterize chitinolytic enzymes found in this study for the bioeconomic production of high-quality chitooligosaccharides from chitin food wastes.

Population genomic analysis reveals geographic structure and climatic diversification for Macrophomina phaseolina isolated from soybean and dry bean across the United States, Puerto Rico, and Colombia.

Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate.

Distinct roles of carbohydrate-binding modules in multidomain β-1,3-1,4-glucanase on polysaccharide degradation.

Lam16A is a novel GH16 β-1,3-1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley β-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against β-1,3(4)-glucan-based polysaccharides. The released products from β-1,3-1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by ~ 85% ± 1.2, enabling the release of up to ~ 0.057 ± 0.0039µg/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by ~ 70.3% ± 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of β-1,3-1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of β-1,3-glucan oligosaccharides for health benefits. KEY POINTS: • Carbohydrate-binding modules strongly influenced the enzyme activity and binding affinity, and further impacted glycoside hydrolase activity. • Lam16A enzymes have sufficient ability to hydrolyze β-1,3-1,4-glucan-based polysaccharides. • Lam16As provide a powerful tool to promote the bioavailability of β-1,3-glucan oligosaccharides.

Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Glycoside hydrolase processing of the Pel polysaccharide alters biofilm biomechanics and Pseudomonas aeruginosa virulence

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.

Compost-derived thermophilic microorganisms producing glycoside hydrolase activities as new potential biocatalysts for sustainable processes

BackgroundThe management of the organic waste recycling process determines the interest in the thermophiles microorganisms involved in composting. Although many microbial enzymes have been isolated and studied for their industrial and commercial uses, there is still a continuous search for microorganisms which could synthesize industrially feasible enzymes, especially when the microbial diversity of cow dung itself makes a potential source of biotechnological enzymes.ResultsThe composting process studied at the Experimental Station of the University of Naples Federico II (Castel Volturno, Caserta, Italy) was characterized by fresh saw dust 40%, bovine manure 58%, and 2% mature compost as raw organic substrates, and its thermophilic phase exceeded a temperature of 55 °C for at least 5 days, thus achieving sanitation. Six microbial strains were isolated and designated as follow: CV1-1, CV1-2, CV2-1, CV2-2, CV2-3 and CV2-4. Based on 16S rRNA gene sequence, HRMAS–NMR spectroscopy, and biochemical investigations, they were ascribed to the genera Geobacillus and Bacillus. All the microbial isolates were qualitatively screened on plates for the presence of hydrolytic activities, and they were quantitatively screened in liquid for glycoside hydrolase enzymes in the extracellular, cell-bound, and cytosolic fractions. Based on these results, strains CV2-1 and CV2-3 were also quantitatively screened for the presence of cellulase and pectinase activities, and pH and temperature optimum plus thermostability of cellulase from CV2-1 were analyzed.ConclusionsThe isolation and the identification of these thermophilic microorganisms such as Geobacillus toebii, Geobacillus galactosidasius, Bacillus composti, Bacillus thermophilus and Aeribacillus composti have allowed the study of the biodiversity of compost, with emphasis on their primary metabolome through an innovative and underutilized technique, that is HRMAS–NMR, also highlighting it as a novel approach to bacterial cell analysis. Subsequently, this study has permitted the identification of enzymatic activities able to degrade cellulose and other polymeric substrates, such as the one investigated from strain CV2-1, which could be interesting from an industrial and a biotechnological point of view, furthermore, increasing the knowledge for potential applicability in different industrial fields as an efficient and environmentally friendly technique.Graphical

Increase of intestinal bacterial sialidase activity exacerbates acute colitis in mice.

The availability of endogenous and dietary carbohydrates in the gastrointestinal tract influences the composition of the gut microbiota. Carbohydrate foraging requires the action of bacterially-encoded glycoside hydrolases, which release mono- and oligosaccharides taken up as carbon sources by multiple microbial taxa. In addition to providing nutrients to the microbiota, the cleavage of host glycans by bacterial glycoside hydrolases may alter the properties of surface glycoproteins involved in cell adhesion and activation processes in the gut lumen. To investigate the impact of bacterial glycoside hydrolase activities on the gut microbial composition and on host glycans during colon inflammation, we increased local glycoside hydrolase activity by supplementing mice with recombinant E. coli expressing specific sialidase, fucosidase and rhamnosidase enzymes during acute colitis induced by dextran sulfate sodium ingestion. Whereas increased fucosidase and rhamnosidase activity did not alter the course of colitis, increased sialidase activity exacerbated disease severity. The effect of increased sialidase activity on inflammation was not caused by changes in the microbial composition given that a similar shift in gut bacteria occurred in all groups of mice supplemented with recombinant E. coli. Increased sialidase activity in the colon of treated mice however significantly altered the distribution of sialic acid on mucosal glycans. Treatment of lamina propria dendritic cells with bacterial sialidase also strongly decreased the density of sialylated ligands to anti-inflammatory siglec lectins, indicating that the remodeling of surface sialylation caused by increased sialidase activity likely accounts for the observed exacerbation of acute colitis in mice.

Characterization of erythromycin-degrading strain Aspergillus sydowii W1 revealed by transcriptome analyses

Erythromycin residual, as a persistent, antibiotic pollutant, poses a serious threat to environmental and human health. Thus, developing effective technologies to eliminate erythromycin from the environment is urgent. In this study, a new fungus W1 identified as Aspergillus sydowii which can utilize erythromycin as its only carbon source was isolated from soil contaminated with erythromycin. The degradation efficiency of erythromycin by strain W1 was 84.05% within seven days under optimal conditions (30 °C, pH 6.0 and 100 mg L−1 erythromycin). The degradation pathway of erythromycin was proposed from four intermediates detected by HPLC-MS, and enzymes involved in erythromycin metabolism in strain W1 were preliminarily inferred. Notably, genomic and transcriptomic analysis also confirmed that strain W1 degrades erythromycin via glycoside hydrolase and esterase activity. This study demonstrated that A. sydowii W1 is a novel erythromycin-degrading strain, which has great potential for removing erythromycin from contaminated soils, and also provides a new perspective for other applications of this particular strain.

PSVIII-16 Metagenomic Analyses of Microbial and Carbohydrate-Active Enzymes in the Rumen of Holstein Steers Supplemented with Seeds of Pharbitis nil

Abstract Our previous research revealed the effects of dietary supplementation of Pharbitis nil seeds (PA) in improving the ruminal methane reduction and digestibility in Hanwoo steers. The objective of this study was to further investigate the effects of PA supplementation on rumen microbial taxonomy, Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology functional categories and the diversity of carbohydrate-active enzymes (CAZymes). Five rumen cannulated Holstein steers (744 ± 35 kg) were fed either basal diet (Control) or basal diet supplemented with PA at 5% dry matter intake (PA5) for two consecutive periods of 40 days in pairwise comparison. Rumen fluid was collected on the last day of each period after 4h of morning feeding. Analysis of rumen microbial metagenome revealed that contigs assigned to taxa Methanobrevibacter millerae decreased (p = 0.090) in PA5 group. Functional annotation of contigs using KEGG database revealed that genes involved in starch and sucrose metabolism were enriched (p = 0.058) in PA5 group compared with control. CAZyme analysis identified genes that encode glycoside hydrolase (GH) accounted for 63.18% and 65.66% of all CAZymes in control and PA5, respectively. The GH2 family (enriched in oligosaccharide-degrading enzymes) genes were greater (p = 0.090) in PA5 than control. Likewise, the abundance of GH26 and GH53 (endo-hemicellulases), GH5_4 (cellulases), and GH13_36 and GH13_42 (α-amylase) were increased (p < 0.1) in PA5 relatively compared with control. Most of the genes were mainly produced by unclassified contigs followed by Cupriavidus oxalaticus and Prevotella ruminicola 23. De novo assembly and binning recovered a total of 107 metagenome-assembled genomes taxonomically assigned to different bacterial phyla and one archaeal phyla. Of these genomes, 39 represented the uncultivated bacteria and 1 represented Lactobacillus genera. This study revealed that PA supplemented steers had increased GH activity, which encode enzymes that efficiently degrade plant cell wall polysaccharides in the rumen thereby increasing the fiber digestibility.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-β-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568.

Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg–1) and kcat/Km value (6.35 mg–1 s–1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg–1) and kcat/Km value (2.83 mg–1 s–1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

The Role of Glycoside Hydrolases in S. gordonii and C. albicans Interactions.

Candida albicans can coaggregate with Streptococcus gordonii and cocolonize in the oral cavity. Saliva provides a vital microenvironment for close interactions of oral microorganisms. However, the level of fermentable carbohydrates in saliva is not sufficient to support the growth of multiple species. Glycoside hydrolases (GHs) that hydrolyze glycoproteins are critical for S. gordonii growth in low-fermentable-carbohydrate environments such as saliva. However, whether GHs are involved in the cross-kingdom interactions between C. albicans and S. gordonii under such conditions remains unknown. In this study, C. albicans and S. gordonii were cocultured in heart infusion broth with a low level of fermentable carbohydrate. Planktonic growth, biofilm formation, cell aggregation, and GH activities of monocultures and cocultures were examined. The results revealed that the planktonic growth of cocultured S. gordonii in a low-carbohydrate environment was elevated, while that of cocultured C. albicans was reduced. The biomass of S. gordonii in dual-species biofilms was higher than that of monocultures, while that of cocultured C. albicans was decreased. GH activity was observed in S. gordonii, and elevated activity of GHs was detected in S. gordonii-C. albicans cocultures, with elevated expression of GH-related genes of S. gordonii. By screening a mutant library of C. albicans, we identified a tec1Δ/Δ mutant strain that showed reduced ability to promote the growth and GH activities of S. gordonii compared with the wild-type strain. Altogether, the findings of this study demonstrate the involvement of GHs in the cross-kingdom metabolic interactions between C. albicans and S. gordonii in an environment with low level of fermentable carbohydrates. IMPORTANCE Cross-kingdom interactions between Candida albicans and oral streptococci such as Streptococcus gordonii have been reported. However, their interactions in a low-fermentable-carbohydrate environment like saliva is not clear. The current study revealed glycoside hydrolase-related cross-kingdom communications between S. gordonii and C. albicans under the low-fermentable-carbohydrate condition. We demonstrate that C. albicans can promote the growth and metabolic activities of S. gordonii by elevating the activities of cell-wall-anchored glycoside hydrolases of S. gordonii. C. albicans gene TEC1 is critical for this cross-kingdom metabolic communication.

Identification of Glycoside Transporters From the Human Gut Microbiome.

Transport is a crucial step in the metabolism of glycosides by bacteria, which is itself key for microbiota function and equilibrium. However, most transport proteins are function-unknown or only predicted, limiting our understanding of how bacteria utilize glycosides. Here, we present an activity-based screening method to identify functional glycoside transporters from microbiomes. The method is based on the co-expression in Escherichia coli of genes encoding transporters and carbohydrate-active enzymes (CAZymes) from metagenomic polysaccharide utilization loci (PULs) cloned in fosmids. To establish the proof of concept of the methodology, we used two different metagenomic libraries derived from human gut microbiota to select 18 E. coli clones whose metagenomic sequence contained at least one putative glycoside transporter and one functional CAZyme, identified by screening for various glycoside-hydrolase activities. Growth tests were performed on plant-derived glycosides, which are the target substrates of the CAZymes identified in each PUL. This led to the identification of 10 clones that are able to utilize oligosaccharides as sole carbon sources, thanks to the production of transporters from the PTS, ABC, MFS, and SusCD families. Six of the 10 hit clones contain only one transporter, providing direct experimental evidence that these transporters are functional. In the six cases where two transporters are present in the sequence of a clone, the transporters’ function can be predicted from the flanking CAZymes or from similarity with transporters characterized previously, which facilitates further functional characterization. The results expand the understanding of how glycosides are selectively metabolized by bacteria and offers a new approach to screening for glycoside-transporter specificity toward oligosaccharides with defined structures.

Oxidative cleavage of polysaccharides by a termite-derived superoxide dismutase boosts the degradation of biomass by glycoside hydrolases.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach.

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

Aberrant Autophagy Impacts Growth and Multicellular Development in a Dictyostelium Knockout Model of CLN5 Disease.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5– cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5– cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5– cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5– cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5– development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5– cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5– slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5– cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.