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Enzyme kinetics

Kristal Jesús-de la Cruz, Carlos Alfonso Álvarez-González, Emyr Peña, José Antonio Morales-Contreras, Ángela Ávila-Fernández
In fishes, trypsins are adapted to different environmental conditions, and the biochemical and kinetic properties of a broad variety of native isoforms have been studied. Proteolytic enzymes remain in high demand in the detergent, food, and feed industries; however, our analysis of the literature showed that, in the last decade, some fish trypsins have been studied for the synthesis of industrial peptides and for specific biomedical uses as antipathogenic agents against viruses and bacteria, which have been recently patented...
April 2018: 3 Biotech
Günseli Kurt-Gür, Emel Ordu
NAD+ -dependent formate dehydrogenases (FDH, EC, providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO2 reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli DH5α-T1 cells. Recombinant GhFDH1 was purified 26...
March 2018: 3 Biotech
Haofeng Gao, Chanjuan Li, Ramesh Bandikari, Ziduo Liu, Nan Hu, Qiang Yong
BACKGROUND: In industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type. RESULTS: A novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8...
March 19, 2018: Microbial Cell Factories
Jian Xiao, Si-Yang Wang, Zi-Jun Zhou, Yao Zhang, Chun-Lei Song, Yi-Yong Zhou, Xiu-Yun Cao
The over-enrichment of lake waters with nitrogen (N) and phosphorus (P) has become a serious environmental problem, but modes of change in stoichiometry and enzymatic regeneration along trophic gradients are largely unknown. Seasonal variations in the kinetics of extracellular aminopeptidase (LAP) and alkaline phosphatase (AP), together with the composition of phytoplankton and concentrations of N and P, were examined from Jun 2013 to September 2014 in a Chinese shallow lake in which two basins had contrasting trophic states...
March 1, 2018: Science of the Total Environment
Konstantinos Feidantsis, Hans O Pörtner, Elisavet Vlachonikola, Efthimia Antonopoulou, Basile Michaelidis
Seasonal temperature changes may take organisms to the upper and lower limit of their thermal range, with respective variations in their biochemical and metabolic profile. To elucidate these traits, we investigated metabolic and antioxidant patterns in tissues of sea bream Sparus aurata during seasonal acclimatization for 1 yr in the field. Metabolic patterns were assessed by determining lactate dehydrogenase, citrate synthase, and β-hydroxyacyl CoA dehydrogenase activities, their kinetic properties and plasma levels of glucose, lactate, and triglycerides and tissue succinate levels...
May 2018: Physiological and Biochemical Zoology: PBZ
Shalini Awasthi, Shalini Gupta, Ravi Tripathi, Nisanth N Nair
Enhanced sampling of large number of collective variables (CVs) is inevitable in molecular dynamics (MD) simulations of complex chemical processes such as enzymatic reactions. Due to the computational overhead of hybrid quantum mechanical/molecular mechanical (QM/MM) based (MD) simulations, especially together with density functional theory (DFT), predictions of reaction mechanism and estimation of free energy barriers have to be carried out within few tens of picoseconds. We show here that the recently developed Temperature Accelerated Sliced Sampling (TASS) method allows one to sample large number of CVs, thereby enabling us to obtain rapid convergence in free energy estimates in QM/MM MD simulation of enzymatic reactions...
March 19, 2018: Journal of Physical Chemistry. B
Stephanie Portillo-Ledesma, Lía M Randall, Derek Parsonage, Joaquín Dalla Rizza, P Andrew Karplus, Leslie B Poole, Ana Denicola, Gerardo Ferrer-Sueta
Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2 and 5, and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system...
March 19, 2018: Biochemistry
Sunil Ghatge, Youri Yang, Woo-Young Song, Tae-Young Kim, Hor-Gil Hur
In the present study, the gene encoding a multicopper oxidase, more precisely a laccase from the thermoalkaliphilic aerobic bacterium Caldalkalibacillus thermarum strain TA2.A1 (CtLac), was cloned and expressed in Escherichia coli. CtLac is a monomeric protein with a molecular weight of 57 kDa as determined by native polyacrylamide gel electrophoresis. The optimum pH and temperature for 2,6-dimethoxyphenol (2,6-DMP) oxidation were 8.0 and 70 °C, respectively. The kinetic constants Km and kcat for 2,6-DMP were of 200 μM and 23 s-1 , respectively...
March 19, 2018: Applied Microbiology and Biotechnology
Sumit Handa, Daniel R Dempsey, Divya Ramamoorthy, Nanci Cook, Wayne C Guida, Tyler J Spradling, Justin K White, H Lee Woodcock, David J Merkler
The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO2 ...
2018: Biochemistry & Molecular Biology Journal
Brian G O'Flynn, Aidan J Hawley, David J Merkler
Crop protection against destructive pests has been at the forefront of recent agricultural advancements. Rapid adaptive evolution has led to insects becoming immune to the chemicals employed to quell their damage. Insecticide resistance is a serious problem that negatively impacts food production, food storage, human health, and the environment. To make matters more complicated are the strict regulations in place on insecticide development, driven by rising public concern relating to the harmful effects these chemicals have on the environment and on society...
2018: Biochemistry & Molecular Biology Journal
Reza Yekta, Gholamreza Dehghan, Samaneh Rashtbari, Rahim Ghadari, Ali Akbar Moosavi-Movahedi
Farnesiferol C (FC) is a natural sesquiterpene coumarin, which includes a widely range of biological activities. In this work, effects of FC on the structure and catalytic function of bovine liver catalase (BLC) was assessed by various spectroscopic and theoretical methods. Kinetic studies showed that FC has a remarkable inhibitory activity on BLC via mixed-type inhibition. The IC50 value as the inhibitory strength of FC was evaluated 1.5 μM. Fluorescence spectroscopy, synchronous fluorescence, CD spectroscopy and UV-vis absorption studies revealed conformational changes in the tertiary and secondary structure of BLC as well as the position of the heme group in the presence of different concentrations of FC...
March 14, 2018: International Journal of Biological Macromolecules
Cláudia S Nóbrega, Bart Devreese, Sofia R Pauleta
The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be - 170 mV, + 133 mV and + 210 mV, respectively, at pH 7...
March 14, 2018: Biochimica et Biophysica Acta
Chen Geng, Tengfei Zhao, Chunxian Yang, Qiaozhuo Zhang, Feng Bai, Junlan Zeng, Fangyuan Zhang, Xiaoqiang Liu, Xiaozhong Lan, Min Chen, Zhihua Liao
N-methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N-methyltransferase (PMT) catalyzes the N-methylation of putrescine to form N-methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger, designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA)...
March 2, 2018: Plant Physiology and Biochemistry: PPB
Matthijs J van Haren, Martin G Thomas, Davide Sartini, David J Barlow, David B Ramsden, Monica Emanuelli, Fábio Klamt, Nathaniel I Martin, Richard B Parsons
The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg NNMT. Although the Km for 4-phenylpyridine was similar to that reported for nicotinamide, its kcat of 9...
March 13, 2018: International Journal of Biochemistry & Cell Biology
Jana Löwe, Aaron A Ingram, Harald Gröger
In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance...
December 5, 2017: Bioorganic & Medicinal Chemistry
Juan Román, Antonio Castillo, Luis Cottet, Andrea Mahn
Myrosinase is a glycosylated enzyme present in the Brassicaceae family that catalyzes the hydrolysis of glucoraphanin to yield sulforaphane, recognized as a health-promoting compound found in cruciferous foods. Broccoli myrosinase has been poorly characterized. In this work, the enzyme was purified from broccoli florets and its kinetic behaviour was analyzed. The cDNA of broccoli myrosinase was isolated and sequenced to obtain the amino acids sequence of the enzyme. A three-dimensional structural model of a broccoli myrosinase subunit was built and used to perform molecular docking simulations with glucoraphanin and other glucosinolates...
July 15, 2018: Food Chemistry
Haotian Wang, Yayuan Li, Xinghui Xia, Xinyue Xiong
Many studies have investigated bioaccumulation and metabolism of polycyclic aromatic hydrocarbons (PAHs) in aquatic organisms. However, lack of studies investigated both processes simultaneously, and the interaction between these two processes is less understood so far. This study investigated the bioaccumulation kinetics of PAHs and metabolic enzyme activities, including total cytochrome P450 (CYPs) and total superoxide dismutase (T-SOD), in zebrafish. Mature zebrafish were exposed to the mixture of phenanthrene and anthracene under constant concentration maintained by passive dosing systems for 16days...
March 2018: Journal of Environmental Sciences (China)
Olivier Turc, François Tardieu
Abortion of reproductive organs is a major limiting factor of yield under water deficit, but is also a trait selected for by evolutionary processes. Youngest reproductive organs must be prone to abortion so older organs can finish their development in case of limited resources. Water deficit increases natural abortion via two developmentally-driven processes, namely a signal from the first fertilized ovaries and a simultaneous arrest of the expansive growth of all ovaries at a precise stage. In maize (Zea mays) subjected to water deficits typically encountered in dryland agriculture, these developmental mechanisms account for 90 % of drought-associated abortion and are irreversible three days after silk emergence...
March 13, 2018: Journal of Experimental Botany
Zia Uddin, Yeong Hun Song, Mahboob Ullah, Zuopeng Li, Jeong Yoon Kim, Ki Hun Park
Diabetes mellitus is one of a major worldwide concerns, regulated by either defects in secretion or action of insulin, or both. Insulin signaling down-regulation has been related with over activity of protein tyrosine phosphatase 1B (PTP1B) enzyme, which has been a promising target for the treatment of diabetes mellitus. Herein, activity guided separation of methanol extract (95%) of Dodonaea viscosa aerial parts afforded nine ( 1 - 9 ) polyphenolic compounds, all of them were identified through spectroscopic data including 2D NMR and HREIMS...
2018: Frontiers in Chemistry
Peter L Cummins, Babu Kannappan, Jill E Gready
The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate...
2018: Frontiers in Plant Science
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