Search Results (31)

The GH43 family of glycosidases represents an important class of industrial enzymes that are widely utilized across the food, pharmaceutical, and various other sectors. In this study, we identified a GH43 family glycoside hydrolytic enzyme, Xyaf313, derived from the plant endophytic fungus Chaetomium globosum DX-THS3, which is capable of transforming several common ginsenosides. The enzyme function analysis reveals that Xyaf313 exhibits dual functionality, displaying both α-L-arabinofuranosidase and β-D-xylosidase activity. When acting as an α-L-arabinofuranosidase, Xyaf313 achieves optimal enzyme activity of 23.96 U/mg at a temperature of 50 °C and a pH of 7. In contrast, its β-D-xylosidase activity results in a slight reduction in enzyme activity to 23.24 U/mg, with similar optimal temperature and pH conditions to those observed for the α-L-arabinofuranosidase activity. Furthermore, Xyaf313 demonstrates considerable resistance to most metal ions and common chemical reagents. Notably, while the maximum enzyme activity of Xyaf313 occurs at 50 °C, it maintains high activity at room temperature (30 °C), with relative enzyme activity exceeding 90%. Measurements of ginsenoside transformation show that Xyaf313 can convert common ginsenosides Rc, Rb₁, Rb₂, and Rb₃ into Rd, underscoring its potential for pharmaceutical applications. Overall, our findings contribute to the identification of a new class of bifunctional GH43 glycoside hydrolases, highlight the significance of plant endophytic fungi as a promising resource for the screening of carbohydrate-decomposing enzymes, and present new candidate enzymes for the biotransformation of ginsenosides. Full article

Valorization of agri-food residues has garnered significant interest for obtaining value-added compounds such as enzymes or bioactive molecules. Rice milling by-products, such as rice bran, have limited commercial value and may pose environmental challenges. Filamentous fungi are recognized for their ability to grow on residues and for their capacity to produce large amounts of metabolites and enzymes of industrial interest. Here, we used filamentous fungi to produce enzyme cocktails from rice bran, which, due to its polysaccharide composition, serves as an ideal substrate for the growth of fungi producing cellulases and xylanases. To this end, sixteen fungal strains were isolated from rice bran and identified at the species level. The species belonged to the genera Aspergillus, Penicillium, and Mucor. The Aspergillus species displayed the highest efficiency in cellulase and xylanase activities, especially A. niger var. phoenicis and A. amstelodami. A. terreus, A. tritici, and A. montevidensis stood out as xylanolytic isolates, while P. parvofructum exhibited good cellulase activity. A. niger var. phoenicis followed by A. terreus showed the highest specific enzymatic activities of α- and β-D-galactosidase, α-L-arabinofuranosidase, α- and β-D-glucosidase, and β-D-xylosidase. Additionally, proteomic analysis of A. terreus, A. niger var. phoenicis, and P. parvofructum exoproteomes revealed differences in enzyme production for rice bran degradation. A. niger var. phoenicis had the highest levels of xylanases and cellulases, while P. parvofructum excelled in proteases, starch-degrading enzymes, and antifungal proteins. Finally, two Penicillium isolates were notable as producers of up to three different antifungal proteins. Our results demonstrate that filamentous fungi can effectively valorize rice bran by producing enzyme cocktails of industrial interest, along with bioactive peptides, in a cost-efficient manner, aligning with the circular bio-economy framework. Full article

Hemp fibers, recognized for their breathability, specific strength, and ultraviolet resistance, are widely utilized in textile manufacturing and composite materials. Bio-degumming is a promising alternative technology to traditional chemical degumming that can be used to produce hemp fibers due to its eco-friendly nature. However, its lower efficiency has hindered its widespread adoption. The unclear and complex structure of the gums leads to a poor understanding on the enzyme types required for bio-degumming, thereby restricting improvements in its efficiency. In this study, the morphological characteristics, polysaccharide composition, and branched structure of hemp stem, roving fibers, and refined fibers were investigated using scanning electron microscopy and laser scanning confocal microscopy in combination with immunofluorescence techniques, with a view to identify the enzymes necessary for the efficient bio-degumming of hemp. The results revealed that the gums were primarily located in the middle lamella, phloem parenchyma, and certain xylem tissues. These tissues showed chunk-like, fence-like, and plate-like shapes, respectively, and tightly wrapped around the fiber bundles. In these tissues, pectin comprised low-esterified homogalacturonan, along with rhamnogalacturonan carrying galactan and arabinan branches. Xylan exhibited acetyl, arabinose, and glucuronic acid branches, while mannan displayed acetyl and galactose branches. Partial xylan and mannan were masked by pectin, and the branching structures impeded their enzymatic removal. As a consequence, the necessary enzymes and their synergistic effects for effective hemp roving degumming were elucidated. Pectin degradation was facilitated by pectate lyase and rhamnogalacturonan-degrading enzymes. Xylan and mannan were effectively removed by endo-xylanase and endo-mannanase, a process necessitating the synergistic action of branched-chain-degrading enzymes, including the esterase, α-L-arabinofuranosidase, α-galactosidase, and α-glucuronidase. This study provided practical strategies to enhance the efficiency of hemp bio-degumming. Full article

The increasing number of uncharacterized proteins in the CAZy database highlights the importance of their functional characterization. Therefore, the substrate and positional specificity of 34 α-L-arabinofuranosidases classified into GH43, GH51, and GH62 families was determined on arabinoxylan, arabinan, and derived oligosaccharides (many enzyme–substrate combinations were examined for the first time) covering all possible kinds of arabinofuranosyl branches using TLC. Arabinoxylan was efficiently debranched by the majority of the tested proteins. Most of them showed AXH-m specificity, acting on 2- or 3-monoarabinosylated substrates, while AXH-d3 specificity (liberation of 3-linked arabinose solely from 2,3 doubly decorated substrates) was found mainly in the subfamily GH43_10, harbouring enzymes of both types. Several GH51 enzymes, however, released arabinose also from a xylooligosaccharide doubly arabinosylated at the non-reducing end. The AXH-m and AXH-d3 specificities correlated well with the dearabinosylation of arabinan and arabinooligosaccharides, which were debranched by all GH51 representatives and some GH43 and GH62 members. The GH51 and GH62 arabinan-debranching enzymes also hydrolyzed debranched arabinan, while within the GH43 family the linear arabinan-degrading ability was found only in the GH43_26 subfamily, comprising specific exo 1,5-α-L-arabinofuranosidases. This study demonstrates a first attempt in the systematic examination of a relationship between CAZy classification and substrate and positional specificities of various α-L-arabinofuranosidases. Full article

Biochemical changes in the cell wall composition and activity of cell wall-modifying enzymes of five apple cultivars, Royal Gala (Gala), Aurora Golden Gala (Aur), Splendour (Spl), Honeycrisp (HC), and Ambrosia (Amb), collected from the 2016 growing season in the early growth phases, namely 40 and 70 days after full bloom (DAFB), at harvest maturity, and after 20 weeks of storage were investigated in relation to the textural changes at harvest maturity through to 20 weeks of storage. Assessing apple texture with a single-point measurement of firmness using a penetration test to a depth of approximately 8 mm in apples may not be an ideal measurement for assessing apple textural quality. Pectin methyl esterase (PME) activity at early developmental stages may be predictive of textural quality after storage. This work also found that β-D-galactosidase (BGAL) and α-L-arabinofuranosidase (AFAS) activities at early developmental stages may be important factors affecting textural quality after storage. Additionally, the degree of methylesterification (DME) assessed with FTIR on apple flesh material at the early developmental stages was strongly positively correlated (r² ≥ +0.891 to +0.963, p ≤ 0.05) with textural quality after storage, including crispness, indicating that FTIR could serve as a rapid screening tool for textural quality at early developmental stages on minimally processed starting material. Full article

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics. Full article

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. Full article

The purpose of this study was to investigate the effects of replacing a portion of whole-plant corn silage with straw on the rumen microbial community structure and carbohydrate-active enzyme activity. The experiment employed a single-factor randomized trial design, with eight late-lactation Chinese Holstein dairy cows being randomly divided into two groups of four replicates each. The control group (CS group) was fed a diet consisting of alfalfa silage and a mixture of alfalfa and whole-plant corn silage, while the experimental group (RS group) received a diet in which one-third of the corn silage was replaced with straw while keeping the other components unchanged. The experiment lasted for a total of 21 days, with a pre-feeding period of 14 days and a formal period of 7 days. The rumen fluid collected on day 21 was used for the rumen fermentation parameters and metagenomic analysis. The concentrations of acetic acid, propionic acid, butyric acid, and total volatile fatty acids (TVFA) in the rumen of RS group cows were significantly lower than those in the CS group (p < 0.01). The ratio of acetic acid to propionic acid was significantly higher in the RS group compared to the CS group (p < 0.01). Metagenomic sequencing revealed that at the genus level, compared to the CS group, the abundance of unclassified bacteria, Bacteroides, Alistipes, Butyrivibrio, Chlamydia, Fibrobacter, unclassified Ruminococcaceae, and unclassified Bacteroidetes in the rumen of RS group cows increased, while the abundance of Eubacterium decreased ([LDA > 3.6], p < 0.05). Compared to the CS group, the enzymatic activities of α-L-arabinofuranosidase (EC3.2.1.55), β-xylosidase (EC3.2.1.37), β-glucosidase (EC3.2.1.21), β-glucosylceramidase (EC3.2.1.45), xylanase (EC3.2.1.8), and arabinanase (EC3.2.1.99) in the rumen of RS group cows increased (p < 0.05). According to the correlation analysis, Alistipes, Bacteroides, and Butyrivibrio showed a significant negative correlation with propionic acid (p < 0.05) and a significant positive correlation with the acetic acid-to-propionic acid ratio (p < 0.05). They also showed a significant positive correlation with GH2, GH3, GH20, GH29, GH43, GH78, GH92, CE1, GT4, β-glucosidase (EC3.2.1.21), α-L-arabinofuranosidase (EC 3.2.1.55), β-xylosidase (EC 3.2.1.37), β-glucosylceramidase (EC 3.2.1.45), xylanase (EC 3.2.1.8), and arabinanase (EC 3.2.1.99) (p < 0.05). In summary, straw can not only alter the composition and structure of the rumen microbiota in cows but also affect the relative abundance of CAZymes at different levels within the rumen. Cows may, thus, potentially improve the degradation efficiency of straw diets by increasing the abundance of certain rumen microbiota and enzymes. Full article

There is no doubt that ruminants have the capability to digest lignocellulosic compounds and to utilize them as an absorbable form of energy by tapping into enzymes produced by the microbial population in their rumens. Among the rumens of various ruminants, this study focused on Korean goat rumens because of their unique digestibility of lignocellulosic biomasses. Therefore, a novel Gene12 gene was screened and unmasked from the constructed rumen metagenomic library of a Korean black goat and expressed in a Bacillus megaterium system. The recombinant protein was distinguished as a novel α-L-arabinofuranosidase enzyme from glycosyl hydrolase family 43 (GH43) for its capability to hydrolyze the non-reducing end of α-1,5-L-arabinofuranose linkages in α-L-arabinofuranosyl groups. The enzyme can also break apart α-L-arabinofuranosidic linkages and act synergistically with other hemicellulolytic enzymes to release α-1,2- and α-1,3-L-arabinofuranosyl groups from L-arabinose-comprising polysaccharides. In silico, phylogenetic, and computational analyses proclaimed that the Gene12 gene encodes a novel carbohydrate-active enzyme possessing a V-shaped indentation of the GH43 catalytic and functional domain (carbohydrate-binding module 6). The recombinant Gene12 protein has shared 81% sequence homology with other members of the GH43 family. Enzymic synopses (optimal pH, temperatures, and stability studies) of the recombinant Gene12 enzyme and its substrate specificity (synthetic and natural substrates) profiling were considered. The recombinant Gene12 α-L-arabinofuranosidase works best at pH 6.0 and 40 °C, and it is stable at pH 4.0 to 7.0 at temperatures of 20 to 50 °C. Additionally, 5-blended β-sheets were identified through a tertiary (3D) structure analysis along with the high substrate specificity against p-nitrophenyl-D-arabinofuranoside (pNPA). The highest substrate specificity of pNPA for Gene12 α-L-arabinofuranosidase indicated its confirmation as an exo-type arabinofuronidase. The results thus propose using the Gene12 protein as an exo-mannered GH43 α-L-arabinofuranosidase (EC 3.2.1.55) enzyme. Full article

In this study, we investigated the effect of exogenous melatonin (MT) on cell wall metabolism leading to Chinese plum (Prunus salicina Lindl.) fruit softening. Exogenous MT treatment increased the endogenous MT content in plum fruits before fruit ripening. However, in mature plum fruits, exogenous MT treatment decreased the fruit hardness, pulp hardness, fruit elasticity, contents of ion-bound pectin, covalently-bound pectin, hemicellulose, and cellulose, and activities of xyloglucan endotransglycosylase/hydrolase and endo-β-1,4-glucanase, and increased the water-soluble pectin content, and activities of pectin methyl esterase, pectin lyase, polygalacturonase, β-galactopyranosidase, and α-L-arabinofuranosidase. Transcriptome analysis revealed that the differentially expressed genes (DEGs) associated with cell wall metabolism in the exogenous MT-treated plum fruits were mainly enriched in the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways. Analysis of these DEGs revealed that exogenous MT treatment affected the expression of genes regulating the cell wall metabolism. Overall, exogenous MT treatment promotes the fruit softening of Chinese plum. Full article

α-l-arabinofuranosidases are glycosyl hydrolases that catalyze the break between α-l-arabinofuranosyl substituents or between α-l-arabinofuranosides and xylose from xylan or xylooligosaccharide backbones. While they belong to several glycosyl hydrolase (GH) families, there are only 24 characterized GH62 arabinofuranosidases, making them a small and underrepresented group, with many of their features remaining unknown. Aside from their applications in the food industry, arabinofuranosidases can also aid in the processing of complex lignocellulosic materials, where cellulose, hemicelluloses, and lignin are closely linked. These materials can be fully converted into sugar monomers to produce secondary products like second-generation bioethanol. Alternatively, they can be partially hydrolyzed to release xylooligosaccharides, which have prebiotic properties. While endoxylanases and β-xylosidases are also necessary to fully break down the xylose backbone from xylan, these enzymes are limited when it comes to branched polysaccharides. In this article, two new GH62 α-l-arabinofuranosidases from Talaromyces amestolkiae (named ARA1 and ARA-2) have been heterologously expressed and characterized. ARA-1 is more sensitive to changes in pH and temperature, whereas ARA-2 is a robust enzyme with wide pH and temperature tolerance. Both enzymes preferentially act on arabinoxylan over arabinan, although ARA-1 has twice the catalytic efficiency of ARA-2 on this substrate. The production of xylooligosaccharides from arabinoxylan catalyzed by a T. amestolkiae endoxylanase was significantly increased upon pretreatment of the polysaccharide with ARA-1 or ARA-2, with the highest synergism values reported to date. Finally, both enzymes (ARA-1 or ARA-2 and endoxylanase) were successfully applied to enhance saccharification by combining them with a β-xylosidase already characterized from the same fungus. Full article

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid. Full article

Plant α-l-arabinofuranosidases remove terminal arabinose from arabinose-containing substrates such as plant cell wall polysaccharides, including arabinoxylans, arabinogalactans, and arabinans. In plants, de-arabinosylation of cell wall polysaccharides accompanies different physiological processes such as fruit ripening and elongation growth. In this report, we address the diversity of plant α-l-arabinofuranosidases of the glycoside hydrolase (GH) family 51 through their phylogenetic analysis as well as their structural features. The CBM4-like domain at N-terminus was found to exist only in GH51 family proteins and was detected in almost 90% of plant sequences. This domain is similar to bacterial CBM4, but due to substitutions of key amino acid residues, it does not appear to be able to bind carbohydrates. Despite isoenzymes of GH51 being abundant, in particular in cereals, almost half of the GH51 proteins in Poales have a mutation of the acid/base residue in the catalytic site, making them potentially inactive. Open-source data on the transcription and translation of GH51 isoforms in maize were analyzed to discuss possible functions of individual isoenzymes. The results of homology modeling and molecular docking showed that the substrate binding site can accurately accommodate terminal arabinofuranose and that arabinoxylan is a more favorable ligand for all maize GH51 enzymes than arabinan. Full article

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4–7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1. Full article

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues. Full article