JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2026
·Vol. 74
·期 33
·26532-26543
·
IF 6.7
Chen Yuehua,Liu Peng,Sheng Binbin,Xing Jiaxin,Ouyang Jia,Zheng Zhaojuan
Lignocellulosic
biomass is an abundant renewable resource that
is rich in aldohexoses and aldopentoses. Efficient biocatalysts for
aldopentose oxidation remain limited, and few enzymes exhibit high
catalytic efficiency toward both aldohexoses and aldopentoses. Here,
a naturally promiscuous pyrroloquinoline quinone-dependent glucose
dehydrogenase (PQQ-GDH) from Pseudomonas fragi NL20W was selected as an engineering template and tailored by semirational
design to expand aldopentose oxidation while preserving native d -glucose activity. The V706P/L723 M mutant exhibited markedly
enhanced catalytic efficiency toward d -xylose and l -arabinose, reaching 8.0- and 2.4-fold those of the wild-type, respectively,
while its efficiency toward d -glucose was 1.3-fold that of
the wild-type. Molecular simulations suggested that the improved catalysis
was associated with an optimized substrate positioning and enhanced
conformational flexibility of the active-site region. This study highlights
the significance of natural enzyme scaffold selection in substrate
scope engineering and expands the application potential of PQQ-GDH
in the sustainable valorization of lignocellulosic sugars.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2026
·
IF 6.7
Dai Jiawei,Gao Yawen,Zhang Lianghui,Wang Shuhao,Lu Yazhou,Bao Yingling,Zhu Linjiang,Chen Hanchi,Chen Xiaolong
Enzyme engineering drives green biocatalysis but lacks
diagnostic
tools to distinguish transport-limited from binding-limited catalytic
bottlenecks, causing unnecessary trial and error. We developed the
combined pocket-tunnel diagnosis (CPTD) framework to identify the
rate-limiting step. It integrates static structural analysis with
molecular dynamics to diagnose geometric and energetic barriers in
the substrate transport and binding. Applying this to glycosyltransferase Bs YjiC for the synthesis of bulky sterol glycosides revealed
a steric tunnel bottleneck. A compact 44 mutant library achieved 5.5-fold,
9.4-fold, and 4.3-fold activity enhancements for DHEA, pregnenolone,
and diosgenin, respectively. Subsequent cavity engineering boosted
diosgenin activity 9.29-fold and expanded the substrate scope to cholesterol.
Comparative validation confirmed diagnostic accuracy, as a pocket-first
strategy failed for both DHEA and pregnenolone and yielded only marginal
gains for diosgenin. In contrast, the same pocket mutations produced
substantially greater enhancements after the tunnel bottleneck was
removed.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2026
·
IF 6.7
Ouyang Binbin,Huang Zhaolin,Song Chenshuo,Yin Dejing,Ni Dawei,Zhang Wenli,Rao Yijian,Xu Wei,Mu Wanmeng
Zearalenone (ZEN) lactonases are promising biocatalysts
for ZEN
detoxification, yet the catalytic mechanism underlying ZEN hydrolysis
remains poorly understood. Here, we combined structural analysis,
quantum-mechanical (QM) calculations, and molecular dynamics (MD)
simulations to elucidate the catalytic mechanism and guide enzyme
engineering. QM and MD analyses identified a near-attack conformation
of the catalytic His245 as essential for proton transfer. Crystal
structure analysis revealed that mutations within the active pocket
enhanced the local hydrophobic microenvironment, thereby optimizing
the reactive conformation through an improved substrate positioning
and catalytic residue alignment. Engineering the hydrophobic microenvironment
significantly enhanced the activity of ZENM toward multiple substrates.
Transfer of the engineered region to another ZEN lactonase, ZHD101,
also significantly improved the hydrolytic activity, supporting the
potential general applicability of this strategy. Hydrophobic microenvironment
engineering might represent a promising strategy for modulating proton
transfer and provide a potential framework for improving the activity
of ZEN lactonases for food and feed detoxification.
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ACS Omega
·
2026
·
IF 5.2
Jingjing Kuang,Yu An,Jianguo Zhu,Kehua Jiang,Fa Sun,Kun Chen
Interleukin-7 (IL7) plays a pivotal role in T cell biology; however, its therapeutic potential is constrained by its short half-life. Conventional chemical biotinylation frequently results in random modifications that compromise its bioactivity. This study endeavors to establish a prokaryotic system for the site-specific biotinylation of IL7 utilizing AviTag and underscores its advantages over random biotinylation in retaining functionality. A recombinant plasmid (pET-Dual-His-IL7-avi-birA) was constructed to coexpress AviTag-fused IL7 and BirA ligase, which was subsequently transformed into Escherichia coli BL21(DE3). Optimal expression conditions were determined as follows: cultivation at 37 °C with shaking at 200 rpm for 12 h following induction with 0.5 mM IPTG in the presence of 80 μM biotin. The expressed protein, predominantly localized in inclusion bodies, was purified using Ni-NTA resin supplemented with 250 mM imidazole. Biotinylation efficiency was confirmed through Western blot analysis and native polyacrylamide gel electrophoresis (PAGE). Functional characterization encompassed T cell proliferation and apoptosis assays employing CCK-8 methodology and flow cytometry, respectively, alongside comparative analyses against chemically randomized biotinylated IL7. Site-specifically biotinylated IL7 (biotin-IL7) was successfully generated and purified. It specifically bound streptavidin and retained T cell proliferative activity comparable to native IL7. Compared with randomly biotinylated IL7, site-specific exhibited stronger induction of T cell proliferation, less interference with antibody binding, and more effective downregulation of T cell apoptosis. The AviTag/BirA-based biotinylation system provides an efficient, economical, and scalable method for producing functional biotin-IL7 with enhanced stability and targeting potential. The key advantages of site-specific over random biotinylation were confirmed: preservation of IL7’s bioactivity, reduction of functional interference, and enhancement of therapeutic efficacy. This approach offers a paradigm for optimizing small therapeutic proteins, particularly in cancer immunotherapy, while highlighting the need to improve soluble expression and biotinylation efficiency.
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Journal of the American Chemical Society
·
2025
·
IF 15.6
Jiawei Guo,Qiaoyu Zhang,Yang Shen,Fangyuan Cheng,Moli Sang,Xuan Wang,Yunjun Pan,Mingyu Liu,Hao-Bing Yu,Bo Hu,Sheng Wang,Liangzhen Zheng,Ce Geng,Chaofan Yang,Lianzhong Luo,Gang Zhang,Lei Du,Yuanning Li,Wei Zhang,Yandong Zhang,Binju Wang,Shengying Li,Xingwang Zhang
Thiolactomycin (1), which features a unique γ-thiolactone ring, is a promising antibiotic candidate that specifically targets bacterial type II fatty acid synthase. Despite extensive studies on its pharmacological activities, modes of action, and chemical synthesis, the enzymatic processes responsible for forming the activity-determining γ-thiolactone ring have remained largely unknown. Here, we resolve this problem by revealing that the condensation and heterocyclization (Cy) domain of the nonribosomal peptide synthetase (NRPS) TlnC (TlnCCy), along with the cytochrome P450 enzyme TlnA, cooperatively enable the γ-thiolactone assembly. TlnCCy mediates an unusual sulfurtransfer reaction to sulfurate the polyketide intermediate, generating a thiocarboxylate intermediate. Subsequently, TlnA acts as a γ-thiolactone synthase, converting the linear thiocarboxylate intermediate into 1 via a distal radical-based cyclization mechanism. These findings not only expand the functional and catalytic repertoires of NRPS Cy domains and P450 enzymes but also highlight a special enzymatic strategy for γ-thiolactone biosynthesis in nature.
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BIORESOURCE TECHNOLOGY
·
2025
·
IF 9
Yi Zheng,Yingbo Yuan,Yuanfei Han,Tianyuan Su,Qingsheng Qi
The annual increase in textile consumption has led to significant waste generation, resulting in severe environmental pollution and resource waste. Particularly for the most common polyester/cotton blended fabrics, the composition is intricate, and recycling is even more challenging. In this study, a recyclable chemical-enzymatic hydrolysis cascade was developed to degrade and achieve closed-loop recycling of blended fabrics. The recyclable organic acid, p -toluenesulfonic acid (TsOH), was employed as a catalyst for hydrothermal treatment. A low concentration (0.5 %) of TsOH efficiently broke down cotton fibers under mild conditions, achieving a microcrystalline cellulose (MCC) conversion rate of over 70 %. The separated polyester fibers were hydrolyzed into monomers by PET hydrolase, achieving a maximum specific space–time yield of 149.6 g TPAeq. L –1 h −1 g enzyme –1 . For blended fabrics with varying proportions, this process exhibits high degradation efficiency and generates no secondary pollution, providing a potential route for the industrialization of textile waste closed-loop recycling.
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Current Protocols
·
2025
·Vol. 5
·期 5
·e70155
·
IF 2.2
Yiming Zhao,Jingjun Hong
The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer . Each histone octamer is composed of two copies each of the histone proteins H2A , H2B , H3 , and H4 . Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from Escherichia coli (E. coli) , their subsequent reconstitution as octamers, and assembly into mononucleosomes in vitro . Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC. Basic Protocol : Purification of recombinant histones and mononucleosome assembly
研究领域:
生物化学
·
分子生物学
·
结构生物学
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Current Protocols
·
2025
·Vol. 5
·期 5
·e70151
·
IF 2.2
Yue Liu,Wansen Tan,Jingjun Hong
Chromatin remodeling serves as a critical mechanism for gene regulation, enabling the replacement of canonical histones with their variants through the assistance of histone chaperones. Our study focused on the process by which the histone variant H2A.Z replaces H2A, mediated by the mammalian SRCAP chromatin remodeling complex subunit hYL1. Previous approaches typically involved separately expressing the two proteins and validating their interaction in vitro . In contrast, we designed an artificial single-chain hH2B-hH2A.Z (hTBZ) construct, co-transformed the hYL1 gene with hTBZ, and co-expressed the proteins in E. coli BL21(DE3). We then performed purification, fast protein liquid chromatography (FPLC), and SDS-PAGE analysis. This approach successfully yielded the complexed proteins, confirming the in vivo interaction between hYL1 and hTBZ. © 2025 Wiley Periodicals LLC. Basic Protocol : Co-transformation of hTBZ/YL1 into BL21 (DE3) competent cells, followed by induction and acquisition of protein
研究领域:
生物化学
·
分子生物学
·
遗传学
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Penghui Zhu,Bochen Pan,Chen Li,Benwei Zhu,Zhong Yao,Yun Sun
Green algal polysaccharides such as ulvan and Enteromorpha polysaccharides (EPs) with versatile physiological activities have drawn increasing attentions as a renewable biomass resource. However, their activities and applications have been greatly limited by its high molecular weight and low solubility. Herein, a novel polysaccharide lyase (PL) family 25 ulvan lyase, termed EPD2, has been identified, heterologously expressed and biochemically characterized to efficient preparation of ulvan and Enteromorpha oligosaccharides. It consisted of 446 amino acids with a molecular weight of 50.28 kDa and shared the highest identity with the ulvan lyases of PL25 family. The EPD2 exhibited optimal activity (1764.95 U/mg and 179.9 U/mg for ulvan and EPs, respectively) at 50 °C. It also exhibited robust salt tolerance and could maintain high activity under high NaCl concentrations (0.4–1.2 M). The molecular docking results indicated that EPD2 produce oligosaccharides with degrees of polymerization (DPs) of 2–4 in an endolytic manner. To expand its applications, EPD2 was then immobilized onto sodium alginate and its structure was characterized. After immobilization, its stability at 40 °C was significantly enhanced. This research enriched the enzyme tools library for degrading Enteromorpha polysaccharide and facilitates the expanded application of these enzymes in preparation of oligosaccharides.
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Frontiers in Immunology
·
2025
·
IF 5.9
Pan Zhou,YanLi Yu,WeiYu Qi,XiaoJuan Wang,YouLi Yu,JianDong Wang,Li Zhang,ZhengQing Yu,TingLi Liu
Introduction: Toxoplasma gondii (T. gondii), as a strict intracellular parasite, can infect nearly all mammals, including humans, posing significant threats to public health. Toxoplasmosis in animals also leads to substantial economic losses in animal husbandry. Currently, no effective treatments are available for toxoplasmosis, creating an urgent need for safe and efficient therapeutics.Methods: In this study, we constructed a subunit vaccine using T. gondii glidesome-associated protein 45 (TgGAP45). To enhance immunogenicity, poly (lactic-co-glycolic acid) (PLGA) nanoparticles were employed as delivery carriers to prepare TgGAP45-PLGA nanospheres. For comparison, two oil adjuvants, Montanide™ ISA 660 VG and Montanide™ ISA 206 VG, were used to formulate TgGAP45-206VG and TgGAP45-660VG emulsions. Following safety evaluation, protective immunity was assessed in animals. Antibody levels, cytokine profiles, dendritic cell (DC) maturation and differentiation, and T lymphocyte proliferation and differentiation were analyzed.Results: The results demonstrated that TgGAP45-PLGA nanospheres induced a mixed Th1/Th2 immune response against T. gondii. Furthermore, parasite burden analysis in spleen and heart tissues revealed that TgGAP45-PLGA nanospheres provided the strongest immunoprotection among the tested vaccines.Discussion: These findings indicate that TgGAP45 delivered via PLGA nanospheres is a promising candidate for preventing acute toxoplasmosis. Further studies and applications are warranted to explore its full therapeutic potential.
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CATALYSIS LETTERS
·
2025
·Vol. 155
·期 7
·1-12
·
IF 2.4
Wang Li,Song YingJie,Guo Yi,Chen Guobing,Zhang Hua,Liang Changhai
Cyanoacetamides(CAAM) are key precursors for the synthesis of agrochemicals and bioactive organic compounds. The activity of nitrile hydrolase (NHase) on nitrile substrates was greatly improved in our previous studies by optimizing the ribosome binding site and α/β subunit expression balance. However, during the NHase-catalyzed hydration of malononitrile, this increased catalytic activity greatly facilitated the further conversion of the intermediate cyanoacetamide to the by-product malonamide, thus preventing the enrichment of cyanoacetamide. To solve this problem, we designed a mutant library of NHase by substrate channel modification based on the strategy of semi-rational design. Among them, the βM40A mutant showed 7.68% higher regioselectivity than wild-type NHase. Structural and mechanistic analyses showed that the mutant (1) remodeled the substrate binding pocket, (2) narrowed the substrate channel, and (3) increased the metal ion-carbonitrile distance by 1.1 Å. Our results suggest that the enzyme’s regioselectivity for the product can be controlled by rational design of key amino acids in the NHase substrate channel, which provides an effective strategy for enrichment of the monocyanamide product. Graphical
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Naying Zhang,Zefeng Ren,Dongsheng Wei,Meng Yang,Meng Niu,Chunxiao Shen,Xuanyan Jin,Maolin Wei,Jaehwan Choi,Myeongsam Park,Zhengqun Li
Superoxide dismutases (SODs) are essential enzymes that protect cells by converting harmful superoxide radicals into oxygen and hydrogen peroxide. However, their application in industrial and medical settings is often limited by poor stability under extreme conditions such as high temperatures, extreme pH, and oxidative stress. In this study, we identified and characterized two highly stable manganese-dependent SODs from extremophiles. These enzymes remained highly active at temperatures up to 80 °C, across a broad pH range (3.0–12), and in the presence of oxidative agents and denaturing chemicals. Structural analysis revealed that their exceptional stability is attributed to tightly folded structures, strong hydrophobic cores, and robust metal-binding sites. Both Os SOD and Gt SOD demonstrated robust catalytic performance, with k ca t k m values of 2.29 × 10 7 and 1.06 × 10 7 M −1 ·s −1 , respectively. In cell-based models, they effectively reduced oxidative stress and inflammation, indicating potential for antioxidant therapy. Additionally, their long-term stability supports their use in cosmetic and pharmaceutical formulations. Overall, these extremophile-derived SODs are highly stable and efficient enzymes with broad potential for industrial, medical, and cosmetic applications.
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