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Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

Icariside I, a bioactive flavonoid derivative derived from Herba epimedii, demonstrates better pharmacological properties compared to its precursor icariin. Enzymatic conversion of icariin to icariside I using α-L-rhamnosidase represents an efficient biotechnological approach. In this study, we characterized a GH78 family α-L-rhamnosidase from Dictyoglomus thermophilum (DthRha) with promising biocatalytic properties. The recombinant DthRha displayed optimal activity at 55 °C and pH 6.0, with remarkable thermostability (retaining > 80% activity after 1 h at 45–65 °C) and pH stability (pH 5.0–7.0). The kinetic parameters Km, kcat and kcat/Km values for pNPR of 0.44 mM, 7.99 s−1 and 18.16 s−1 mM−1, respectively. Notably, DthRha exhibited good organic solvent tolerance, retaining > 50% activity after 4 h in 10% DMSO. Applied in a DMSO cosolvent system, DthRha achieved 92.3% conversion of icariin to icariside I within 4 h under optimized conditions. Interestingly, elevating the substrate concentration to 10 mM resulted in a consistently high icariin conversion of 95.8%. The enzymatic hydrolysis method can be applied to the industrial production of Icariside I. Furthermore, DthRha not only cleaves the α-1,2 glycosidic bond between glucoside and rhamnoside in compounds like naringin, but also exhibits tolerance to organic solvents, making it suitable for the hydrolysis of other poorly soluble flavonoids.

Activation of fatty acids as acyl-adenylates by fatty acid-AMP ligase (FAAL) is a well-established process contributing to the formation of various functional natural products. Enzymatic characterization of FAALs is pivotal for unraveling both the catalytic mechanism and its role in specific biosynthetic pathways. In this study, we recombinantly expressed and characterized a novel FAAL derived from marine Pseudoalteromonas citrea ( Pc FAAL). Pc FAAL was a cold-adapted neutral enzyme, demonstrating optimal activity at 30 °C and pH 7.5. Notably, its specific activity relied on the presence of Mg 2+ ; however, higher concentrations exceeding 10 mM resulted in inhibition of enzyme activity. Various organic solvents, especially water-immiscible organic solvents, demonstrated an activating effect on the activity of Pc FAAL on various fatty acids. The specific activity exhibited a remarkable 50-fold increase under 4% (v/v) n-hexane compared to the aqueous system. Pc FAAL displayed a broad spectrum of fatty acid substrate selectivity, with the highest specific activity for octanoic acid (C8:0), and the catalytic efficiency ( k cat / K m ) for octanoic acid was determined to be 1.8 nM −1 ·min −1 . Furthermore, the enzyme demonstrated biocatalytic promiscuity in producing a class of N-acyl amino acid natural products, as verified by LC-ESI MS. Results indicated that the Pc FAAL exhibits promiscuity towards 10 different kinds of amino acids and further demonstrated their potential value in the biosynthesis of corresponding functional N-acyl amino acids.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis -host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 μM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

Malate dehydrogenase (MDH) is a key energy metabolic enzyme with distinct coenzyme specificity for either NAD+ or NADP+ in all domains of life. Here, we characterize a novel MDH from the bloom-forming cyanobacterium Microcystis aeruginosa PCC7806 (MaMDH), which displays dual-coenzyme specificity with comparable efficiency for both NAD+ and NADP+, albeit with a slight preference for NAD+. MaMDH exists as a 72.1 kDa homodimer with a subunit mass of 36.2 kDa in solution. Kinetic measurements yielded Km values of 33.140 μM for NAD+ and 113.200 μM for NADP+, with a kcat ratio (NAD⁺/NADP⁺) of 3.64. The enzyme exhibited optimal activity at pH 8.0 and 40 °C, along with notable thermostability, retaining over 90% activity after incubation at 70 °C for 20 min. Through structure-guided mutagenesis of the predicted coenzyme-binding motif, we shifted MaMDH cofactor preference from NAD+ toward NADP+, supporting the hypothesis that dual-specificity MDHs may represent evolutionary intermediates in the emergence of NADP+-dependent chloroplast MDHs. This study provides new insights into the molecular evolution mechanisms of coenzyme specificity within the MDH family.

Fishscale bamboo rhombic-spot, caused by Neostagonosporella sichuanensis , poses a significant threat to Phyllostachys heteroclada in Sichuan province. Based on genomic analysis, four cutinase genes, NsCut1–NsCut4, were identified, cloned, and functionally validated. Bioinformatics analyses revealed that the proteins encoded by these genes possess secretory functions, lack transmembrane domains, and contain conserved cutinase domains highly homologous to those in other fungi. Recombinant proteins expressed via a prokaryotic system exhibited strong hydrolytic activity against glycerol tributyrate and bamboo white cream at 40°C and pH 8.0, while signal peptide and subcellular localization analyses confirmed their secretory function and localization to the cell wall. Gene knockout experiments were performed to construct deletion strains ΔNsCut and corresponding complemented strains ΔNsCut+ . Notably, ΔNsCut1 and ΔNsCut3 resulted in reduced pigmentation, decreased spore production, and increased sensitivity to NaCl, H 2 O 2 , and Congo red, along with reduced pathogenicity—indicating that these genes play key roles in metabolic and reproductive processes, oxidative stress responses, and the maintenance of cell wall integrity. In contrast, ΔNsCut2 and ΔNsCut4 did not exhibit significant differences compared to the wild type. This work advances our understanding of the role of cutinases in the pathogenic interaction between N. sichuanensis and P. heteroclada , providing a theoretical basis for further exploration of the pathogen’s underlying mechanisms.

ObjectiveExtracellular fructosyltransferase (FTase, E.C.2.4.1.9) from Aspergillus oryzae IPT-301 was immobilized on silica gel by adsorption and biochemically characterized aiming at its application in the transfructosylation reaction of sucrose for the production of fructooligossaccarides (FOS).ResultsThe transfructosylation activity (AT) was maximized by the experimental design in function of the reaction pHs and temperatures. The AT of the immobilized enzyme showed the kinetics behavior described by the Hill model. The immobilized FTase showed reuse capacity for six consecutive reaction cycles and higher pH and thermal stability than the soluble enzyme.ConclusionThese results suggest a high potential of application of silica gel as support for FTase immobilization aiming at FOS production.

Zearalenone (ZEN) is a toxic secondary metabolite of Fusarium sp. commonly found in wheat, corn, and other crops. In addition to economic losses, ZEN can seriously endanger the health of both humans and livestock, thus presenting an urgent need for ZEN-detoxifying enzymes that function in the extreme heat or pH conditions of industrial fermenters. Here, we identify and characterize the activity of the ZEN-degrading enzyme from Exophiala spinifera, ZHD_LD, which shares 60.15% amino acid identity and a conserved catalytic triad with the well-characterized ZEN-detoxifying protein ZHD101 from Clonostachys rosea. Biochemical activity and stability assays indicated that purified recombinant ZHD_LD exhibited high activity against ZEN with optimal reaction conditions of 50 ℃ and pH 7.0–10.0. Structural modeling of the ZHD_LD active site and comparison with ZHD101 revealed its likely mechanism of ZEN degradation. This research provides an industrially valuable candidate enzyme for ZEN detoxification in food and livestock feed.

Tannase, as a type of tannin−degrading enzyme, can catalyze the hydrolysis of ester and depside bonds in gallotannins, thereby releasing gallic acid and glucose. Based on this reaction mechanism, Tannase can effectively improve the problems of bitter taste, weak aroma, and tea cheese in tea infusion, and is therefore widely used in the tea industry. However, due to high production costs, difficulties in purification and recovery, and insufficient understanding of Tannase properties, the large−scale application of Tannase is severely limited. Therefore, the sources of Tannase and the effects of fermentation temperature, pH, stirring speed, time, carbon, and nitrogen sources on the preparation of Tannase are described in this study. The advantages and disadvantages of various methods for measuring Tannase activity and their enzymatic characterization are summarized, and the concentration and purification methods of Tannase are emphasized. Finally, the application of Tannase to reduce the formation of tea precipitate, enhance antioxidant capacity, increase the extraction rate of active ingredients, and improve the flavor of the tea infusion is described. This study systematically reviews the production, characterization, purification, and application of Tannase to provide a reference for further research and application of Tannase.

The thermostable β-glucosidase gene from Thermotoga petrophila DSM 13995 was cloned and overexpressed in Escherichia coli. The activity of the recombinant β-glucosidase was 21 U/mL in the LB medium. Recombinant β-glucosidase was purified, and its molecular weight was approximately 81 kDa. The optimal activity was at pH 5.0 and 90 °C, and the thermostability of the enzyme was improved by Ca(2+). The β-glucosidase had high selectivity for cleaving the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1, which produced the pharmacologically active minor ginsenoside 20(S)-Rg3. In a reaction at 90 °C and pH 5.0, 10 g/L of ginsenoside Rb1 was transformed into 6.93 g/L of Rg3 within 90 min, with a corresponding molar conversion of 97.9%, and Rg3 productivity of 4620 mg/L/h. This study is the first report of a GH3-family enzyme that used Ca(2+) to improve its thermostability, and it is the first report on the high substrate concentration bioconversion of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 by using thermostable β-glucosidase under high temperature.