Collagenolytic proteases have attracted increasing attention due to their ability to hydrolyze collagen and their relevance in biotechnological processes. In this study, a collagenolytic protease produced by Mucor subtilissimus UCP 1262 was characterized through an integrated evaluation of its biochemical properties, thermal-inactivation thermodynamics, and conformational behavior. The enzyme was produced by solid-state fermentation using wheat bran and purified by DEAE-Sephadex A50 ion-exchange chromatography. Optimal collagenolytic activity was observed at pH 7 and 40 °C, and approximately 50% of the initial activity was retained after 24 h at 30-40 °C, indicating moderate thermal stability. Fluorescence spectroscopy revealed marked pH- and temperature-dependent changes in fluorescence intensity and emission maxima, with incomplete structural recovery after cooling. Thermodynamic analysis further supported the occurrence of temperature-dependent destabilization. Overall, the results provide new insight into the functional and conformational stability of the collagenolytic protease from M. subtilissimus UCP 1262 and support its further investigation for biotechnological applications under mild processing conditions.
Glycosylation, one of the most significant and prevalent post-translational modifications, engenders a diverse array of protein structures, with over half of eukaryotic proteins assuming glycosylated forms. Given the limitations of alternative deglycosylation methods, identifying and characterizing novel glycosidases is important for understanding the diverse roles of glycans. In this study, the enzymatic activities of three recombinant endo-β-N-acetylglucosaminidases (ENGases) from Bifidobacterium pullorum subsp. pullorum (EndoBP), Bifidobacterium kashiwanohense (EndoBK), and Bifidobacterium bohemicum (EndoBbo) were assessed using ribonuclease B as a model glycoprotein. Furthermore, the optimal pH and temperature conditions for each enzyme were elucidated. Released N-glycans were quantified by a phenol-sulphuric acid assay, and the resulting N-glycan compositions were profiled by MALDI-MS. Moreover, comparative structural models were generated to provide structural context for the conserved GH18 catalytic regions of the enzymes. Based on the apparent kinetic analysis of the three enzymes on a glycoprotein mixture (whey, bovine IgG, bovine lactoferrin, and soy protein), Km values, expressed on a total protein mass basis, were 3.44, 7.18, and 2.85 mg/mL and Vmax values were 2.29 x 10-2, 2.52 x 10-2, and 1.35 x 10-2 mg/mL×min for EndoBP, EndoBK, and EndoBbo, respectively. MALDI-TOF/TOF-MS profiling of whey-derived N-glycans revealed distinct enzyme-dependent patterns, with 31, 21, and 10 N-glycan compositions detected after treatment with EndoBP, EndoBK, and EndoBbo, respectively. EndoBP exhibited the broadest observed glycan-release profile, whereas EndoBbo produced a comparatively narrower profile under the conditions examined. These findings highlight distinct glycan-release behaviors among the three ENGases and support the enzymatic recovery of structurally diverse, value-added N-glycan fractions from whey as a potential whey-valorization strategy.
Collagenolytic proteases have attracted increasing attention due to their ability to hydrolyze collagen and their relevance in biotechnological processes. In this study, a collagenolytic protease produced by Mucor subtilissimus UCP 1262 was characterized through an integrated evaluation of its biochemical properties, thermal-inactivation thermodynamics, and conformational behavior. The enzyme was produced by solid-state fermentation using wheat bran and purified by DEAE-Sephadex A50 ion-exchange chromatography. Optimal collagenolytic activity was observed at pH 7 and 40°C, and approximately 50% of the initial activity was retained after 24h at 30–40 °C, indicating moderate thermal stability. Fluorescence spectroscopy revealed marked pH- and temperature-dependent changes in fluorescence intensity and emission maxima, with incomplete structural recovery after cooling. Thermodynamic analysis further supported the occurrence of temperature-dependent destabilization. Overall, the results provide new insight into the functional and conformational stability of the collagenolytic protease from M. subtilissimus UCP 1262 and support its further investigation for biotechnological applications under mild processing conditions.
The application of steam-exploded green coconut fiber (GCF) (an agro-industrial waste) was studied as a support for the immobilization of lipases, using lipase B from Candida antarctica (CALB), lipase from Pseudomonas fluorescens (PFL) or the engineered lipase Eversa (EL) as model enzymes. The achieved immobilization yield was 82.01 ± 2.02%, 87.62 ± 4.34%, and 77.47 ± 1.88% for GCF-PFL and GCF-CALB in 24 h, and GCF-EL in 48 h, respectively. The biocatalysts' activities (U/mg) and recovered activities in relation to the soluble enzymes (%) were 10.22 ± 0.96 U/mg and 26.37 ± 2.25%, 9.22 ± 0.17 U/mg and 49.84 ± 2.30%, 27.23 ± 0.21 U/mg and 9.48 ± 0.11% for GCF-CALB, GCF-PFL, and GCF-EL, respectively. After the immobilization, the enzymes were subjected to modification with glutaraldehyde (GA) or/and polyethylenimine (PEI). The modification with both GA and PEI was the best achievement as it reduced the desorption of lipases from the support and substantially increased their thermostability (for GCF-CALB-GA-PEI: stabilization factor (SF) > 154.8 at 60 ºC; for GCF-PFL-GA-PEI: SF > 58.6 at 75 ºC and GCF-EL-GA-PEI: SF > 172.6 at 80 ºC). The maximum enzyme load was determined to be 5 mg/g for CALB biocatalysts, 10 mg/g for PFL and EL. This study shows an alternative use of this agroindustrial, with the environmental relevance that it presents.
Glucose dehydrogenase (GDH) is a key enzyme for NAD(P)H cofactor regeneration in industrial biocatalysis. However, conventional engineering approaches are frequently constrained by limited catalytic efficiency and excessive enzyme loading, which collectively compromise process economics and hinder large-scale deployment. In this study, we performed structure-guided rational design by targeting three functionally discrete domains of GDH, including the substrate-binding region, the cofactor-binding pocket, and the interdomain communication interface. An engineered variant GDH-M6 was constructed, which manifests a 35-fold enhancement in catalytic efficiency relative to the wild-type enzyme. In the biocatalytic synthesis of the pivotal chiral intermediate for R-lipoic acid, GDH-M6 reduced enzyme loading by more than 90% and allowed a doubling of the substrate concentration. As a result, overall reaction productivity was substantially increased and the GDH-M6 outperformed wild-type GDH as well as all previously reported mutants under comparable conditions. Notably, the domain-engineering paradigm established herein provides a broadly applicable toolkit for augmenting the catalytic performance of dehydrogenases, and offers a structural blueprint for resolving analogous kinetic bottlenecks that commonly arise in NAD(P)⁺-dependent enzymes utilized for industrial cofactor regeneration.
Soybean isoflavones exist predominantly as glycosides (e.g., daidzin, genistin), requiring enzymatic hydrolysis by β-glucosidases (EC 3.2.1.21) to release bioactive aglycones (e.g., daidzein, genistein) with enhanced bioavailability. In this study, a novel glycoside hydrolase family 1 (GH1) β-glucosidase gene (ChaBGL) derived from the halophilic marine bacterium Celeribacter halophilus was successfully cloned and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme (ChaBGL) displayed maximal activity at 45 °C and pH 6.0. ChaBGL exhibited broad halotolerance across multiple salt types, retaining 97.5 ± 4.4% activity at 1000 mM NaCl, 76.5 ± 5.6% at 1000 mM KCl, 79.5 ± 4.5% at 1000 mM Na2SO4, and 83.2 ± 5.2% at 500 mM K2SO4. ChaBGL demonstrated high catalytic efficiency towards the substrate p-nitrophenyl-β-D-glucopyranoside (pNPG), achieving a specific activity of 101.3 ± 4.0 U/mg. Kinetic analysis revealed a Km of 0.17 ± 0.02 mM and an apparent kcat of 87.8 ± 3.5 s-1. Notably, ChaBGL demonstrated high efficacy in hydrolyzing natural soybean isoflavone glycosides. Within 60 min at 30 °C, it achieved near-complete hydrolysis of daidzin (95.4 ± 0.34%) and genistin (∼100%), resulting in substantial increases in the yields of bioactive daidzein (6.73 ± 0.15-fold) and genistein (9.78 ± 0.31-fold). Consequently, the percentage of aglycones in the total isoflavonoids surged from an initial 10.3 ± 0.8% to 96.5 ± 0.07%. These findings collectively highlight ChaBGL's high catalytic efficiency in hydrolyzing glycosidic bonds and its significant potential for diverse industrial applications, particularly in the nutritional, veterinary, and pharmaceutical sectors.
Ginsenoside Rh2, a potential anticancer agent originally isolated from the medicinal plant Panax ginseng, has low natural abundance and is difficult to extract, making biosynthetic production a promising alternative. UDP-glycosyltransferases (UGTs) that specifically catalyze protopanaxadiol (PPD) glycosylation to produce Rh2 have been widely identified in plants and microorganisms. In this study, we report that Barbarea vulgaris UGT73C21 effectively catalyzes this reaction. The enzyme was expressed in Escherichia coli and purified to electrophoretic homogeneity via Ni2⁺-affinity chromatography. It exhibited optimal activity at pH 8.0 (100 mM HEPES) and 45 °C. Mn2⁺, Mg2⁺, and Ca2⁺ significantly enhanced enzyme activity, whereas other tested metal ions reduced it. The kinetic parameters Km, Vmax, kcat, and kcat/Km were determined as 231.92 μM, 5.68 μM min-1, 0.053 s-1, and 228 M-1 s-1, respectively. In a reaction containing 0.2 mg/mL UGT73C21, 1 mM PPD, and 5 mM UDP-Glucose in HEPES buffer (pH 8.0), 0.55 mM Rh2 was produced within 1 h, corresponding to a 55% conversion rate. These results demonstrate that B. vulgaris UGT73C21 is an efficient biocatalyst for ginsenoside Rh2 biosynthesis.
Efficient utilization of natural alginate is restricted by its high viscosity. Alginate lyase degrades alginate via β-elimination to reduce viscosity, supporting its development and application. However, natural alginate lyases exhibit poor thermostability, limiting their application in high-temperature environments. Enhancing the thermostability of natural alginate lyase would better meet the demands of industrial production. We cloned and characterized RwAly5A, a PL-5 family alginate lyase from Ralstonia wenshanensis 56D2. Recombinant RwAly5A exhibited maximal catalytic activity at pH 8.0, with robust tolerance over the pH range of 4.6-10.6. Its maximum activity is observed at 50 °C, and although the protein remains fairly stable at 0-40 °C, its thermostability deteriorates rapidly at temperatures above 50 °C. Additionally, RwAly5A exhibited optimal activity at 100 mM NaCl. Its activity was promoted by a broad spectrum of cations, including monovalent (K+, NH4+, and Li+) and divalent (Ca2+ and Mg2+) ions. In contrast, Mn2+, Ba2+, Fe3+, and EDTA suppressed the enzyme, and SDS almost completely abolished its function. Based on molecular dynamics (MD) simulations, residues predicted to be located in the flexible-loop region were chosen as targets for proline substitution, and the resulting variants were subjected to thermostability mechanism studies. Proline substitutions were introduced in flexible loops, generating D20P, D227P, and A229P mutants, which exhibited half-lives at 50 °C that increased to 1.14‑, 4.69‑, and 2.28‑fold, respectively, while retaining most of the catalytic activity. These findings provide new strategies for improving alginate lyases for commercial applications.
This paper provides a critical review of pectinases, focusing on structural diversity, advances in production technologies, industrial limitations, and emerging optimization strategies. In addition to the canonical right-handed parallel β-helix fold, several source-dependent structural features are highlighted, including Fn3-like accessory domains in bacterial enzymes, plant PME pro-domains in pectin methylesterases (PMEs), and naturally occurring bifunctional enzymes that couple de-esterification with depolymerization. Recent progress in pectinase development is also summarized, encompassing solid-state fermentation using agro-industrial residues, heterologous expression systems, protein engineering, and modern immobilization platforms, all of which contribute to improved enzyme yield, stability, and reusability. Two major industrial constraints remain prominent: the incomplete degradation of highly methyl-esterified pectin and the limited stability of enzymes under harsh operational conditions, including elevated temperature, extreme pH, solvent exposure. This review further emphasizes that effective solutions increasingly rely on multidisciplinary integration, including enzyme consortium design, bifunctional or co-expression systems enabling cascade reactions, sequence- and structure-guided engineering approaches such as directed evolution, advanced immobilization strategies, and sustainable low-cost fermentation substrates. These developments position pectinases as biocatalysts for greener and more efficient processes across food, fiber, and health-related industries.
This study developed a highly efficient immobilized phospholipase D (PLD) system using a magnetic chitosan-based carrier with the natural crosslinking agent genipin (Fe3O4@CS-Gnp-PLD). Systematic characterization confirmed successful PLD immobilization, with chitosan's abundant amino and hydroxyl groups providing stable binding sites that enhanced both enzyme loading and stability. In the sodium deoxycholate-based mixed micelle system, the Fe3O4@CS-Gnp-PLD achieved an 8.05-fold higher phosphatidylserine (PS) conversion rate than free PLD, owing to enhanced substrate solubility and improved enzyme accessibility. The immobilized enzyme demonstrated exceptional operational stability, retaining 80% activity after 6 reuse cycles and 72% activity after 12 days of storage at 4 °C. This integrated platform, combining chitosan-based enzyme immobilization with a mixed micelle system, offers an efficient, stable, and recyclable approach for sustainable PS biomanufacturing.
Endo-β-1,4-mannanases (EC 3.2.1.78) hydrolyze β-1,4-mannan backbones, enabling the conversion of mannan-rich biomass into value-added products such as mannose and mannan-oligosaccharides (MOS). In this study, an endo-β-1,4-mannanase produced by Aspergillus niger code 1234 under submerged cultivation was purified and biochemically characterized. The purification strategy included ultrafiltration (10 kDa), followed by size-exclusion and ion-exchange chromatography using Sephadex® G-75 and DEAE-Sephacel™, respectively. The purified enzyme (AnMan5A-like) was confirmed by SDS-PAGE and mass spectrometry. AnMan5A-like has an estimated molecular mass of 41.2 kDa, an optimum activity at 62 ºC and pH 3.9, and a specific endo-β-1,4-mannanase activity of 79.30 IU/g of protein. The enzyme exhibited remarkable thermostability, retaining more than 95% of its residual activity after 72 h at 50 °C and 60 °C, respectively. Kinetic analysis revealed a Km of 2.52 mg/mL and a Vmax of 2.20 µmol/min/mL using Locust Bean Gum as the substrate. Enzymatic hydrolysis assays demonstrated the production of MOS with degrees of polymerization up to six. Structural analysis suggested a catalytic cleft capable of accommodating five subsites and favoring mannopentaose as a transient substrate that is rapidly cleaved into mannobiose and mannotriose. These results highlight the potential of functional oligosaccharides production via conversion of mannan-rich biomass using a stable endo-β-1,4-mannanases from A. niger code 1234. Its high catalytic performance, combined with thermostability under acidic conditions, makes AnMan5A-likea promising biocatalyst for industrial applications in the food, animal feed, and biorefinery sectors.
L-Fucose is a physiologically important monosaccharide widely present in various biomass resources, especially microalgae. Metabolically engineered microbial systems have gained growing interest for L-fucose production due to their high yield and industrial scalability. In the biosynthetic pathway, α-L-fucosidase catalyzes the terminal step of L-fucose formation. Here, we report the identification and characterization of a novel α-L-fucosidase derived from Desertivirga arenae (GenBank: WP_207534398.1). The recombinant His-tagged enzyme, purified via nickel-affinity chromatography, displayed optimal activity at pH 7.0 and 30 °C. Its activity was strongly suppressed by Cu2 + and Fe2+, whereas Mn2+, Ca2+, and Mg2+ exerted only negligible stimulatory effects. Thermal stability assessment revealed half-lives (t1/2) of 29.51 min at 35 °C, 7.74 min at 40 °C, and 5.60 min at 45 °C. When heterologously expressed in the 2'-fucosyllactose-producing Escherichia coli chassis MGC064 using the pET-22b(+) vector, this enzyme facilitated efficient L-fucose biosynthesis, reaching a titer of 5.58 g/L. While its titer is lower than that of the benchmark AfcA, the substantially shorter gene size and favorable biochemical properties position this novel enzyme as a promising candidate for future engineering and industrial application.
In this study, the efficacy of β-carotene produced from the isolated yeast strain Rhodotorula alborubescens, for evaluating the in vitro antioxidant, anti-inflammatory, and cytotoxic activities were evaluated. In vitro anti-oxidant activities against various radicals, including hydroxyl, superoxide anion, nitrogen oxide, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric ion reducing anti-oxidant power (FRAP) and total antioxidant assay-cupric reducing antioxidant capacity (CUPRAC) were determined and showed good free radical scavenging (FRS) activity, with the FRS value (%) ranging from 20.01 ± 4.20% (DPPH, 1000 µg/mL) to 65.66 ± 6.08% (reactive nitrogen oxide, 50 µg/mL), however significant reducing activity was observed in the CUPRAC assay (IC50: 0.7794 ± 0.44 µg/mL). Also, the down-regulation was observed when IL-4, IL-5, and IL-13 interleukins were profiled for anti-inflammatory properties. In-vitro cytotoxic potential was also evaluated on human leukemia monocytic cell THP-1 cell line by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. In-vitro cytotoxic assay resulted in an IC50 value of 3.116 ± 0.55% with observed morphological changes, loss of membrane integrity, and shrinkage of the cells. Notably, β-carotene treatment significantly reduced the medium fluorescence intensity (MFI) levels, contributing to the redox balance protection. These results suggested the accessibility of Rhodotorula alborubescens as anti-oxidant, anti-allergic and cytotoxic, which can also be further studied for therapeutic, food and cosmetic industries application.
Diketopiperazine (DKP) dimers are one of the important microbial natural products owing to their structural diversity and a broad spectrum of biological properties. However, few studies on fungal P450-catalyzed DKP dimerization have been reported. Herein, a novel cytochrome P450 monooxygenase DkpdB was originally discovered in the marine-derived fungus Aspergillus sydowii MNP-2 and shown to catalyze the formation of the DKP dimer WIN 64821 from cyclo(Trp-Phe) through a proposed C3-C3' oxidative coupling reaction. Heterologous expression in Aspergillus nidulans RJMP1.5 together with in vivo and yeast microsomal assays confirmed this activity, and its substrate-binding mode is predicted by structural modeling and molecular docking analyses. Furthermore, site-directed mutagenesis identified E180 and T181 as critical residues for DkpdB-catalyzed dimerization of cyclo(Trp-Phe) by molecular docking. These findings provide new insights into fungal P450-catalyzed DKP dimerization and expand the repertoire of functionally characterized fungal P450 dimerases.
A set of rubber oxygenases was discovered through phylogenetic analysis and AI-based structural modeling of complexes of the putative enzymes with a substrate mimicking cis-1,4-polyisoprene. Sixteen candidate proteins were selected from thermophilic microorganisms, all sequence-related to the Latex clearing protein from Streptomyces sp. K30 (LcpK30). Sequence truncation and solubility tags were then evaluated to enhance protein expression, with the SUMO tag proving to be the most effective. Including LcpK30, nine heme-containing oxygenases were successfully expressed in E. coli NEB 10-beta cells, purified (35-157 mg L-1 yield) and characterized. Steady-state kinetics revealed significant rubber latex-degrading properties for six of them, with the truncated SUMO-fused LcpK30 (SUMO-LcpK30T) showing activity in agreement with literature. Notably, the catalytic efficiencies of all the expressed homologs lay within one order of magnitude and the oxygenase from Thermomonospora echinospora was found to be particularly promising in terms of activity, especially at high latex concentrations (more than 1% w/v). The analysis of reaction mixtures by both HPLC and HPLC-MS confirmed the oxidation of cis-1,4-polyisoprene to form the expected isoprenoid oligomers (n = 2-12), whose distribution was consistent with the usual endo-type cleavage pattern in all but one case. This bioprospecting effort afforded a platform of new rubber-degrading enzymes with diverse efficiencies and product profiles, capable of adapting to targeted applications.
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) is an enzyme with its unique capability to transform starch and related substrates into cyclodextrins (CDs). Cyclodextrins are widely used in various industries (food, pharmaceuticals, textiles, and cosmetics). The present study focuses on the purification method of native CGTase from Alkalihalobacillus trypoxylicola KRM and enzyme characterization. The CGTase was purified to homogeneity by ammonium sulfate precipitation followed by starch adsorption and gel filtration. The purified CGTase demonstrated a specific activity of 2733.46 U/mg, corresponding to a 33.2-fold purification. Optimal activity was found at pH 8.0 (Tris HCl buffer) and at 50 °C temperature. CGTase was strongly inhibited by Fe3 +, Zn2+, Pb2+ and Al3+, while some metal ions (Ca2+, Na+, K+ and Mg2+) exerted stimulating effect. A metal chelating agent had a strong inhibitory effect, suggesting that metal ions may contribute to enzyme activity or structural stability. The detergent Triton X-100 was found to be lethal to the CGTase. CGTase showed temperature-dependent loss of activity with increasing first-order deactivation rates and Arrhenius analysis indicated an activation energy of ∼78.7 kJ/mol. The Km (1.83 mg/ml), and Vmax (833.33 μmol/min/ml) values were measured using a Lineweaver-Burk plot. To the best of our knowledge, this is the first report of a CGTase purified from Alkalihalobacillus trypoxylicola KRM. The enzyme exhibited moderate thermal stability and favorable catalytic properties under alkaline conditions.
Vibrio cholerae is an important foodborne pathogen that causes severe diarrhea and poses a serious threat to public health. In this study, we developed a novel visual biosensor platform that integrates recombinase-aided amplification (RAA) with lateral flow assay (LFA) technology for the rapid and sensitive detection of the Vibrio cholerae outer membrane protein W (OmpW) gene. The system used multiple phosphorylated primers in the RAA reaction to generate DNA amplicons of different lengths. Subsequently, the amplification products were treated with λ exonuclease, and the resulting single-stranded DNA fragments were hybridized with FAM- and biotin-labeled probes immobilized prior to LFA detection, producing a visible colorimetric signal. The proposed assay achieved a detection limit of 57.2 CFU/mL. Compared to conventional methods, the proposed sensor platform offers several advantages, including rapid detection, the elimination of thermal cycling, and operational simplicity, making it suitable for rapid and specific detection of V. cholerae in resource-limited settings.
The gastrointestinal tracts of aquatic animals provide a unique environment for microbial growth. Proteases derived from microorganisms in aquatic taxa are particularly valuable owing to their stability under various temperature and salinity conditions. The aim of this study was to isolate and identify protease-producing bacteria from the intestines of abalone (Haliotis discus hannai) and to evaluate their potential applicability to fishery waste degradation. Specifically, bacteria with high protease activity were isolated from the abalone intestine and identified through 16S rRNA sequencing, followed by the optimization of culture conditions for protease production using response surface methodology (RSM). In total, 15 bacteria were isolated from the intestines of abalone, including six bacteria with protease activity. Among these, T14 showed the highest protease activity at 2.42 ± 0.19 units/mL and was selected for protease production. T14 displayed 99.71% similarity to Vibrio fluvialis NBRC 103150. The optimal culture conditions were pH 7.85, 29.49 °C, and 2.69% NaCl, as determined through RSM modeling. The predicted and validated protease activities were 3.20 and 3.19 ± 0.12 units/mL, respectively. PMSF showed the strongest inhibition, suggesting that strain T14 mainly exhibited serine protease-like activity. The crude enzyme from strain T14 was applied to representative fishery waste materials, including fish skin, bones and intestines. SDS-PAGE analysis showed decreased intensities of several protein bands and the appearance of lower-molecular-weight bands after treatment, indicating partial protein degradation. These results suggest that the crude enzyme from strain T14 has potential for the biological pretreatment and partial degradation of fishery waste materials.
