JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Qiong Wang,Mei Zhao,Hossain M. Zabed,Xinrui Tang,Jiaojiao Chen,Jiayin Wang,Haoyang Li,Xianghui Qi
The discovery of novel heat-resistant lipases has the potential to broaden their applications in the food industry and other fields. This study identified a novel heat-resistant lipase PFHL from Pseudomonas fluorescens HK44 based on data-driven mining, which was subsequently expressed in Escherichia coli for its molecular insights into substrate specificity. The purified PFHL demonstrated optimal activity at 60 °C and pH 7.0 with a half-life of 2.52 h and retained 48.23% of its relative activity at 90 °C. The enzyme exhibited a substrate preference for medium-chain fatty acid esters, displaying a catalytic efficiency kcat/Km of 134.31 mM–1·min–1 toward 4-nitrophenyl laurate (pNPC12), 1.45-fold higher than that for 4-nitrophenyl palmitate (pNPC16). Additionally, molecular docking and molecular dynamics simulations were performed to elucidate the mechanism underlying its substrate specificity and catalytic efficiency. The results revealed that pNPC12 fit the catalytic pocket better than pNPC16, and the complex exhibited greater stability during the simulation. Up to seven hydrogen bonds were formed, and the substrate was able to rapidly reach and position itself within the catalytic pocket. These findings offer valuable insights into PFHL’s substrate preferences, laying the groundwork for its rational design and further optimization to enhance its suitability for industrial applications in the future.
研究领域:
生物技术
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酶学
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结构生物学
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工业酶
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食品科学
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计算生物学
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DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2024
·
IF 9.7
Yizhen Zhang,Yu-Cai He,Cuiluan Ma
Lignin is a plentiful and readily accessible renewable resource. Vanillylamine is a crucial raw material used to synthesize pharmaceuticals and high-value furan compounds that can be acquired by aminating lignin-derived vanillin (Van). However, effectually achieving the biocatalytic synthesis of vanillylamine has remained challenging. In this study, a dimethyl sulfoxide (DMSO)-H 2 O (1:9, vol/vol) bioreaction medium was constructed, and a recombinant E. coli ATA1012 carrying ω-transaminase from Caulobacter sp. D5 was used as the ω-transaminase biocatalyst to acquire the effectual biocatalytic synthesis of vanillylamine. Under optimized bioreaction conditions (37 ℃ and pH 7.5) by supplementary of isopropylamine (IPA) (Van/IPA = 1:5, mol/mol), 80–100 mM Van could be effectually converted by ATA1012 whole cells in DMSO-H 2 O (1:9, vol/vol) within 12 h, yielding 91.2 %–95.4 % vanillylamine, with >99 % selectivity. An efficient amination process was developed using ATA1012 with superior transaminase catalytic activity and substrate tolerance to effectively convert Van to vanillylamine in a DMSO-H 2 O medium.
研究领域:
生物技术
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生物化学
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有机化学
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药学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Ziqi Liang,Qiao Zhou,Yicheng Li,Xiaoqing Liu,Yiwen Shen,Jian Tian,Xiaolu Wang,Xing Qin,Yuan Wang,Huiying Luo,Xiaojun Yang,Bin Yao,Tao Tu
Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction of self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization to regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic optimization of reaction conditions, including cosolvents, cell concentrations, and plasmid selection, to enhance catalytic performance. Under optimized conditions, the heme domain Vdh-K1 achieved a 91.6% conversion efficiency and was subsequently selected for chimeric enzyme assembly. By employing local energetic frustration analysis to evaluate protein flexibility and allosteric dynamics, we identified chimeric P450 variants with highly frustrated linkers. The optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, catalytic activity, and coupling efficiency, achieving a yield of 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli whole-cell catalysis─the highest reported yield to date. This work underscores the utility of computational frustration analysis in refining linker dynamics for multidomain enzymes and establishes a scalable, cost-effective framework to advance P450s systems for industrial biosynthesis of high-value compounds.
研究领域:
生物化学
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工业生物技术
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酶工程
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产品关联
DOI
PubMed
高影响因子
Processes
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2024
·Vol. 12
·期 9
·2006
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IF 2.8
Wei He,Benshun Chen,Bin Yin,Jianren Ye,Yucai He
Simvastatin is a widely used statin medication that is commonly prescribed to lower cholesterol levels and reduce the risk of cardiovascular events. It is marketed under the brand name Zocor and is known for its effectiveness in treating high cholesterol and managing cardiovascular disease. Monacolin J is an important precursor used to synthesize simvastatin and is mainly produced by chemical methods in industry. Here, monacolin J was synthesized through an enzymatic method under optimized reaction conditions. One recombinantEscherichia coliBL21 (DE3) strain containing lovastatin hydrolase (encoded by CDV55_102090) fromAspergillus turcosuswas constructed, which effectively transformed 100 g/L of lovastatin to monacolin J within 3.5 h at pH 8.0 and 30 °C, with a conversion rate of >99.8%. Furthermore, the T5010, the temperature at which the residual activity was half of the initial enzymatic activity after 10 min of heat treatment, was >50 °C, indicating the tremendous potential of this bioprocess for synthesizing monacolin J at an industrial scale.
研究领域:
生物化学
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制药工程
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工业微生物学
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DOI
高影响因子
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
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2025
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IF 3.1
Lin Ge,Yu Xia,Wenxin Xu,Ruobing Jia,Tingting Zhang
Gastrodin, the primary bioactive constituent of Gastrodia elata , possesses numerous remarkable pharmacological properties. In this investigation, UDP-glycosyltransferase from Rauvolfia serpentina (RsUGT) was expressed, subsequently purified and characterized. The maximum yield of the enzyme was 17.57 mU/ml and possessed a relative molecular weight of approximately 77.7 kDa. Utilizing GST affinity resin, RsUGT was purified 20.8-fold, with an overall recovery rate of 58.6% and specific activity of 79.2 mU/mg. The optimal temperature and pH for RsUGT was identified as 40°C and 10.0, respectively. Notably, 2% DMSO could increase the RsUGT activity by 12.15%. The Michaelis-Menten constants K M and V max were determined to be 0.50mM and 171.60 mU/mg. By optimizing the conditions for the enzymatic biosynthesis of gastrodin by RsUGT, the highest gastrodin production was 285.35 mg/l, accompanied by a molar conversion rate of 99.67%. In addition, the conditions of gastrodin biosynthesis by recombinant strain BL-RsUGT were also studied. The highest gastrodin production was 225.99 mg/l, and the corresponding p HBA conversion rate was 98.00%. These findings confirmed the promising potential of RsUGT in the production of gastrodin.
研究领域:
植物学
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药理学
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生物化学
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DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
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2024
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IF 16.6
Wang Qing,Zhang Jie,Zhao Zhe,Li Yichen,You Jiajia,Wang Yi,Li Xiangfei,Xu Meijuan,Rao Zhiming
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the ‘dock’ of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
研究领域:
微生物学
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遗传学
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合成生物学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2024
·
IF 3.7
Qing Wang,Jiajia You,Yichen Li,Jie Zhang,Yi Wang,Meijuan Xu,Zhiming Rao
In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT74) can increase the mutant frequency of the target gene by 1.12 × 104-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT74 system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT74 to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.
研究领域:
合成生物学
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分子遗传学
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DOI
PubMed
高影响因子
Journal of Chemical Information and Modeling
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2025
·
IF 5.3
Binbin Chen,Jiahui Huang,Yingchun Liu,Lirong Yang,Qi Wang,Haoran Yu
The catalytic mechanism of a pyridoxal 5′-phosphate (PLP)-dependent UstD was herein studied in atomic detail, employing the computational hybrid QM/MM methodology. UstD is a PLP-dependent enzyme that catalyzes the decarboxylative aldol reactions between l-aspartate and aldehyde or ketone derivatives to form γ-hydroxy-α-amino acids. In the reaction catalyzed by UstD, the loss of CO2 renders the C–C bond-forming reaction effectively irreversible, which makes UstD a special case among the enzymes catalyzing the C–C bond-forming reactions. This enzyme is currently seen as the optimal approach for the regioselective synthesis of γ-hydroxy-α-amino acids, which are very difficult to obtain by standard chemical methods. The results obtained herein showed that the catalytic mechanism of UstD might follow two paths to occur in three phases: (1) decarboxylation of substrate l-aspartate, (2) C–C bond formation by addition of aldehyde, and (3) the regeneration of catalytic sites. Although Path A and Path B showed a negligible difference in the energy barrier of the rate-determining step, Path A involves three additional steps in the overall pathway compared with Path B, which makes the reaction proceed more readily through Path B. According to the QM/MM energy profile of Path B, the rate-limiting step of the catalytic process is the decarboxylation of the side chain of l-aspartate, which has a calculated energy barrier of 19.19 kcal/mol. Two crucial residues, H263 and Y257, were identified to interact with the substrate aspartic acid. The knowledge about the transition states, intermediates, key residues, and protein conformational changes along the reaction path will be valuable for engineering UstD to improve the synthesis of γ-hydroxy-α-amino acids that serve as building blocks of various high-value chemicals such as antidiabetics and nutritional supplements.
研究领域:
生物化学
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计算生物学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Zhuangzhuang Huang,Junru Zhou,Jialing Wang,Sheng Xu,Cheng Cheng,Jiangfeng Ma,Zhen Gao
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.
研究领域:
生物化学
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酶工程
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计算生物学
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产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2024
·
IF 4.8
Jingjing Dong,Yanli Wang,Xiaoxiao Li,Ying Chen,Rui Fan,Nana Wang,Zhengyu Jin,Yuxiang Bai
The starch-converting GH70 α-glucanotransferases, as the influential enzymatic tools for the synthesis of structurally diverse α-glucans, are of great value in the food industry. In this work, a new GtfB-type α-glucanotransferase from Fructilactobacillus sanfranciscensis Gs2 (FsGs2 GtfB) was mined from the NCBI database, which is highly conserved in four conserved regions I-IV for GtfB enzymes but possessed distinct characteristics in some residues at acceptor subsites +1/+2. Biochemical characterizations revealed that the FsGs2 GtfB enzyme demonstrated a novel 4,6-α-glucanotransferase activity, which was able to cleave the (α1 → 4) linkages in maltooligosaccharide/starch substrates and mainly synthesize branching (α1 → 4,6) linkages in the gelatinized system, resulting in the generation of low-molecular-mass short-clustered (α1 → 4,6)-glucans. Nevertheless, FsGs2 GtfB acted as a quasi-hydrolase instead of transglucosylase in the granular system, causing a notable decrease in the molecular mass of granular wheat starch and a tendency towards shortened chain lengths. Hence, the FsGs2 GtfB enzyme represents an additional potent enzymatic tool of reassembling the (α1 → 4) and (α1 → 6) linkages in starch molecules and enriches the diversity of α-glucans that can be synthesized.
研究领域:
食品科学
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生物化学
·
酶工程
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DOI
高影响因子
JOURNAL OF DRUG DELIVERY SCIENCE AND TECHNOLOGY
·
2025
·
IF 4.9
Feifei Wang,Dianlong Jia,Qingcui Song,Xiaomeng Lu,Guozi Xia,Jun Li,Xiaodong Jia,Mingliang Gu,Fengjiao Yuan,Zhengping Wang,Zhangyong Xia
Resistance against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death of cancer cells is a major obstacle in clinical application of TRAIL. Bortezomib (BTZ) is the first-generation proteasome inhibitor to enhance TRAIL-mediated apoptosis and overcome tumor cell resistance to TRAIL, therefore we developed a novel nanodrug called LSBTZ-TRAIL to co-deliver TRAIL and BTZ via liposomes. TRAIL was coupled to the surface of the liposome by DSPE-PEG 2000 -NHS, while BTZ was encapsulated in the internal aqueous phase. LSBTZ-TRAIL was characterized with a particle size of 147.00 ± 1.300 nm, polydispersity index (PDI) of 0.056 ± 0.005 and zeta potential of -8.17 ± 0.600 mV. LSBTZ-TRAIL did not change the secondary structure of TRAIL, and LSBTZ-TRAIL was stable in 50% fetal bovine serum and tumor microenvironment. In vitro, the cell killing capacity of LSBTZ-TRAIL was 1.16-6.22 times that of TRAIL + BTZ. Mechanistically, LSBTZ-TRAIL induced apoptosis of tumor cells by up-regulating the death receptor (DR4/5) and activating the caspase signaling pathway. In vivo and in vitro uptake experiments, LS-TRAIL accumulated more in tumor cells or tissues than LS. In vivo , the antitumor effect of LSBTZ-TRAIL was 2.62 times that of TRAIL + BTZ, and prolonged the survival and blood circulation time of mice. In addition, safety evaluation showed that LSBTZ-TRAIL had no acute toxic effects in vivo . These results suggested that the liposomal co-delivery system of TRAIL and BTZ significantly enhance their antitumor effects and provide a new approach to overcome TRAIL-resistance.
研究领域:
肿瘤学
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药学
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分子生物学
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DOI
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
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2024
·Vol. 41
·期 1
·1-12
·
IF 4
Li Yang-Nan,Zhu Chen-Yuan,Xu Chen-Lu,Yu Shen,Huan Tong,Zhang Ye-Wang
A heparinase III ( Ns Hep-III) from Niabella sp . was identified, cloned, and expressed as soluble form in E. coli BL21 (DE3). With heparin as substrate, the maximum activity of Ns Hep-III of 90.0 U·mg − 1 was achieved at pH 7.1 Tris-HCl (containing 15 mM Mg 2+ ) and 30 o C. The half-life of Ns Hep-III was determined to be 5 h at 30 o C. The interactions between Ns Hep-III and substrates were studied by molecular docking. The combination of Ns Hep-III and a heparinase I from Bacteroides eggthii ( Be Hep-I) was employed to cleave heparin. Analysis of the enzymatic products of Ns Hep-III by SAX-HPLC showed seven different modified disaccharides, indicating that Ns Hep-III has a wide range of substrate specificity. The results of GPC analysis demonstrated that the average molecular weight of the product of heparin cleavage by the combination of Ns Hep-III/ Be Hep-I was reduced to 3969 Da, which accounted for 90% of all the components, and complied with the requirements of the European Pharmacopoeia. Ns Hep-III has notable activity and efficiency in cleaving heparin, which is potentially useful for the industrial production of low molecular weight heparin.
研究领域:
生物化学
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药学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）