JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Xiao-Ting Zhou,Ke-Yan Wei,Shan Li,Qi Shen,Ya-Ping Xue,Yu-Guo Zheng
Nitrilases provide a sustainable route for directly converting nitriles to carboxylic acids. However, efficient biocatalytic synthesis of the agrochemical intermediate 3,6-dichloropicolinic acid(3,6-DCPA) from 3,6-dichloropicolinonitrile (3,6-DCPN) remains unreported. We identified a novel nitrilase from Rhodococcus pyridinivorans (RpNIT) and developed the integrated Feature Cluster-Multi-Force field-Evolutionary Co-Localization (FMEC) strategy to simultaneously enhance activity, thermostability, and specificity. FMEC employs hierarchical clustering, molecular dynamics simulations, and multiforce field algorithms, identifying 19 candidate residues. Using Parallel Iterative Saturation Mutagenesis, we generated the quintuple mutant M5a-DGPAT. This variant exhibited a 17.2 °C increase in melting temperature and a 3.3 × 104-fold improvement in hydrolytic efficiency (kcat/Km) over wild-type RpNIT, while abolishing hydratase activity. Structural analysis attributed the superior performance to an optimized rigidity-flexibility balance, refined substrate binding pocket geometry, and strengthened interaction networks. M5a-DGPAT achieved complete hydrolysis of 347.9 mM 3,6-DCPN to 3,6-DCPA under mild conditions, demonstrating significant industrial potential. This study establishes a robust computational framework for designing highly active, stable, and specific engineered enzymes.
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Nature Communications
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2025
·Vol. 16
·期 1
·1-16
·
IF 15.7
Kang Wei,Wu Zhiyue,Huang Xinzhi,Qi Hongbin,Wu Jiaxuan,Wang Jiahui,Li Jing,Wu Sijin,Kang Byung-Ho,Li Bo,Ma Juncai,Xue Chuang
Biomolecular condensates are dynamic cellular compartments that concentrate proteins and enzymes to regulate biochemical reactions in time and space. While these condensates can enhance enzyme activity, how this function changes as condensates age remains poorly understood. Here, we design synthetic catalytic condensates that selectively recruit enzymes to investigate this temporal evolution. We show that catalytic condensates exhibit time-dependent activity: they initially accelerate enzymatic reactions but gradually lose efficiency due to the transition from liquid-like to solid-like states. This aging process, characterized by protein aggregation and loss of selective barriers, impairs enzyme function both in vitro and living cells. We further demonstrate that small molecules which influence aging dynamics can modulate catalytic efficiency of condensates. Our findings show that condensate aging as a key regulator of enzymatic activity and provide crucial insights for designing functional synthetic condensates.
研究领域:
分子生物学
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生物化学
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细胞生物学
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合成生物学
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ENZYME AND MICROBIAL TECHNOLOGY
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2025
·
IF 3.7
Yingying Zhu,Dawei Ni,Zeyu Li,Zhebin Hao,Liang Wang,Wanmeng Mu
Indigo is an important blue pigment widely used in textile, food, and medicine industries. Biological production of indigo attracts increasing attention recently. Cell factory production of indigo encounters the problem of the toxicity of the precursor indole. Enzymatic production is the alternative biological approach, however, NADPH regeneration should be solved. In this study, flavin-containing monooxygenase from Methylophaga aminisulfidivorans was used for enzymatic production of indigo from indole and formate dehydrogenase from Pseudomonas sp. 101 was co-expressed for NADPH regeneration. Indigo production was enhanced by combination of molecular modification, promoter engineering, and translation initiation region engineering. Finally, 0.183 g/L of indigo was produced from 0.5 g/L of indole and 0.5 mM of sodium formate, with the conversion ratio of 32.5 %. This study demonstrates a feasible and effective strategy for enzymatic production of indigo.
研究领域:
生物催化
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酶促合成
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代谢工程
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工业微生物学
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应用生物化学
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AICHE JOURNAL
·
2025
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IF 4
Hanchi Chen,Shijie Lu,Liying Zha,Yazhou Lu,Qixin Wang,Linjiang Zhu,Xiaolong Chen
This study presents an approach combining molecular dynamics (MD) simulations and near-attack conformation (NAC) analysis to mine and engineer UDP-glucosyltransferases. Using the MD-NAC strategy, we identified that Bs YjiC from Bacillus subtilis possesses menthol glycosylating activity for the first time, and it exhibited a 28% higher k cat / K M value compared to the known l- menthol glycosylating enzyme, Bl YjiC from Bacillus licheniformis . The strategy was further employed to modify Bs YjiC by preventing nonproductive substrate motion and stabilizing NACs through engineering key residues identified by MD. Based on this strategy, 14 positive mutants were identified out of 16 candidates, and the double mutant A76D/V108L (M2) demonstrated a 10.64-fold increase in k cat / K M value compared to the starting point. Whole-cell cascade reactions with sucrose synthase yield 160 mM glucoside after catalyzing for 96 h, demonstrating its application merit. This work underscores MD-driven enzyme engineering as a promising strategy to overcome limitations of static structure-based approaches.
研究领域:
结构生物学
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计算生物学
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酶工程
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生物化学
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合成生物学
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WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
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2025
·Vol. 41
·期 8
·1-13
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IF 4.2
Jiang Cheng,Li Luli,Li Jialu
Chitinolytic enzymes are capable of hydrolyzing the cell walls of pathogenic fungi, disrupting the exoskeletons of insect pests, and producing N-acetyl-β-D-glucosamine oligomers or monomers. In this study, a new β-N-acetylhexosaminidase gene encoding an enzyme from glycoside hydrolase family 20 (GH 20) was identified from the biocontrol fungus Chaetomium globosum W7. The corresponding protein was designated CgNagase20 and was heterologously expressed in Escherichia coli . The molecular mass of the protein was approximately 66.7 kDa, and this enzyme exhibited maximal activity at 55 °C and pH 4.0. Heavy metal ions, particularly Fe 3+ , Ag + , and Hg 2+ , were found to strongly inhibit CgNagase20 activity. CgNagase20 exhibits typical substrate specificity of β-N-acetylhexosaminidase (EC 3.2.1.52), acting on N-acetylglucosides and N-acetylgalactosides, with monosaccharides as the sole end products. The Vmax and Km values for CgNagase20 when acting on 4-nitrophenyl N-acetyl-β-D-glucosaminide (pNP-βGlcNAc) were calculated to be 118.85 µmol/min/mg protein and 1.59 mM, respectively. Additionally, CgNagase20 demonstrated antifungal activity, significantly inhibiting spore germination and hyphal growth of Fusarium sporotrichioides , the causative agent of potato dry rot. In conclusion, CgNagase20 represents the first β-N-acetylhexosaminidase identified and characterized from C. globosum , with potential applications in both the industrial and agricultural sectors.
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Molecular Catalysis
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2025
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IF 4.9
Meichen Zhu,Qinzhe Lin,Mengning Zhong,Jian Dong,Leyi Zhao,Qiyun Wu,Huilin Ren,Wenting Du,Jun Li
Chiral amines serve as vital building blocks in pharmaceutical synthesis, yet their stereoselective production remains a significant challenge. Biocatalytic approaches harness the inherent stereospecificity of enzymes to enable precise, sustainable, and highly selective construction of stereocenters. Here, we engineer an imine reductase (IRED) to catalyze the asymmetric synthesis of (3 S )-3-{[(1 R )-1-(naphthalen-1-yl)ethyl]amino}-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine, the key chiral intermediate of Evocalcet . Computational enzyme design of PcIRED-M3 identified the W219L variant, which enhanced catalytic efficiency 8.9-fold and achieved >99 % diastereomeric excess ( d.e. ) versus 63.15 % in wild-type. The W219L substitution presumably reduces steric hindrance by extending the residue-substrate naphthyl distance from 2.1 Å to 4.7 Å while concurrently improving hydride transfer efficiency. This improvement is also owing to the optimized π-σ orbital alignment with the decreased spacing (from 6.2 Å to 4.9 Å) between the imine carbon and the C-4 position of NADPH’s nicotinamide ring. Reaction parameters optimization (30 % DMSO, K 2 HPO 4 -KH 2 PO 4 buffer pH 7.5, 35 °C) achieved 62.7 % conversion in 48 h. This work establishes the first enzymatic route to Evocalcet intermediates and identifies W219 as a stereochemical gatekeeper residue that allosterically coordinates substrate-cofactor geometry.
研究领域:
生物催化
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酶工程
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有机合成
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药物化学
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计算酶设计
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Yuvaraj Ravikumar,Abdullah Arsalan,Xinrui Tang,Guoyan Zhang,Xianghui Qi
l-Arabinose isomerase (l-AI) catalyzes d-galactose to produce the rare, industrially important sugar d-tagatose. Enzyme stability is vital for its application in industrial processes, and rational design-based protein engineering methods have been employed to improve its stability. This study employed molecular dynamics simulations (MDS)-guided rational redesign of flexible regions to improve the thermostability of mesophilic Bifidobacterium adolescentis l-AI (Ba-l-AI). Comparative MDS (200 ns, 340–360 K) of thermophilic l-AIs (Geobacillus kaustophilus Gk-l-AI and Thermotoga maritima Tm-l-AI) and mesophilic Ba-l-AI identified flexible regions in Ba-l-AI via RMSF and ionic interaction. Five stabilizing mutations (G78C, K46R, N187R, K112R, and N190R) were introduced to provide rigidness. MDS of the mutant revealed reduced RMSF/RMSD, improved compactness, and enhanced conformational stability across temperatures, supported by free-energy landscape analysis resembling thermophilic profiles. Experimentally, the mutant exhibited superior thermal stability (72 °C), broader pH/temperature tolerance, and efficient d-tagatose production. The redesigned Ba-l-AI’s rigid backbone and stability highlight its potential for industrial d-tagatose biosynthesis.
研究领域:
生物催化
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蛋白质工程
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结构生物学
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工业生物技术
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计算生物学
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酶学
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Synthetic and Systems Biotechnology
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2025
·
IF 4.4
Yuke Qin,Shuting Hu,Yibin Yang,Xiaoqiang Jia
The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P T7-cadO using the Cadmium ions (Cd 2+ )-binding protein binding site cadO with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd 2+ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the lacI and lac operator ( lacO ) as the signal amplifier to construct a dual-input promoter P T7-cadO-lacO-cadO and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of “CadR-P J23100 -P T7-cadO-lacO-cadO -mRFP1-LacI”, with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd 2+ (0.00001-0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd 2+ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd 2+ with a final concentration of 0.001-0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors’ circuits.
研究领域:
生物传感器
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合成生物学
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环境监测
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分子生物学
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基因工程
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Qiang Wang,Hossain M. Zabed,Mei Zhao,Xianghui Qi
Laccases, as multicopper oxidases, play a pivotal role in lignin degradation and hold broad industrial promise in biorefineries, bioremediation, textiles, and pulp and paper processing. However, their use is limited by poor stability under harsh operational conditions. Here, we designed a novel thermostable laccase (WCotA) from Bacillus amyloliquefaciens by combining data-driven mining with rational engineering. WCotA displayed remarkable thermostability and pH tolerance, with an optimal activity at 85 °C and a half-life of 1.0 h. To further improve thermostability, a combinatorial mutation library was constructed and screened, yielding mutant M2. M2 exhibited a prolonged half-life of 2.25 h at 85 °C and enhanced activity of 101.05 U/mg, outperforming the wild type. Structural and molecular dynamics analyses elucidated the mechanisms underlying its improved thermostability. This work provides a robust strategy for laccase discovery and engineering, advancing their sustainable application in industrial processes.
研究领域:
酶工程
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分子生物学
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生物催化
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工业微生物学
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蛋白质工程
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Journal of Environmental Chemical Engineering
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2025
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IF 7.2
Wenwei Tang,Meihuan Tong,Hui Liao,Peiqi Zhang,Xinping Zeng
Aureomycin (CTC) is a commonly used tetracycline antibiotic that causes water and soil pollution and greatly endangers the environment and human health. Microbial degradation to remove residual antibiotics in wastewater is of growing interest. Due to its unique structure and excellent oxidative properties, biogenic manganese oxides (BMO) has been widely studied in the field of water pollutant removal. However, the current preparation of BMO generally suffers from low yield and insufficient production efficiency. In this paper, an efficient manganese-oxidizing bacterium Bacillus sp. D-1 was isolated. Manganese-oxidizing engineering bacteria were constructed to achieve rapid and sufficient BMO production, and the structure and function of the BMO was characterized. SEM was used to characterize the layered structure of the nanoparticles. XPS and XRD were used to determine that the mixture was composed of manganese species with three valence states. BMO was used to degrade 10 mg·L −1 CTC, and the CTC removal rate reached 93.2 % in 24 h, with an optimal reaction pH of 7.6. Finally, LCMS and ECOSAR were used to analyze the oxidative degradation products of CTC and their biological toxicity. Therefore, this paper successfully constructed a high-performance manganese-oxidizing engineering bacterium, revealed the efficient degradation mechanism of CTC by BMO, provided a new strategy for the green treatment of combined pollution of manganese and antibiotics in water bodies, and realized the continuous removal of manganese and water pollutants.
研究领域:
环境微生物学
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环境工程
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分子生物学
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污染物生物修复
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水处理技术
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高影响因子
ACS Applied Materials & Interfaces
·
2025
·
IF 8.2
Ziyu Kuai,Yibing Huang,Fengjie Hao,Wantong Jiang,Mengmeng Niu,Yu qin,Ao Song,Yuran Deng,Youzhong Wan,Quan Luo
Atomic precision synthesis and modulation of nanozymes are critical for evaluating their performance and developing functional applications. Herein, we developed an in situ strategy for the controlled synthesis of gold clusterzymes (AuCEs) within the confined cavities of the protein scaffolds. Using a stable dodecameric protein (SP1) as a ligand, we fused a short peptide containing the CCY sequence (SP1-CCY) to the N-terminus of SP1, which is located within its cyclic structure. In this design, cysteine and tyrosine synergistically chelate and reduce Au ions, while the confined protein structure restricts cluster growth, leading to the formation of a 25-gold atom (Au25) nanocluster with strong fluorescence and peroxidase-like activity. Unlike chemical ligands, protein scaffolds offer atomic-level precision and allow for site-specific modulation of the local environment through rational mutations, such as L9D, L9I, and E73I/L9I. This allows precise control of both fluorescence and enzymatic activity via protein engineering. Additionally, heteroatom doping of the Au core with copper endows the AuCEs with significant superoxide dismutase-like activity. Antioxidant assays demonstrate that the optimized AuCEs exhibit potent reactive oxygen species scavenging activity, providing significant protection to Caenorhabditis elegans from oxidative stress and highlighting their potential as therapeutic candidates for aging-related diseases.
研究领域:
纳米医学
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生物材料学
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分子生物学
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老年病学
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抗氧化治疗
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纳米酶
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转化医学
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MOLECULAR NEUROBIOLOGY
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2025
·
IF 4.3
Chen Xue,Zhou Xing-Yue,Lan Cai,Fu Hai-Jun,Li Zhi-Chao,Chen Meng-Yi,Wen Yong-Ping,Yu Lu,Qin Da-Lian,Wu An-Guo,Wu Jian-Ming,Zhou Xiao-Gang
Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by pathological protein aggregation and oxidative stress, leading to progressive neurodegeneration. Enhancing autophagy, the primary intracellular pathway for clearing misfolded proteins, represents a promising therapeutic strategy. In this study, we identify Araloside A (ARA), a triterpenoid saponin derived from Aralia elata , as a potent autophagy inducer that alleviates AD- and PD-related pathology. In neuronal cell models, ARA promotes autophagosome formation, increases LC3-II and Beclin-1 levels, and decreases P62, indicating enhanced autophagic activity. Mechanistic investigations reveal that ARA directly binds to Raf, MEK, and ERK proteins and activates autophagy in a Raf/MEK/ERK-dependent manner. This activation facilitates the clearance of APP, total Tau, phosphorylated Tau, and α-synuclein, thereby reducing cytotoxicity. Furthermore, in transgenic Caenorhabditis elegans models of AD and PD, ARA treatment alleviates protein aggregation and behavioral deficits via ERK-dependent autophagy. Together, these findings identify ARA as a natural compound that enhances autophagic clearance of neurotoxic aggregates via Raf/MEK/ERK pathway activation, offering promising therapeutic insights for neurodegenerative proteinopathies.
研究领域:
神经科学
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药理学
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分子生物学
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神经退行性疾病
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细胞生物学
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天然产物化学
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高影响因子
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