Mechanisms of enzymatic glycoside hydrolysis (2023)

For an efficient use of lignocellulosic materials in ruminants, the investigation of effective enzymes, especially bifunctional enzymes, is of crucial importance. In this study, a new stable bifunctional cellulase-xylanase protein from the bison rumen metagenome, CelXyn2, was expressed and characterized. The enzyme showed optimal activity at pH 6.0 and 45°C. The remaining endoglucanase and xylanase activities were 90.6% and 86.4%, respectively, after a 60-minute pre-incubation at 55°C. Hydrolysis of rice straw, wheat straw, sheep grass and sugar beet pulp by CelXyn2 demonstrated the ability to degrade cellulose and hemicellulose polymers. Treatment with CelXyn2 improved hydrolysis of agricultural residues with a significant increase in total gas, lactate and volatile fatty acid production. The results of 16S rRNA and real-time PCR showed that the effect on the rumen microbial community in vitro depends on the fermentation substrates. This study demonstrated that CelXyn2 can enhance lignocellulose hydrolysis and in vitro rumen fermentation. These properties make CelXyn2 a promising candidate for agricultural applications.

Enzyme activity on the synthetic substrate (str-Nitrofenil-β-D-glucopyranosid (strNPG)) sorted by specificity (k_Cat/K_M), placed Bgl-3 (recombinantP. carotovorumsubsp.carotovorum-ß-glucosidase expressed inEscherichia coli) in the group of ß-glucosidases, while the order according to the number of traffic (k_Cat) placed Bgl-3 at the top of the group, indicating its potential for high activity in biomass processing. Here the role of Lys211, His212, Arg172 and Asp114 in substrate recognition and stabilization of the glucose moiety in catalysis due to interactions with the hydroxyls of the substrate C1–C6 via hydrogen bonding is proposed, as well as the role of two methionines, Met255 and Met322, in the hydrophobic stabilization. However, enzyme inactivation due to enzyme nucleophile ion pair dissociation and acid base in the pH range >8 and <5 may affect the formation of the enzyme intermediate complex, suggesting that the latter is rate-limiting in Bgl3 catalysis. Asp290 and Glu517 in the substrate binding clefts are the proposed nucleophiles and catalytic acids/bases. The proximity of His212 and His522 stabilizes the enzymatic glycosyl intermediate through hydrogen bonding.

Cyclopelitol aziridines have found wide application as mechanism-based covalent and irreversible inhibitors of retention glycosidases. These compounds, as well as their parent cyclophellitol (a natural product that retains the β-glucosidase inactivator), utilize the mechanism of action of retention of glycosidases, which process their substrate by forming a transient covalent intermediate. In contrast, inverting glycosidases, another large family of glycosyl hydrolases, do not utilize such a covalent intermediate, and thus useful scaffolds for mechanism-based inhibitor design have yet to be discovered. In this work, we investigate the chemical processes that enable the assembly of cyclitol aziridine, in which an aziridine electrophile is exocyclically bound, more distal than the anomeric carbon—and thus supposedly closer to the nucleophile of the inverting glycosidase active site. The developed chemistry enabled the synthesis of a directed library of diverse contentsN‐substituted α‐ and β‐glucopyranose‐configured cyclitol aziridines for future evaluation as inhibitors or inactivators of α‐ and β‐glucosidases.

More and more studies are focusing on the degradation of chondroitin sulfate (CS) to improve its biological activity. The paper mainly introduces CS decomposition methods and their mechanisms. Studies have shown that different degradation methods can lead to different structures of low molecular weight chondroitin sulfate (LMCS). LMCS were prepared by β-elimination reaction, hydrolysis reaction, hydrogen abstraction reaction, and deamination reaction. The degradation of CS is influenced by two aspects: the structure of CS (disaccharide composition and molecular weight) and the influence of degradation conditions (temperature, pH, degradation promoters, auxiliary conditions and time). LMCS with different structures have different biological activities. In addition, degradation could also alter CS metabolism, for example through the effects of absorption and gut microbiota. Therefore, choosing an appropriate decomposition method for the development and use of CS is very important.

Enzymes are an environmentally friendly, economical and effective alternative to chemical catalysts for sustainable industrial development. Amylases are starch-digesting enzymes that hydrolyze the glycosidic bonds in starch to glucose, maltose, maltotriose, and dextrin. The use of α-amylase in starch-based industries has been widespread for decades and there are few microbial sources for efficient production of this enzyme. Modern strain improvement strategies such as biotechnological approaches and applications of genetic engineering have improved the efficiency of α-amylase production and its biochemical activity. α-Amylases account for about 30% of the world's enzyme production and find diverse applications in various industries such as the food industry, detergent industry, textile and paper industry. With the emergence of new frontiers in biotechnology, the range of applications of amylase has expanded to many other areas such as clinical, medicinal, and analytical chemistry. The use of low-grade agro-industrial residues as a substrate is currently in focus to improve the economic viability of production and solve the problems of their disposal and pollution. The advent of metagenomic approaches and the application of protein engineering techniques will provide new sources of α-amylases with design features such as better thermostability and chemical tolerance suitable for specific industrial applications. It is expected that amylases will continue to open up new opportunities in biotechnology research and related industries for product and process development.

TmCel5B is a lichenase belonging to the glycoside hydrolase 5 family of subfamily 36 (GH5_36). To gain insight into the active site of this subfamily of multifunctional endoglycanases, we determined the crystal structure of TmCel5B in complex with the iminosugar 1-deoxynojiromycin (DNJ). DNJ is attached to the -1 subsite and forms a network of noncovalent interactions with the acid/base residue Glu139, the nucleophile Glu259, and other residues conserved in the GH5 family. The catalytic site displayed a Glu-Arg-Glu triad of catalytic glutamates found only in the GH5_36 subfamily. A structural comparison of the active sites of GH5_36 homologues revealed divergent residues and loop regions that are likely molecular determinants of homologue-specific properties. Furthermore, a comparative analysis of the binding mode of iminocyclitol complexes of GH5 homologues revealed the structural basis of their binding to GH5 glycosidase, in which the binding site of subsites, ligand interactions with specific conserved residues, and electrostatic interactions of catalytic glutamates with ring nitrogen key.

Archives of Biochemistry and Biophysics, Volume 540, Numbers 1–2, 2013, p. 117-124

β-Xylosidases are involved in xylan biodegradation and release xylose from the non-reducing end of xylooligosaccharides. MushroomPenicillium purpurogenumsecretes two enzymes with β-D-Xylosidase activity belonging to the family of 43 glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-l-arabinofuranozidaza/ksilobiohidrolaza aktivna na p-nitrofenil-α-l-Arabinofuranozid (pNPAra) in p-Nitrophenyl-β-D-Ksilopiranozid (pNPXyl) s aK_Mof 0.65 and 12mM, respectively. The other, β-D-Xylosidase 1 (XYL1), is only active on pNPXyl with aK_Mfrom 0.55 mM. Derxyl1The gene is expressed inshepherd figs, cleaned and characterized. The properties of both enzymes were compared to explain their difference in substrate specificity. Structural models for each protein were created using homology modeling tools. Molecular docking simulations were used to analyze the interactions that determine the protein's affinity for both ligands. The structural analysis shows that the active complexes (ABF3–pNPXyl, ABF3–pNPAra, and XYL1–pNPXyl) possess specific interactions between the substrate and catalytic residues that are absent in the inactive complex (XYL1–pNPAra), while other interactions with noncatalytic residues come in all complexes. . pNPAra is a competitive inhibitor of XYL1 (K_And=2.5 mM), confirming that pNPAra binds to the active site but not to the catalytic residues.

International Journal of Biological Macromolecules, Band 170, 2021, p. 196-206

Marine microalgae are promising sources of new glycoside hydrolases (GH), which are of great value for biotechnical and industrial applications. Although many GH1 family β-glucosidases have been extensively studied, studies on microalgal β-glucosidases are rare and no structure of algal β-glucosidases has been reported. Here we report a biochemical and structural study of the GH1-β-glucosidase derived from BGLN1Nannochloropsis ozeanica, oily microalgae. The phylogenetic analysis of BGLN1, together with the known structures of the GH1 β-glucosidases, showed that BGLN1 is branched at the root of the eukaryotic part of the phylogenetic tree. BGLN1 showed greater activity against laminaribiosis compared to cello-oligosaccharides. Unlike most other GH1 β-glucosidases, BGLN1 is partially inhibited by metal ions. The crystal structure of BGLN1 showed that BGLN1 has a typical (α/β)₈-Barrel fold with variations in loops and N-terminal regions. BGLN1 contains additional residues at the N-terminus that are essential for maintaining protein stability. BGLN1 has a more acidic substrate-binding pocket than other β-glucosidases, and variations outside of the conserved -1 site determine substrate specificity. These results suggest that microalgal GH enzymes may exhibit unique structural and functional features that will provide new insights into carbohydrate synthesis and metabolism in marine microalgae.

Protein Expression and Purification, Vol. 129, 2017, p. 9-17

A-l- Fucosyl residues are commonly found in oligosaccharides, polysaccharides and glycoconjugates, which play fundamental roles in various biological processes. α-l- Fucosidases, glycoside hydrolases to catalyze the removal of α-l-fucose, may serve as desirable tools in the study and modification of biomolecules containing fucose. In this study, α-l-Fucosidase named Alf1_Wf was purified from a marine bacteriumWenyingzhuangia fucanilyticaby a combination of chromatographic methods. The Alf1_Wf sequence was identified by proteome analysis from the predicted bacterial proteome. Recombinant Alf1_Wf with a 6xHis tag was expressed inE coliand showed α-l-Fucosidase activity. Sequence annotation revealed that Alf1_Wf contains a combination of a GH29 catalytic domain and a CBM35 accessory domain. Alf1_Wf was confirmed to be a member of the GH29-A subfamily based on phylogenetic analysis. In addition, the biochemical and kinetic properties of the enzyme were determined. Determination of the substrate specificity showed that Alf1_Wf is able to hydrolyze the α1,4-fucosidic bond and the synthetic substrate pNP-fucose. In addition, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidases and meanwhile discovered a new multidomain structure of the glycoside hydrolase. The knowledge gained from this study should support further research and application of α-l-Fucosidase.

New Biotechnology, Volume 62, 2021., p. 68-78

The use of retention glycoside hydrolases as synthetic tools for glycochemistry is very recent and is the focus of significant research. Due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (t/h) the rational technique of restraining glycoside hydrolases to generate transglycosylases remains a challenge. Therefore, to better understand the factors promoting transglycosylation in GH51, the α-l-arabinofuranozidaza izThermobacillus xylanilyticus,Research into the active site of this enzyme has continued. In particular, the properties of two mutants, F26L and L352M, located close to the active site were characterized using kinetic and 3D structural analysis as well as molecular dynamics simulations. The results show that the presence of L352M in the context of the triple mutant (which also contains R69H and N216W) induces changes at both the donor and acceptor subsites, the latter being due to a domino effect. Overall, the R69H-N216W-L352M mutant shows excellent transglycosylation activity (70% yield, 78% transfer rate and reduced secondary hydrolysis of the product). This study also reconfirmed the central role of the conserved residue R69. The R69H mutation affects both the catalytic nucleophile and the acid/base, including their flexibility, and has a critical impact onT/Hseptum. Finally, the results show that increased loop flexibility in the acceptor subsites leads to new interactions with the acceptor, particularly with the hydrophobic binding platform composed of N216W, W248 and W302.

Bioresource Technology, Volume 335, 2021, Article 125261

Lytic polysaccharide monooxygenases (LPMOs) emerged ten years ago and have been described as enhancers of biomass degradation because they play an extremely important role in elucidating the scheme of enzymatic hydrolysis of biomass. These are oxidative enzymes that require the donation of electrons from partners during the catalytic action on the cellulose backbone. Commercial cellulase preparations are largely derived from robust fungal sources, hence fungal LPMOs have been discussed (AA9). The characterization of LPMOs suffers from the numerous complications discussed, and challenges in detecting LPMOs in secretomes have also been highlighted. This Review focuses on the importance of LPMO for biomass hydrolysis, making it a key ingredient in the commercially available cellulolysis cocktail for biomass degradation, and also discusses the challenge of its routine analysis. It also highlights several important points that are helpful in expressing catalytically active recombinant AA9 LPMOs.

Biochimica et Biophysica Acta (BBA) – General Topics, Volume 1850, Number 9, 2015, pp. 1953-1961