BIOCHEMICAL ENGINEERING JOURNAL
·
2010
·
IF 2.193
Gangfeng Ren,Hongwei Yu
After analyzing protein surface structure, the opposite side of the activity site of esterase BioH was observed to be hydrophilic. Hence subsequently, a hydrophilic-modified solid support was selected to immobilize enzyme through oriented adsorption. The immobilized enzyme remained 86% of its original activity under the acylation of 1-phenylethanol, much more than those immobilized on other solid supports with low hydrophilicity (20–35.6%), as the mode of the immobilization could facilitate enzyme to expose its active site to the substrate. This demonstrated that the strategy to select solid support based on the analysis of protein surface structure was successful. It was also noticed that the substrate of the reaction was obviously adsorbed onto the support, therefore the adsorption effect could not be ignored and it was necessary to obtain the adsorption isotherm of the substrate for a more authentic result. The parameter K m shifted from 884.56 to 296.09 mM after immobilization. This was possibly mainly caused by the increment of substrate concentration around the enzyme due to the substrate adsorbing on the support. It could be another explanation for why those parameters were changed after enzyme immobilization in addition to some existing proposed reasons such as stabilizing conformation, facilitating dispersion of enzyme, etc.
研究领域:
生物技术
·
酶学
·
蛋白质工程
✓ 已验证
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2024
·
IF 4.1
Kehao Yuan,Zongchao Huo,Ya`ning Zhang,Zuran Guo,Yucan Chang,Yunming Jin,Lining Gao,Tong Zhang,Yanwei Li,Qinyuan Ma,Xiuzhen Gao
During the deamination and amination processes of meso -diaminopimelate dehydrogenase ( meso -DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso -DAP during deamination . Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso -DAP. However, some H154 variants showed enhanced k cat / K m values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved k cat / K m values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.
研究领域:
生物化学
·
蛋白质工程
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2021
·
IF 5.279
Juan Han,Hui Feng,Jiacong Wu,Yuanyuan Li,Yang Zhou,Lei Wang,Peng Luo,Yun Wang
Today, we are seeking an efficient biotransformation of cellulosic material into sustainable biochemical products to meet the increasing global energy demand. Herein, we report the fabrication of multienzyme hybrid nanoflowers (ECG-NFs) by co-immobilizing three recombinant enzymes (cellobiohydrolase (CBH), endo-glucanase (EG), and β-glucosidase (BG)) integrating a binary tag composed of elastin-like polypeptide (ELP) and His-tag to act as a tri-enzyme biocatalyst, which catalyzes the hydrolysis of cellulose into glucose. The prepared ECG-NFs exhibited excellent performance in terms of pH stability, thermal stability, storage stability, and catalytic efficiency compared to free multienzyme system. Notably, ECG-NFs could be recycled for up to eight consecutive runs. The Km and kcat/Km values for ECG-NFs were 9.33 g L–1 and 0.0051 L min–1 g–1, respectively, which were better than those of the free multienzyme system, indicating a better substrate affinity. Finally, the overall enzyme activity of ECG-NFs increased by 1.12 times and the degradation efficiency of ECG-NFs was superior to the free multienzyme system, which revealed that ECG-NFs could facilitate an effective one-pot hydrolysis of cellulose into glucose.
研究领域:
生物化学
·
生物技术
·
材料科学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
·
2021
·
IF 6.291
Zhijian Ke,Qian Zhu,Wankui Jiang,Yidong Zhou,Mingliang Zhang,Mingli Jiang,Qing Hong
Carbaryl is the representative of carbamate insecticide. As an acetylcholinesterase inhibitor, it poses potential threat to humans and other non-target organisms. Agrobacterium sp. XWY-2, which could grow with carbaryl as the sole carbon source, was isolated and characterized. The carH gene, encoding a carbaryl hydrolase, was cloned from strain XWY-2 and expressed in Escherichia coli BL21 (DE3). CarH was able to hydrolyze carbamate pesticides including carbaryl, carbofuran, isoprocarb, propoxur and fenobucarb efficiently, while it hydrolyzed oxamyl and aldicarb poorly. The optimal pH of CarH was 8.0 and the optimal temperature was 30 ℃. The apparent K m and k cat values of CarH for carbaryl were 38.01 ± 2.81 μM and 0.33 ± 0.01 s −1 , respectively. The point mutation experiment demonstrated that His341, His343, His346, His416 and D437 are the key sites for CarH to hydrolyze carbaryl.
研究领域:
环境科学
·
微生物学
·
生物化学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Tianyu Wang,Jianxing Feng,Hao Sun,Yanmin Liang,Ting Du,Jie Dan,Jianlong Wang,Wentao Zhang
Metal aerogel with porous structure and interlocking pore channel can provide abundant active sites to boost the peroxidase (POD)-like activity for high-performance colorimetric analysis of food quality. Herein, CuBi bimetallic aerogels with different atomic ratio (Cu 1 Bi 9 , Cu 5 Bi 5 and Cu 9 Bi 1 ) were fabricated by a facile one-step reduction strategy, and the POD-like activity of the synthesized CuBi bimetallic aerogels was studied. The specific activity of Cu 9 Bi 1 aerogel was significantly enhanced due to the Bi-doping strategy. Through comparing the K m values we find that CuBi bimetallic aerogel can be used as a cheap alternative to equivalent noble metal materials for biosensor applications. Then, the optimized nanozyme was exploited to establish a colorimetric method for the total antioxidant capacity (TAC) by utilizing ascorbic acid (AA) as a representative antioxidant model. The linear range and limit of detection (LOD) were determined to be 300–900 μM and 59.4 μM, respectively, the wide detection range simplifies sample pre-treatment steps. Additionally, the method also demonstrated good feasibility for the TAC of food samples. The current study expands new fields for the application of metal aerogels and successfully developed macroscopic nanozymes with a convenient application, facile preparation and low cost.
研究领域:
纳米技术
·
分析化学
·
食品科学
·
材料科学
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2022
·Vol. 414
·期 22
·6611-6620
·
IF 4.478
Tang Yulian,Lv Xue,Gou Wenxin,Zhou Xuemei,Hao Junkai,Feng Jing,Qi Yuji,Hu Lei,Yan Zhengquan
Dopamine (DA) is an important neurotransmitter; however, any excess or deficiency of DA will cause several diseases in humans. To monitor DA efficiently and conveniently, a Ag nanozyme strengthened by bioactive folic acid (FA@AgNPs) was developed by homogeneous redox assembly. After the microstructure and performance were characterized in detail, it was noted that the proposed FA@AgNPs possessed superior peroxidase-like activity due to the ultra-small Ag nanoparticles and multiple amino, hydroxyl, and aromatic rings in FA. FA@AgNPs accelerated the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) with a low Michaelis constant ( K m ) and high maximal reaction rate ( V max ). Importantly, the characteristic absorbance intensity of FA@AgNPs-TMB-H 2 O 2 at 652 nm ( A 652 ) was exclusively deteriorated in the presence of trace DA, accompanied by a visual color change from blue to colorless. Under the optimized conditions (pH 4.0, 300 μL 1.5 mM TMB, 300 μL 1.0 M H 2 O 2 and incubated for 30 min at room temperature), there expressed an excellent linear relationship between lg A 0 / A 652 and c DA from 1.0 ×10 −8 to 6.67×10 −6 mol/L with a low limit detection of 7.1×10 −10 mol/L (S/N=3). When applied for monitoring of DA in real fruit juice and pharmaceutical samples, the recovery was between 96.6% and 104.9%, with RSD less than 2.2%. The enhanced peroxidase-like activity of the FA@AgNP system and its selective recognition mechanism for DA are also proposed. Graphical abstract
研究领域:
纳米技术
·
生物化学
·
分析化学
·
神经科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOORGANIC CHEMISTRY
·
2024
·
IF 4.5
Xiao-Fang Zhang,Hong-Fei Li,Hui Liu,Fang-Lu Wei,Jiao-Xian Du,Ji-Kai Liu,Juan He,Tao Feng
Twenty-nine sesquiterpenoids, including pseudoguaiane-type ( 1 – 11 ), eudesmane-type ( 12 – 23 ), and carabrane-type ( 24 – 29 ), have been identified from the plant Carpesium abrotanoides . Of them, compounds 1 – 4 , 12 – 15 , and 24 – 27 , namely carpabrotins A–L, are twelve previously undescribed ones. Compound 3 possessed a pseudoguaiane backbone with a rearrangement modification at C-11, C-12 and C-13, while compound 4 suffered a carbon bond break between the C-4 and C-5 to form a rare 4,5- seco -pseudoguaiane lactone. Compounds 1 – 3 , 5 , 13 – 16 and 25 – 27 exhibited anti-inflammatory activity by inhibiting NO production in LPS-induced RAW264.7 macrophages with IC 50 values less than 40 μM, while compounds 1 , 2 , 5 , 13 , 14 , 16 , and 25 – 27 showed significant inhibitory activity comparable to that of dexamethasone. The anti-atopic dermatitis (AD) effects of compounds 5 and 16 were tested according to 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in KM mice, and the results revealed that the major products 5 and 16 improved the histological features of AD-like skin lesions and mast cell infiltration in mice. This study suggested that sesquiterpenoids in C. abrotanoides should play a key role in its anti-inflammatory use.
研究领域:
药理学
·
植物化学
·
炎症研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2021
·
IF 6.057
Weiwei Tang,Andrew Gordon,Hui-ying Wang,Ping Li,Jun Chen,Bin Li
The development of in situ methods for the analysis and visualization of enzyme activity is of paramount importance in drug discovery, research, and development. In this work, the functionalized and array patterned indium tin oxide (ITO) glass slides were fabricated by non-covalent immobilization of amphipathic phospholipid-tagged peptides encompassing the thrombin cleavage site on steric acid-modified ITO slides. The fabricated peptide arrays provide 60 spots per slide, and are compatible with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) measurement, free matrix peak interference, and tolerance to repeated aqueous washing. The peptide arrays were used for the investigation of thrombin activity and screening for its potential inhibitors. The thrombin activity and its Michaelis-Menten constant (K m ) for immobilized peptide substrate was determined using developed MALDI MS peptide array. To investigate the applicability and effectiveness of peptide arrays, the anti-thrombin activity of grape seed proanthocyanidins with different degrees of polymerization (DP) was monitored and visualized. MALDI MS imaging results showed that the fractions of proanthocyanidins with the mean DP of 4.61–6.82 had good thrombin inhibitory activity and their half-maximal inhibitory concentration (IC 50 ) were below 10 μg/mL. Therefore, the developed peptide array is a reliable platform for the discovery of natural thrombin inhibitors .
研究领域:
药物发现
·
分析化学
·
药学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2021
·
IF 4.821
Wenfei Dong,Guo Chen,Lin Zhang,Haiyan Cao,Wenbing Shi,Hongmei Lan,Hao Zhou
The active site of natural horseradish peroxidase (HRP) comprises iron coordinated equatorially by a porphyrin ring and axially by the imidazole group of His170. The low water solubility of porphyrin rings inspired us to investigate the potential of the iron-imidazole group as peroxidase mimic. Herein, iron-imidazole (Fe-MIM) was one-pot constructed using ascorbic acid stabilized Fe 2+ and 2-methylimidazole in the presence of zinc acetate at room temperature in aqueous medium, along with the formation of ZIF-8 framework around Fe-MIM sites. Fe-MIM/ZIF-8 was characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffractometry, X-ray photoelectron spectroscopy, and Brunauer–Emmett–Teller surface measurements. Fe-MIM/ZIF-8 exhibits high peroxidase-like activity, efficiently catalyzing the redox reaction between H 2 O 2 and 3, 3′, 5, 5′-tetramethylbenzidine, as made evident by the appearance of a UV–Vis signal at 652 nm. Fe-MIM/ZIF-8 displays higher catalytic activity than the material (Fe + MIM) prepared by ascorbic acid stabilized Fe 2+ and MIM, due to its rich biomimetic architecture and large BET surface area. The K m of Fe-MIM/ZIF-8 for H 2 O 2 was 28 μM, which is 132 and 98 times lower than those of HRP and hemin (iron III porphyrin), respectively. This low K m value indicates the strong affinity of Fe-MIM/ZIF-8 for H 2 O 2 , which proves the superiority of biomimetic Fe-MIM sites and suggests Fe-MIM/ZIF-8′s potential for H 2 O 2 -related biosensing. The highly sensitive colorimetric methods for H 2 O 2 and H 2 O 2 -related glucose detection has been proposed. The applicability of this method to glucose detection in serum samples was tested with satisfactory results.
研究领域:
材料科学
·
分析化学
·
生物化学
·
纳米技术
✓ 已验证
产品关联
DOI
高影响因子
BIOORGANIC CHEMISTRY
·
2022
·
IF 5.307
Hua-Tao Liu,Chun-Yue Weng,Shen-Yuan Xu,Shu-Fang Li,Ya-Jun Wang,Yu-Guo Zheng
Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase Ls CR for the synthesis of (1 S )-2-chloro-1-(3,4-difluorophenyl) ethanol (( S )-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a “small and smart” mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant Ls CR M3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) Ls CR, the catalytic activity of Ls CR M3 was increased by 4.7 times, the catalytic efficiency k cat / K M value was increased by 2.9 times, and the half-life t 1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% ( v / v ) isopropanol, Ls CR M3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e. ) in 11 h with a space–time yield (STY) as high as 809 g/L∙d.
研究领域:
酶工程
·
生物催化
·
化学生物学
·
药物化学
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xue Wang,Futing Wen,Liu He,Jiangyu Su,Ping Jiang,Daiping He
Defects engineering as an effective strategy was widely utilized to modulate catalytic performance for heterogeneous catalysts through tuning surface electronic surface and creating more active sites. In this work, porous Co–Mn oxide nanosheets with abundant oxygen vacancies (OVs-CMO NSs) were prepared via a simple “one-stone-two-birds” method. OVs were verified by various characterizations and found more favorable for O 2 absorption and activation to produce more reactive O2•- radical, resulting in an improved oxidase-like activity in initiating 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation , with a lower K m value of 0.00717 mM and a higher V max of 2.086 × 10 −7 M/s. Since heparin (Hep) shows an inhibition effect on OVs-CMO NSs-TMB chromogenic system due to strong absorption, a colorimetric sensing platform for Hep detection was established. The present colorimetric detection shows high sensitivity, good selectivity and reliability for normal goat serum. This work not only provides a high-performance non-precious metal oxidase mimic prepared by simple surface defect engineering, but also builds a sensitive colorimetric method for Hep detection.
研究领域:
材料科学
·
纳米技术
·
分析化学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Journal of Materials Chemistry B
·
2020
·Vol. 8
·期 30
·6459-6468
·
IF 5.344
Xiaodan Zhang,Yuwan Lu,Qiumeng Chen,Yuming Huang
A self-templated strategy was adopted to design hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides via the Mo or W doping of ZIF-67, and subsequent pyrolysis under an atmosphere of air at a low temperature of 450 °C. The hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides displayed tunable oxidase-like and peroxidase-like activities able to efficiently catalyze the oxidation of TMB to generate a deep blue color in the absence or presence of H2O2. Relative to that of the un-doped Co3O4, the oxidase mimic activity of the Mo-doped Co3O4 increased to 1.3 to 2.1-fold, while its peroxidase mimic activity increased to 7.1 to 19.9-fold, depending on different Mo doping amounts. The oxidase mimic activity of the W-doped Co3O4 increased to 2.1 to 2.3-fold, while its peroxidase mimic activity increased to 4.8 to 5.9-fold, depending on the different W doping amounts. The Mo- and W-doped Co3O4 nanohybrid exhibited both higher O2 and H2O2 activating capability, and their H2O2 activating capacity was superior to the O2 activating capability. Furthermore, the Mo- and W-doped Co3O4 nanohybrids exhibited similar O2 activating abilities, while the Mo-doped one displayed a higher H2O2 activating capability than the W-doped one. The discrepant peroxidase-like nature of Mo- and W-doped Co3O4 nanohybrids is likely attributed to their different catalytic mechanisms. The peroxidase-like activity of Mo-doped Co3O4 is highly related to the ˙OH free radical, while that of W-doped Co3O4 is likely ascribed to the electron transfer between TMB and H2O2. The Km values of Co3O4/MoO3 for TMB and H2O2 were 0.0352 mM and 0.134 mM, which were 3.2- and 1.9-fold lower than that of pure Co3O4, respectively. A Co3O4/MoO3-based colorimetric platform was developed for the determination of H2O2 in the 0.1–200 μM range, with a limit of detection of 0.08 μM (3σ). Based on the thiocholine (TCh) inhibition of the excellent peroxidase-like activity of Co3O4/MoO3 and the TCh generation via acetylcholinesterase (AChE) catalyzed hydrolysis of acetylthiocholine chloride (ATCh), the colorimetric platform was extended to screen AChE activity and its inhibitor.
研究领域:
纳米材料
·
酶模拟
·
分析化学
·
生物传感器
·
催化
✓ 已验证
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）