Analytica
·
2022
·Vol. 3
·期 1
·36-53
·
IF 0
Andrey A. Chudin,Elena V. Kudryashova
Reversed micelles are helpful to solubilize otherwise insoluble membranotropic or membrane-bound enzymes in their functional form, thus enabling activity assay and inhibition analysis. However, in the case of redox enzymes, this task is further complicated by the necessity to select an appropriate electron-acceptor (EA) which, ideally, should be compatible with spectrophotometric measurements in reversed micelles. Here, we have identified such an EA and successfully used it in a reversed micellar environment to assay the activity of two homologous enzymes from mitochondria:l-galactone-1,4-lactone dehydrogenase (EC 1.3.2.3) fromArabidopsis thaliana(AtGALDH) and galactonolactone oxidase (EC 1.3.3.12) fromTrypanosoma cruzi(TcGAL), differing in their membranotropic properties, with TcGAL being almost insoluble in water and particularly difficult to assay. Furthermore, we have demonstrated the possibility to use this assay for inhibition analysis, with an elucidation of the mechanism and inhibition parameters, which otherwise could not be possible. In order to perform inhibition analysis, we improved the approach for the determination of activity of such membrane enzymes based on a reversed micellar system as membrane matrix, necessary for the functioning of membrane enzymes. A number of electron acceptors (EA) were tested for AtGALDH and optimal conditions of activity determination for AtGALDH were found. The suggested method was successfully applied to the study of the inhibition of AtGALDH by lycorine, and the mixed competitive mechanism of inhibition of AtGALDH by lycorine was determined. The developed approach to inhibitor analysis was applied for TcGAL, insoluble in water membrane, and the method provides new opportunities for searching effective inhibitors that may be potential drugs. Indeed, galactonolactone oxidase fromTrypanosoma cruzi(TcGAL) and AtGALDH are homologues, and the inhibition of TcGAL stops the vital biosynthesis of vitamin C in parasiteTrypanosoma cruzifrom causing Chagas disease. The approach proposed can be applied for the screening of inhibitors of AtGALDH and TcGAL, as well as to study properties of other membrane enzymes including determination of the mechanisms of inhibition, structure and catalytic properties, the impact of membrane components (for example lipids), and so on.Keywords:determination of membrane enzyme activity;spectrophotometric method;membrane enzymes;l-galactone-1,4-lactone dehydrogenase;Arabidopsis thaliana;galactonolactone oxidase;Trypanosoma cruzi;lycorine;drugs;Chagas disease
研究领域:
生物学
·
生物化学
·
药理学
·
寄生虫学
✓ 已验证
DOI
Bioresources and Bioprocessing
·
2021
·Vol. 8
·期 1
·1-10
·
IF 4.578
Sun Chuanqi,Li Zonglin,Ning Xiao,Xu Wentian,Li Zhimin
Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo , and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract
研究领域:
生物化学
·
生物技术
·
酶学
✓ 已验证
DOI
PubMed
高影响因子
BIOMACROMOLECULES
·
2023
·
IF 6.2
Xin Qiao,Xiaoliang Wang,Haixu Chen,Yan Huang,Shangsong Li,Luxuan Li,Yinyong Sun,Xiaoman Liu,Xin Huang
Modulated membrane functionalization is a necessary and overarching step for hollow microcompartments toward their application as nanoreactors or artificial cells. In this study, we show a way to generate phospholipid hybrid proteinosomes that could show superposed virtues of liposomes and proteinosomes. In comparison to pure proteinosomes, both the membrane fluidity and permeability are improved obviously after forming the phospholipid hybrid proteinosomes. Specifically, the integration of phospholipids also endows the hybrid proteinosomes demonstrating a stepwise release of the encapsulants of FITC-dextran (70 and 150 kDa) triggered sequentially by phospholipase and protease, and then a modulated cascaded enzymatic reaction between two different populations of proteinosomes are achieved. Therefore, it is anticipated that such constructed phospholipid hybrid proteinosomes could be employed as an improved microcompartmental model for further advanced artificial cell design toward achieving logic signal communication within the various artificial cellular populations as well as potential applications in the field of microreactors.
研究领域:
纳米技术
·
生物化学
·
生物物理学
·
细胞生物学
·
化学工程
✓ 已验证
DOI
高影响因子
Nanoscale
·
2022
·Vol. 14
·期 41
·15340-15347
·
IF 8.307
Lin Tian,Zijun Huang,Weidan Na,Yuanyuan Liu,Shuai Wang,Yu He,Wenjing Cheng,Tianzi Huang,Zhao Li,Tongxiang Li
The biocatalytic design of nanomaterials with enzyme-like activity is considered a reliable and promising toolkit for the generation of diagnostic agents in complex biological microenvironments. However, the preparation of nanomaterials while maintaining a high catalytic activity in tumor cells (pH 6.0–6.5) poses a prominent challenge. Herein, we constructed a biomimetic enzyme-trigged dual-mode system with colorimetry at 652 nm and photothermal biosensors to detect glutathione based on hollow MnO2-nanosheet-decorated Ag nanowires (Ag@MnO2) as an oxidase-like nanozyme. As expected, Ag@MnO2 catalyzed the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the absence of H2O2, leading to a blue-colored oxidized TMB (oxTMB) that displayed oxidase-like activity in pH 6.0. Interestingly, the portable dual-mode colorimetry and photothermal method for GSH was developed based on the redox reaction between GSH and oxTMB. This detection method exhibited a wide linear range of 0.1–55 μM for GSH with a low detection limit of 0.08 μM. This work highlights a new insight into nanotechnology by taking advantage of biomimetic design in biological analysis.
研究领域:
生物化学
·
纳米医学
·
诊断学
✓ 已验证
DOI
高影响因子
METABOLIC ENGINEERING
·
2017
·
IF 8.142
Lu Dai,Fei Tao,Hongzhi Tang,Yali Guo,Yaling Shen,Ping Xu
Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as “specialists”. However, some enzymes are “generalists” that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols , including 1,2-butanediol, 1,2,4-butanetriol, erythritol , 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays , and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.
研究领域:
生物化学
·
合成生物学
✓ 已验证
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2013
·
IF 3.334
Juan Zhang,Lin Xiao,Yucai Yang,Zhaoxia Wang,Genxi Li
In this paper, we find that the effect of lignin on pancreatic lipase (PL) is dependent on reaction medium and substrate used. Experimental results reveal that lignin can gradually bind to PL to form a PL–lignin complex, resulting in an increased activity of the enzyme. The binding process is spontaneous and the PL–lignin complex formation is an endothermic reaction induced by hydrophobic and electrostatic interaction. There is a non-radiation energy transfer from PL to lignin during the binding process, and the binding of lignin to PL conforms to a secondary exponential decay function. Moreover, the α-helix content of the enzyme will be changed and the rigidity of its side chain will be enhanced due to the formation of lignin–PL complex. This study has not only provided the activation effect of lignin on PL, but also given an insight into the interaction between lignin and the enzyme, which would benefit the application of lignin in the pharmacy and food industry, as well as other fields.
研究领域:
生物化学
·
酶学
·
食品科学
✓ 已验证
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2017
·Vol. 184
·期 9
·3145-3151
·
IF 4.58
Wang Lei,Yang Wen,Li Tengfei,Li Duo,Cui Zhimin,Wang Yan,Ji Shunli,Song Qinxin,Shu Chang,Ding Li
The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.
Schematic presentation of the photometric thrombin assay based on a triple enzyme-mimetic activity of combined nanomaterials (consisting of GO, AuPtNPs and the G-quadruplex/hemin DNAzyme) and two aptamers TMB: 3,3,5,5-tetramethylbenzidine, TMB ox : 3,3,5,5-tetramethylbenzidine oxide, AuPtNP: gold/platinum nanoparticles).
研究领域:
生物化学
·
医学检测
·
纳米生物学
✓ 已验证
DOI
高影响因子
VETERINARY MICROBIOLOGY
·
2022
·
IF 3.246
Hongyan Luo,Mengru Li,Zhishuang Yang,Dekang Zhu,Pei Li
A novel chromosomally-located β-lactamase gene, bla RASA-1 , was identified in Riemerella anatipestifer RA-CH-1. The RASA-1, encoded by bla RASA-1 , was a class A extended-spectrum β-lactamase (ESBL), which shared 42.7% and 40.5% identities with the RAA-1 and CGA-1 β-lactamase, respectively. Overexpression of RASA-1 in Escherichia coli confers broad resistance to β-lactams and the purified native RASA-1 revealed ESBL-like hydrolysis activity. Blasting in GenBank showed that bla RASA-1 was exclusively detected in Riemerella anatipestifer . Moreover, sequence analysis revealed that this gene was located within the multi-resistance region of Riemerella anatipestifer genome.
研究领域:
微生物学
·
抗药性研究
✓ 已验证
DOI
高影响因子
Analytical Methods
·
2014
·Vol. 6
·期 17
·6789-6795
·
IF 1.938
Wei Li,Zhao Gao,Rongxin Su,Wei Qi,Libing Wang,Zhimin He
Pepsin is an active digestive enzyme present in the acidic environment of animal stomachs, and has been widely used to prepare bioactive peptides in the food industry. In this work, a simple fluorescence sensor for scissor-based detection of pepsin activity was developed by using lysozyme-stabilized gold nanoclusters (AuNCs@Lyz) in aqueous media. Under acidic conditions (pH 3.0), enzymatic digestion of AuNCs@Lyz with pepsin results in a significant decrease of fluorescence intensity. Notably, its acidic environment not only helps maintain the maximum fluorescence of gold nanoclusters, but also ensures the highest enzymatic activity of pepsin. In addition to offering high selectivity because of the unique proteolytic action of pepsin under acidic conditions, this facile method provides high sensitivity. With the sensing system, the linear range for pepsin detection is found to be 1 μg mL−1 to 100 μg mL−1, with a detection limit of 0.256 μg mL−1 at a signal-to-noise ratio of 3. Furthermore, the AuNCs@Lyz based fluorescent sensing system could find applications in highly sensitive and selective detection of pepsin in food and biological samples.
研究领域:
生物化学
·
食品科学
·
分析化学
✓ 已验证
DOI
高影响因子
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
·
2021
·Vol. 102
·期 5
·1919-1926
·
IF 3.639
Jiandong Shen,Qixing Jiang,Wei Zhang,Yanshun Xu,Wenshui Xia
BACKGROUND Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l -3-carboxy- trans -2,3-epoxy-propionyl- l -leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.
研究领域:
食品科学
·
生物化学
✓ 已验证
DOI
PubMed
高影响因子
LANGMUIR
·
2021
·
IF 3.882
Yixian Luo,Dou Jin,Wenjin He,Jianzhong Huang,Aicheng Chen,Feng Qi
The opal-like SiO2 microcarriers with different pore diameters named opal-SiO2I and opal-SiO2II were synthesized and utilized as microcarriers to immobilize Rhizopus oryzae lipase (ROL) and Aspergillus oryzae α-amylases (AOA). ROL and AOA can be more stably immobilized on the cross-linked SiO2 opals by neopentyl glycol diglycidyl ether (NGDE), which is the first attempt to use it as a cross-linking agent compared with glutaraldehyde. According to the morphology analysis, multiple layers of SiO2 monodisperse microspheres were regularly packed and formed an opal-like structure, and enzymes were well scattered and immobilized throughout the SiO2 opals. The results showed that the performance of enzymes immobilized on opal-SiO2II with a larger specific surface area was much better than that of opal-SiO2I. The enzyme activity of ROL@opal-SiO2II and AOA@opal-SiO2II cross-linked with 1% NGDE increased 5.32 and 9.32 times compared with their free counterpart, respectively. Furthermore, pH and thermal stability and reusability of ROL/AOA@opal-SiO2II were significantly improved and higher than those of ROL/AOA@opal-SiO2I and free enzymes. This study provides an easily obtained microcarrier opal-SiO2II, which shows potential for efficient different enzyme immobilizations and further industrial applications.
研究领域:
生物化学
·
材料科学
·
酶工程
✓ 已验证
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2019
·
IF 8.456
Chuanxia Chen,Wendong Liu,Pengjuan Ni,Yuanyuan Jiang,Chenghui Zhang,Bo Wang,Jinkai Li,Bingqiang Cao,Yizhong Lu,Wei Chen
Enzyme-like activity and efficiency of nanomaterials are strongly controlled by their size, composition, and structure, and hence the structural parameters need to be optimized. Here, we report that two-dimensional Pd nanoplates enclosed by {100}-facets [{100}PdSP@rGO] exhibit substantially enhanced intrinsic oxidase-like activities relative to the {111}-facets ones and Pd nanocubes in catalyzing the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine. By taking ascorbic acid 2-phosphate as the substrate, which transforms to ascorbic acid in the presence of acid phosphatase (ACP), the {100}PdSP@rGO could be used as an efficient nanozyme for colorimetric ACP detection without resorting to destructive H2O2. A good linear relationship from 0.01 to 6.0 mU/mL with a detection limit of 8.3 μU/mL is obtained, which is better than most previously reported ACP assays. Importantly, the excellent assay performance could be successfully applied to ACP determination in serum samples with high accuracy. This study demonstrates that the enzyme-like activity of nanomaterials could be finely tuned simultaneously by controlling their exposed crystal facets and high specific surface area.
研究领域:
纳米酶学
·
生物分析化学
·
分子诊断
·
纳米材料学
·
临床检验医学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）