Catalysts
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2024
·Vol. 14
·期 12
·911
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IF 3.8
Shen Yu,Xue-Yong Li,Wen-Jie Si,Ye-Wang Zhang
Formolase, a thiamine pyrophosphate (TPP)-dependent enzyme, catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. It has many important functions in the biosynthesis of carbon-based compounds and utilization of CO2. However, the enzyme has low activity and stability in the catalytic process, resulting in high cost in the applications. To improve the stability, formolase was immobilized onto magnetic nanoparticles, which were designed to have functional epoxy groups for covalently binding the enzyme. In the immobilization, effects of pH, temperature, and cofactor TPP on the immobilization were investigated and optimized. The results showed that the retention activity of immobilized formolase was highly related to TPP. In the presence of TPP, the specific activity of the immobilized formolase was 6.8 times higher than that without TPP. The optimal immobilization conditions were as follows: a temperature of 20 °C, a pH of 7.0, an immobilization time of 8 h, and an enzyme loading of 20 mg/g. Molecular docking was used to analyze the effect of TPP on the stabilization of the enzyme in the immobilization, which indicated that TTP could stabilize the enzyme structure during the immobilization. The stabilization effect of TPP could be a reference in the immobilization of other enzymes with TPP as the cofactor.
研究领域:
生物化学
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酶工程
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CURRENT ISSUES IN MOLECULAR BIOLOGY
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2024
·Vol. 46
·期 9
·9916-9927
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IF 2.8
Miao Tian,Yuan-Yuan Xu,Yang-Nan Li,Shen Yu,Yi-Lin Wang,Xiao-Lai Ma,Ye-Wang Zhang
Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.
研究领域:
生物工程
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酶学
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医药生物技术
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FOOD CHEMISTRY
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2024
·
IF 8.5
Weijun Leng,Ying Li,Xin Liang,Li Yuan,Xiuting Li,Ruichang Gao
Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp 31 , Ser 72 , Asn 121 , Asp 471 , and Glu 485 were crucial for maintaining PG5-myosin interaction, with Ser 72 and Asn 121 making significant energy contributions.
研究领域:
食品科学
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酶工程
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蛋白质工程
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计算生物学
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·Vol. 90
·期 9
·
IF 3.9
Yuan Li,Shu-Fang Li,Lin Zhang,Ya-Ping Xue,Yu-Guo Zheng
Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L−1 d−1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis.
研究领域:
生物技术
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酶工程
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生物化学
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化学生物学
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Cell Systems
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2024
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IF 9
Runtao Zhu,Jiao Zhang,Lin Wang,Yunfeng Zhang,Yang Zhao,Ying Han,Jing Sun,Xi Zhang,Ying Dou,Huaxiong Yao,Wei Yan,Xiaozhou Luo,Junbiao Dai,Zhuojun Dai
Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper’s transparent peer review process is included in the supplemental information .
研究领域:
生物技术
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生物医学工程
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合成生物学
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Jingyu Zhao,Huanhuan Sun,Gege Wang,Qi Wang,Yipeng Wang,Qingbin Li,Shuangyu Bi,Qingsheng Qi,Qian Wang
Two-component systems (TCSs) sensing and responding to various stimuli outside and inside cells are valuable resources for developing biosensors with synthetic biology applications. However, the use of TCS-based biosensors suffers from a limited effector spectrum, hypersensitivity, low dynamic range, and unwanted signal crosstalk. Here, we developed a tailor-made Escherichia coli whole-cell γ-aminobutyric acid (GABA) biosensor by engineering a chimeric GABA chemoreceptor PctC and TCS. By testing different TCSs, the chimeric PctC/PhoQ showed the response to GABA. Chimera-directed evolution and introduction of the insulated chimeric pair PctC/PhoQ*PhoP* produced biosensors with up to 3.50-fold dynamic range and good orthogonality. To further enhance the dynamic range and lower the basal leakage, three strategies, engineering of PhoP DNA binding sites, fine-tuning reporter expression by optimizing transcription/translation components, and a tobacco etch virus protease-controlled protein degradation, were integrated. This chimeric biosensor displayed a low basal leakage, a large dynamic range (15.8-fold), and a high threshold level (22.7 g L–1). Finally, the optimized biosensor was successfully applied in the high-throughput microdroplet screening of GABA-overproducing Corynebacterium glutamicum, demonstrating its desired properties for extracellular signal biosensing.
研究领域:
合成生物学
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生物技术
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分子生物学
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微生物学
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PubMed
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ACS Sustainable Chemistry & Engineering
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2024
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IF 7.1
Tong Zhang,Shubin Li,Lei Chen,Weiwen Zhang,Tao Sun
Cyanobacteria are known to be photoautotrophic cell factories capable of converting CO2 into valuable chemicals. The newly discovered marine cyanobacterium Synechococcus sp. PCC 11901 (hereafter PCC 11901) offers several advantages like rapid growth, high biomass, and high salinity tolerance, representing a promising chassis. To promote its application, we developed genetic toolboxes applicable to PCC 11901 in this study. First, a cobalamin (VB12)-independent chassis was constructed, allowing for cheaper cultivation. Second, genome copy numbers and transformation methods were, respectively, measured and optimized. Then, 14 neutral sites were identified and characterized within the genome PCC 11901, providing locations for genetic integration of exogenous cassettes. Subsequently, promoter libraries were developed, reaching an expression range of approximately 800 folds for constitutive promoters and an induction fold of up to approximately 400 for inducible promotors, respectively. As a proof of concept, natural production of the total lipid and phycocyanin was investigated using VB12-independent chassis, which realized an increase of 14.91% with lipid content compared with that of the wild-type strain. Further, we engineered the synthetic pathways of glucosylglycerol (GG) into PCC 11901 using the established toolboxes, reaching 590.41 ± 21.48 mg/L for GG production and self-sedimentation in photoreactors with the highest OD750 nm at 17.57 ± 0.77. Finally, the GG-producing strain grew well in seawater, reaching 324.50 ± 5.34 mg/L in shaking flask, which provided new strategies for cyanobacteria cultivation and production. Our work here made it possible to develop the fast-growing PCC 11901 as efficient carbon-neutral cell factory in the future.
研究领域:
合成生物学
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微生物学
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生物工程
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
·
IF 8.5
Xiaotong Xu,Xiaoshuang Yan,Xuerong Jin,Juyi Li,Qiaoxia Hu,Dong-Hyun Ahn,Chao Zhong
Colanic acid (CA), an exopolysaccharide synthesized by bacteria, is known for its potential to extend the lifespan of nematodes. However, its high viscosity poses practical application challenges, which can be mitigated by employing colanic acid-degrading enzymes (CAEs). This study presents the expression and characterization of a novel CAE from Escherichia phage phi92, used for the production of CA oligosaccharides (CAOSs). Optimal CAE expression conditions were identified as induction at 20 °C for 24 h with 0.1 mM IPTG. The enzyme showed maximal activity at 55 °C and pH 6.0, with stability in the range of 4–50 °C and pH 4.0–7.0. Structural characterization of CAOSs was performed using GC–MS, LC–MS and NMR spectroscopy, revealing the composition of hexasaccharide and dodecasaccharide units, with CAE cleaving the β-1,4 glycosidic linkage between glucose and fucose. CAOSs were found to mitigate oxidative stress and inflammation induced by H 2 O 2 in RAW 264.7 macrophages, inhibiting NO and MDA production while enhancing CAT activity. Additionally, CAOSs modulated the mRNA expression of pro-inflammatory (IL-1β, COX-2) and anti-inflammatory (IL-10, HO-1) factors. This study deepens the understanding of CAE and facilitates the preparation of CA oligomers for applications in food, cosmetics, and pharmaceuticals.
研究领域:
微生物学
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生物化学
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医药科学
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BIOORGANIC CHEMISTRY
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2024
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IF 4.5
Muhammad Waheed Iqbal,Xinrui Tang,Tahreem Riaz,Shahid Mahmood,Yufei Zhang,Mei Zhao,Junhua Yun,Jia Li,Xianghui Qi
6-Deoxy- l -sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l -fucose was identified. The genes for l -fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d -tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli , developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co 2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (T m ) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l -fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l -fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes.
研究领域:
生物技术
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酶学
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生化工程
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食品科学
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PubMed
高影响因子
Current Protocols
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2025
·Vol. 5
·期 12
·e70284
·
IF 2.2
Jinwei Zhai,Wansen Tan,Lei Ji,Jingjun Hong
This article mainly describes a fermentation and purification method for expressing recombinant collagen protein in Escherichia coli . The method comprises constructing engineered bacteria expressing human type III collagen and adopting a strategy of feeding in batches for high-density fermentation. The rapid proliferation of bacterial cells is promoted at 37°C, and then the culture is inoculated into a fermentation tank with different carbon sources for growth. When glycerol is used as the main carbon source, the yield of recombinant collagen protein can reach 0.25 to 0.40 g/L. The method allows exploration of the differences in recombinant collagen production with different carbon sources in order to identify the most suitable fermentation medium component. The human type III collagen produced by the method has the typical structure of collagen, with high cell adhesion and the stability of tissue structure. Therefore, it can be used as the raw material for various collagen products, especially facial fillers, dressings, freeze-dried fibers, and gels. © 2025 Wiley Periodicals LLC. Basic Protocol : Fermentation and purification of recombinant human type III collagen expressed in Escherichia coli
研究领域:
生物技术
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分子生物学
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蛋白质工程
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工业微生物学
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PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
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2025
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IF 4
Yuhan Zhao,Xiaojie Zhou,Mei Li,Xinghui Qiu
The Asian tiger mosquito Aedes albopictus transmits several arboviruses, including chikungunya, Zika, and dengue, posing serious public health threats. The excessive use of insecticides has led to increasing resistance to commonly applied insecticides in this species. We previously observed that enhanced expression of a CYP6Z8 homolog (AalbCYP6Z8) was associated with insecticide resistance in two field populations of Ae. albopictus ; however, its capacity in insecticide metabolism had not been biochemically characterized. In this study, to explore the potential role of AalbCYP6Z8 in insecticide metabolism, a fully functional enzyme system was reconstituted in vitro by using Escherichia coli –produced recombinant AalbCYP6Z8 and NADPH-dependent cytochrome P450 reductase along with cytochrome b 5. Our metabolism assays showed that CYP6Z8 was capable of metabolizing organophosphates (temephos and malathion) and carbamates (propoxur and bendiocarb), but not pyrethroids (deltamethrin and permethrin). Mass spectrometry analyses tentatively identified the structures of specific metabolites, revealing that AalbCYP6Z8 transforms organophosphates through oxidative desulfurization and carbamates through dealkylation and hydroxylation. This work demonstrates that AalbCYP6Z8 is a versatile metabolizer of acetylcholinesterase-inhibiting insecticides.
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Cell Communication and Signaling
·
2025
·Vol. 24
·期 1
·6
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IF 8.9
Yang Xinyi,Ning Tingting,Zhang Wanru,Hou Songyuan,Su Jiayi,Li Wenkun,Wu Jing
Background Gastric microbiota dysbiosis is closely linked to chronic inflammation and carcinogenesis. Fusobacterium nucleatum ( Fn ), an opportunistic gastrointestinal pathogen, is implicated in perturbing the immune system and form an inflammatory microenvironment that fuels the occurrence and progression of gastric cancer (GC). However, the underlying mechanisms remain elusive. We aimd to investigate the impacts of Fn on GC cells and the crosstalk between GC cells and mast cells (MCs). Methods Fn abundance and MC infiltration were assessed in clinical GC tissues. Cell-based functional experiments, including wound healing, transwell, and ELISA, were performed using conditioned medium-based co-culture systems to explore how Fn influenced GC cell migration and the crosstalk with MC. A murine lung metastasis model was employed to evaluate Fn -driven metastasis and the effects of metronidazole and Reparixin. Results We showed that Fn and MCs were enriched and positively correlated in clinical GC tissues compared to paracancerous tissues. Fn and MCs exhibited tumor-promoting properties by facilitating GC cell migration. Besides, Fn and GC cells can recruit MCs and facilitate its activation. This crosstalk between GC cells and MCs was further enhanced by Fn , thereby amplifying this positive feedback loop between them. The production of CXCL8 in response to Fn stimulation was a significant mediator. Suppression of CXCL8 in GC cells weakened Fn ’s promoting effects on GC cell migration, MC recruitment and activation. Similarly, knocking down CXCL8 in MCs reversed the Fn -induced feedback effect of MCs on the highly invasive phenotype of GC cells. Mechanistically, the exoprotein Gbp of Fn activated CypA-NF-κB pathway in both GC cells and MCs, leading to the upregulation of CXCL8 expression. In addition, the in vivo assays substantiated Fn ’s ability to enhance tumor progression by facilitating cell migration, upregulating CXCL8 expression, and inducing MC infiltration and activation. However, these effects were reversed by metronidazole, a highly Fn -susceptible antibiotic, and Reparixin. Conclusions In summary, this study suggests that Fn contributes to GC progression by promoting tumor cell migration, MCs recruitment and activation. Simultaneously, the enhanced CXCL8-mediated crosstalk between GC cells and MCs plays a vital role in the pro-cancer effect of Fn.
研究领域:
肿瘤学
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微生物组学
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免疫肿瘤学
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分子肿瘤学
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炎症与癌症
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信号转导
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高影响因子
危险品化学品经营许可证（带存储）