198,958 research outputs found

    Insolubilization process increases enzyme stability

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    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes

    Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

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    Horseradish peroxidase (HRP) is a commonly used enzyme in many biotechnological fields. Improvement of HRP stability would further increase its potential application range. In the present study, 13 single- and three double-mutants of solvent exposed, proximal lysine and glutamic acid residues were analysed for enhanced H2O2 stability. Additionally, five single- and one pentuple-consensus mutants were investigated. Most mutants displayed little or no alteration in H2O2 stability; however, three (K232N, K241F and T110V) exhibited significantly increased H2O2 tolerances of 25- (T110V), 18- (K232N), and 12-fold (K241F). This improved stability may be due to an altered enzyme-H2O2 catalysis pathway or to removal of potentially oxidisable residues

    In Vitro Stability of Phytase from Recombinant Bacteria E. Coli BL21 (DE3) EAS1-AMP

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    The objective of the research was to inquire the Km, Vm, activity, intracellular phytase stability exposed to pH variation, temperature variation and protease (pepsin and pancreas) in vitro. The phytase was produced from recombinant bacteria E. coli BL21(DE3) EAS1-AMP using 1.5 mM IPTG as inducer. Intracellular enzyme was extracted via freeze shock and centrifugation. Pure enzyme was acquired through NI-NTA agarose column. The enzyme was then tested for Km, Vm, phytase activity and stability against pH, temperature and protease. Treatment levels for stability against protease were P0: without protease, P1: addition of pepsin, P2: addition of pepsin and pancreas, and the data were statistically analyzed using analysis of variance of one-way Completely Randomized Design. Crude intracellular phytase had Vm 6.39 υM/sec, Km 34.82 υM, and 277 units activity. Intracellular phytas was stable at pH 4–6 and 0–550 C. Protease level influenced the activity of intracellular phytase (P<0.05). Intracellular phytase was stable against pepsin but not pancreas. Keywords: Km, Vm, activity, intracellular phytase, pH, temperature, protease

    Characterization of thermophilic β-Glucosidase of rhizospheric bacterial strain (LSKB15) isolated from Cholistan Desert, Pakistan

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    Fifty thermophilic bacterial strains isolated from rhizospheric soil of Cholistan desert, Pakistan, and designated as LSKB01-LSKB50 were screened for β-glucosidase gene (bgl) belonging to glycoside hydrolase family 1 (GH 1) using PCR technique. Subsequently, the same strains were screened for extracellular β-glucosidase production using esculin as substrate. All fifty strains were shown to be amplified for conserved region of bgl gene and to secrete extracellular β-glucosidase. One strain (LSKB15) secreted relative high amount of this enzyme as indicating by size of ferric-esculetin precipitate. This strain was further cultivated on cellulose containing media and β-glucosidase was purified by ammonium sulfate, dialysis and gel filtration chromatography. The purified enzyme showed an optimal temperature of 60°C and an optimal pH of 7. It also showed excellent temperature and pH stability retaining > 90% activity after incubation for 2 h at pH 5-8 and 40-60°C. Finally, the purified enzyme was run on Native-PAGE and subsequently incubated in phosphate buffer containing 5 mM of 4-methylumbelliferyl-β-D-glucoside (4-MUG) for 15 min at 50°C and visualized by UV light as white band. We concluded that thermophilic LSKB15 β- glucosidase may work with other cellulase to degrade available cellulose synthesized by plant and the properties exhibited by it such as high temperature and pH stability pointed out its potential industrial importance

    Ultrasensitive bioluminescent determinations of adenosine triphosphate (ATP) for investigating the energetics of host-grown microbes

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    Stability and optimal concentrations of reagents were studied in bioluminescence assay of ATP levels. Luciferase enzyme was prepared and purified using Sephadex G-100. Interdependencies between enzyme and luciferin concentrations in presence of optimal Mg are illustrated. Optimal ionic strength was confirmed to be 0.05 M for the four buffers tested. Adapted features of the R- and H-systems are summarized, as well as the percentages of ATP pools released from representative microbes by heat and chloroform

    The Analysis of Hydrophobic Interaction on Aspergillus Niger Xylanase Enzyme Thermal Stability

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    Xylanase is a type of enzyme that has an important role in the industrial field. One measure that can be done to improve the thermostability of an enzyme is by protein engineering. The mutation of the protein can be done by studying protein structures through molecular dynamics simulation approach. In this research, thermal stability analysis on Aspergilus niger Wild Type Xylase (AnX) was performed which aims to study the thermal stability characteristics of xynalase enzyme from Aspergillus niger through molecular dynamics simulation approach. AnX molecular dynamics simulation was performed by NAMD (Not Just Another Molecular Dynamic) software at 300 – 500 K. The research was focused on the study of enzyme thermal stability characteristics in order to get the information of residues accountable for such characteristics. The selection of residues to be mutated was based on hydrophocic interaction analysis. Then from that, the design of xylanase enzyme mutant with better thermostability than wild type xynalase enzyme was made in order to provide design reference for more stable xylanase mutation design which can be implemented in wet experiments for of Aspergillus niger Xylanase enzyme genetic engineering. The enzyme was unfolded at 500 K at 9.5 ns. The residues responsible of the thermal stability were based on hydrophobic interaction analysis in Alanin at residue 60. This residue is located in segmen/chain 3. The best mutant is Alanin 60 residue mutant which is replaced by Methionin and ∆∆Gsolv of -21.10345 was obtained. Thus, Ala60Met is the most stable mutant which might increase the thermal stability of Aspergillus niger Xylanase Enzyme