The industrial applications of enzymes are limited due to the activity-stability trade-off, which implies that the improvement of thermostability often accompanies decreased activity. This study presents a dual-strategy approach to simultaneously improve the catalytic efficiency and thermostability of α-galactosidase galV from Anoxybacillus vitaminiphilus WMF1. Our integrated method combines computational analysis with enzyme property prediction to selectively target and modify the catalytic region and residues that are distant from the active site. We identified and experimentally validated mutations that improve activity without compromising stability and further increased thermostability through additional distant-site mutations. The resulting mutant enzyme variant N549Q/T550N/Y634F demonstrated a 6.2-fold increase in catalytic efficiency and a 3.2-fold improvement in the half-life at 65 °C. Molecular dynamics (MD) simulations supported the structural basis for the observed enhancements. This approach offers a refined strategy for engineering α-galactosidases with improved industrial applicability, overcoming the traditional trade-offs between enzyme activity and stability. Hydrolytic activity toward raffinose family oligosaccharides (RFOs) was validated using soymilk as a model substrate, demonstrating significant practical potential.

Chronic non-healing wounds requires dressings that combine flexibility, infection control, and responsive environments, a need unmet by current options. In this study, we developed a dual-dynamic-network hydrogel using covalent Schiff base bonds and non-covalent Al3+-carboxyl coordination with oxidized dextran (ODex), polyethyleneimine-modified gelatin (PEI-Gel), polyacrylic acid (PAA), and aluminum ions (Al3+), achieving three synergistic functions critical for clinical application. The DPA (ODex/PEI-Gel/PAA) hydrogel exhibits tissue-like deformability, with an elongation capacity of 920.9 ± 1.2 % and a tensile strength of 0.54 ± 0.3 MPa, while preserving structural integrity under cyclic stress conditions. In vitro tests demonstrated the hydrogel's antibacterial efficacy, with MIC90 values of 25, 20, and 6 mg/mL against Escherichia coli, Staphylococcus epidermidis, and S. saprophyticus, respectively. The hydrogel's pH-responsive drug release mechanism facilitates intelligent therapeutic delivery, achieving over 90 % cumulative release of emodin at pH 3.0 within 168 h. Overall, this work highlights the promising potential of the antibacterial hydrogel drug delivery platform with superior mechanical characteristics for the treatment of chronic cutaneous wounds.

The economic viability of cellulose biotransformation remains constrained by high enzyme costs, with processive endoglucanases emerging as promising candidates due to their dual-function hydrolysis mechanism. However, comprehensive kinetic and synergistic analyses of these enzymes are notably limited. This study investigates the kinetic properties of GH5 processive endoglucanase (M3-1) through various kinetic models. Inverse Michaelis-Menten analysis revealed M3-1's superior substrate recognition capacity, demonstrating 95.5 % productive binding site coverage compared to 48.8 % in non-processive endoglucanases. This enhanced efficiency is attributed to M3-1's distinctive structural features, particularly its open and deep cleft configuration. Pre-steady-state kinetics identified substrate association as the rate-limiting step, providing crucial direction for enzyme engineering efforts. Synergistic studies with cellobiohydrolase (CBH) demonstrated remarkable degradation synergy (DS value up to 8.2 on filter paper) and improved substrate resistance compared to traditional EG/CBH combinations. We propose a novel bidirectional degradation mechanism for the M3-1/CBH system, operating both inside-out and outside-in. The effectiveness of M3-1/CBH combination was further enhanced by up to 320 % through the addition of nonionic surfactants and expansin. These findings advance our understanding of processive endoglucanases and their potential applications in biomass conversion.

Glucansucrases are extracellular enzymes capable of synthesizing diverse α-glucan polymers and oligosaccharides, including the industrially relevant mutan. The mutI-encoded mutansucrase (MUT-I) from Leuconostoc pseudomesenteroides G29 was biochemically characterized as a robust biocatalyst for α-glucan engineering. Recombinant MUT-I (165.1 kDa) demonstrated optimal activity at pH 5.5/30 °C with a high substrate affinity (Km = 3.46 mM) and catalytic efficiency (kcat = 10.35 s-1). It synthesized water-insoluble mutan (91% α-1,3 linkages; Mw = 644,083 g/mol) exhibiting exceptional thermal stability (degradation onset 287.65 °C) and compact fibrous morphology. The enzyme displayed dual functionality: (1) generating gluco-oligosaccharides through maltose-dependent acceptor reactions and (2) introducing α-1,3 branches into dextrans (74% and 14% branching efficiency for dextran T3 and T70, respectively). The deep learning tool AlphaFold 3 identified conserved catalytic residues (Asp476, Glu514, and Asp587) governing polymerization dynamics. This study establishes MUT-I's potential for sustainable production of structurally defined α-glucans as biodegradable alternatives to synthetic polymers in food and biomedical applications.

Dextran, a polysaccharide with critical pharmaceutical applications, requires precise molecular weight control for optimal functionality. Traditional chemical synthesis methods face challenges in efficiency and environmental sustainability. Here, we present a combinatorial strategy integrating enzyme engineering and soft nanoconfinement to achieve one-step biosynthesis of tailored dextran. Twelve recombinant dextransucrases were designed by swapping domains V/IV between the processive enzyme DSR-OK (producing ultra-high molecular weight dextran) and the distributive enzyme DSR-MΔ2 (yielding low molecular weight dextran). Among these variants, CZ8 demonstrated superior catalytic activity, synthesizing 40 kDa dextran directly from sucrose. To further enhance chain elongation, lipid-based nanomaterials, alcohol ethoxylate (AEO) and polyethylene glycol (PEG, 20 kDa), were incorporated into the reaction system. Under optimized conditions (125 g/L sucrose, 4 U/mL CZ8, 5 wt% AEO, or 1 wt% PEG), the molecular weight of dextran increased to 70 kDa, attributed to nanoconfinement-induced spatial restriction favoring polymerization. Notably, the AEO nanomaterial spontaneously separated via gravitational settling within 24 h, achieving >99 % removal efficiency without energy-intensive purification. Structural analysis confirmed dextran's α-(1,6) backbone, with minor α-(1,3) branching under nanoconfinement. This study provides a green, scalable platform for dextran production, emphasizing enzyme-nanomaterial synergy for precise molecular weight control and sustainable downstream processing.

Xylo-oligosaccharides (XOS) have gained significant attention for their extensive health-boosting characteristics. However, the tedious procedures involved in lignocellulose pretreatment and XOS purification, coupled with the low efficiency of enzymatic hydrolysis, constrain the broader commercialization of XOS. In this study, the effects of fusion order and linker composition on enzyme activity and thermostability of fusion proteins consisting of a GH11 xylanase mutant (XYNm) and an expansin (EXLX) were investigated. The results indicated that the reconstructed protein XYNm-R2-EXLX exhibited 2.6-fold activity higher than EXLX-R2-XYNm. Furthermore, the denaturation thermodynamics evaluation demonstrated a significant enhancement in the thermostability of XYNm-R2-EXLX. Additionally, the reconstructed fusion protein showed a notable affinity adsorption efficiency on corncob, reaching a remarkable 95.5%. Subsequently, the fusion enzyme-corncob complex underwent direct hydrolysis, and a yield of 58.7 g XOS/kg corncob was obtained by employing RSM optimization techniques. Finally, a membrane-assisted refining process was implemented, resulting in the recovery of 68.2% (w/w) of the initial XOS (X2-X4) with a purity of 96.2%. Here, a chemical free and eco-friendly bioprocess for production of low-degree polymerization XOS was developed, eliminating the need for xylan separation from corncob and simplifying the process for product purification. These findings exhibited the great potential in XOS (X2-X4) production.

Synthetic methylotrophic Yarrowia lipolytica was constructed to convert methanol into biomass components and succinic acid.

Succinate is the end product of anaerobic metabolism of Escherichia coli, and its over-production needs abundant reducing force. Electro-fermentation (EF) is a novel biotechnology to steer and control fermentative processes by supplying extra electrons. However, E.coli is a non-electroactive strain which needs the support of electron shuttle in EF. Here, membrane engineering strategies of "Building bridges" via screening direct electron transport pathway and "Digging tunnels" via screening membrane porins were developed to improve the transmembrane transport of electron during the cathodic electro-fermentation (CEF). As a result, the total electron quantity during electro-fermentation was increased from 1.21 mmol to 7.90 mmol, and succinate yield was increased by 23.3% when these strategies simultaneously were applied to the succinate candidate E. coli Suc260. Hence, this study provides a reference mode for designing and constructing non-electroactive bacteria for electrofermentation of reductive metabolites.

Dextran, a variant of α-glucan with a significant proportion of α-(1,6) bonds, exhibits remarkable solubility in water. Nonetheless, the precipitation of dextran has been observed in injection vials during storage. The present study aimed to establish a technique for generating insoluble dextran and analyze its structural properties. Additionally, the potential for positively ionizing IS-dextran with polyethyleneimine was explored, with the ultimate objective of utilizing IS-dextran-PEI as a promising support for enzyme immobilization. As a result, IS-dextran was obtained by the process of slow evaporation with an average molecular weight of 6555 Da and a yield exceeding 60%. The calculated crystallinity of IS-dextran, which reaches 93.62%, is indicative of its irregular and dense structure, thereby accounting for its water insolubility. Furthermore, positive charge modification of IS-dextran, coupled with the incorporation of epichlorohydrin, resulted in all zeta potentials of IS-dextran-PEIs exceeding 30 mV, making it a promising supporting factor for enzyme immobilization.

生物被膜是微生物附着在载体材料表面的高度有组织的微生物群体,主要由菌体和微生物的胞外分泌物构成.与浮游细菌相比,生物被膜内的微生物对抗菌剂、恶劣环境及宿主免疫防御机制的抗性显著增加,还具有自增殖、可重复利用等优点.近年来,生物被膜在污水处理、工业发酵、食品工程和生物制药等领域受到越来越多的关注.载体材料的理化性质对生物被膜的构建具有重要影响.有机高分子材料因价廉、具有较轻的比重和密度、易于表面改性等特点,成为介导生物被膜构建的优选载体材料.本文对水凝胶、高分子分离膜、聚合物纤维膜及移动床生物膜反应器(MBBR)载体等几大类功能高分子材料促进生物被膜构建及其生物转化应用研究进行综述,以期为相关科研工作者提供参考.

Escherichia coli has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO2 emissions. Here, we aim to address this issue by reducing CO2 emissions of recombinant E. coli during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of E. coli E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of nuoA and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD+ and ATP supplies. The results demonstrate that E23 (ΔnuoA, NOG) exhibited decreased CO2 emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD+ and ATP supply can prompt E. coli to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration.

Methanol has become an attractive substrate for the biomanufacturing industry due to its abundant supply and low cost. The biotransformation of methanol to value-added chemicals using microbial cell factories has the advantages of green process, mild conditions and diversified products. These advantages may expand the product chain based on methanol and alleviate the current problem of biomanufacturing, which is competing with people for food. Elucidating the pathways involving methanol oxidation, formaldehyde assimilation and dissimilation in different natural methylotrophs is essential for subsequent genetic engineering modification, and is more conducive to the construction of novel non-natural methylotrophs. This review discusses the current status of research on methanol metabolic pathways in methylotrophs, and presents recent advances and challenges in natural and synthetic methylotrophs and their applications in methanol bioconversion.

重组蛋白在大肠杆菌中表达时,往往面临着形成包涵体的问题,而重组蛋白若是分泌至周质空间则基本解决了这一问题,周质空间的周质蛋白不仅能帮助重组蛋白正确折叠还有利于二硫键的生成.信号肽是一段由15-30个氨基酸组成,被融合在重组蛋白N端的短肽,按照结构、功能的不同可以划分为N区、H区和C区,具有引导重组蛋白转运至细胞周质空间的作用.本文综述了信号肽的结构组成、作用机理和基本分泌途径,讨论了信号肽的高效转运和筛选方法,总结了在大肠杆菌中重组蛋白融合信号肽实现周质表达的新进展,并对未来高效信号肽选择方面的研究进行了探讨.

An extracellular thermostable xylanase (XynNTU) from Paenibacillus campinasensis NTU-11, consisted of a glycoside hydrolase (GH) family 11 catalytic domain, a Gly/Pro-rich linker sequence (LS) and a family 6 carbohydrate-binding module (CBM6), was identified and expressed in E. coli BL21. The purified XynNTU had a specific activity of 2750 U/mg and an optimal activity at 60 degrees C and pH 7.0, and retained a residual activity of 58.4% after incubation (60 degrees C, 48 h). Two truncated mutants, CBM6-truncated form XynNTU-CDLS, CBM6 and linker-truncated form XynNTU-CD, possessed similar values of optimum pH and temperature as the native XynNTU. XynNTU-CD displayed a lower thermostability than XynNTU, whereas for XynNTU-CDLS, more than 90% of residual activity was remained (60 degrees C, 48 h), indicating that this enzyme presented a higher thermo-stability than that of the majority of reported GH11 xylanases. Furthermore, XynNTU and two mutants main-tained more than 70% of residual activity at pH values of 5-9. Kinetic measurements suggested that CBM6 had a crucial function in the ability of the enzyme to bind and hydrolyze xylan substrates, while LS had a relatively mild influence. Collectively, a noticeable thermostability and a high specific activity of XynNTU and its truncated form XynNTU-CDLS highlights their potentials for diverse industrial applications.

Escherichia coli AFP111 was previously engineered for succinate production by eliminating byproducts of synthesis pathways. Still, the succinate yield is limited due to the insufficient NADH supplement, when fed with glucose. Microbial electrolysis cell (MEC) allows microorganisms to perform unbalanced fermentation by establishing polarized cathode interaction.

Exopolysaccharides can be produced by various bacteria and have important biological roles in bacterial survival depend on molecular weight, linkage, and conformation. In this study, Leuconostoc pseudomesenteroides G29 was identified and found to produce two types of exopolysaccharides from sucrose including soluble and insoluble alpha-glucans. By regulation of pH above 5.5, soluble alpha-glucan production was increased to 38.4 g.L-1 from 101.4 g.L-1 sucrose with fewer accumulation of lactic acid and acetic acid. Simultaneously, the quantity of thick white precipitate, that is insoluble alpha-glucan, was also increased. Then, alpha-glucans were prepared by enzymatic reaction with crude glucansucrases from the supernatant of G29 fermentation broth and purified for structure analysis. Based on the integration analysis of FT-IR and NMR, it was observed that soluble a-glucan is a highly linear dextran with alpha-1,6 glycosidic bonds while the insoluble alpha-glucan has 93% of alpha-1,3 and 7% of alpha-1,6 glycosidic bond. The results extend our understanding of exopolysaccharides production by L. pseudomesenteroides, and this water insoluble alpha-1,3-glucan might have potential application as biomaterials and/or biochemicals. (C) 2021 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd. All rights reserved.

Background: The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes , a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD + . Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle. Results: MFC assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. Firstly, an acid tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV. Conclusions: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Nano-metallic materials are playing an important role in the application of medicine, catalysis, antibacterial and anti-toxin due to their obvious advantages, including nanocrystalline strengthening effect, high photo-absorptivity, high surface energy and single magnetic region performance. In recent years, with the increasing consumption of global petrochemical resources and the aggravation of environmental pollution, nanomaterials based on bio-based molecules have aroused great concern. Bio-based molecules refer to small molecules and macromolecules directly or indirectly derived from biomass. They usually have good biocompatibility, low toxicity, degradability, wide source and low price. Besides, most bio-based molecules have unique physical, chemical properties and physiological activity, such as optical activity, acid/alkali amphoteric property, hydrophilic property and easy coordination with metal ions. Thus, the corresponding nano-materials based on bio-based molecules also have unique functions, such as anti-inflammatory, anti-cancer, anti-oxidation, antiviral fall blood sugar and blood fat etc. In this paper, we give a comprehensive overview of the preparation and application of nano-metallic materials based on bio-based molecules in recent years.

Iron dextran is a common anti-anemia drug, and it requires low molar mass dextran as substrate. In this work, we selected 11 amino acid residues in domain A/B of DSR-MΔ2 within a 5-angstrom distance from sucrose for site-directed mutagenesis by molecular docking. Mutation of Q634 did not affect the enzyme catalytic activity, but showed an obvious impact on the ratio of low molecular weight dextran (L-dextran, 3,000–5,000 Da) and relatively higher molecular weight dextran (H-dextran, around 10,000 Da). L-dextran was the main product synthesized by DSR-MΔ2 Q634A, and its average molecular weight was 3,951 Da with a polydispersity index <1.3. The structural characterization of this homopolysaccharide revealed that it was a dextran, with 86.0% α(1→6) and 14.0% α(1→4) glycosidic linkages. Moreover, L-dextran was oxidized with NaOH and chelated with ferric trichloride, and an OL-dextran-iron complex was synthesized with a high iron-loading potential of 33.5% (w/w). Altogether, mutation of amino acids near the sucrose binding site of dextransucrase can affect the chain elongation process, making it possible to modulate dextran size.