Insolubilization process increases enzyme stability

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

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

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

Assaying activity and assessing thermostability of hyperthermophilic enzymes

There is now a wide variety of intra- and extra-cellular enzymes available from organisms growing above 75°C, and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95°C will involve measurement below the optimal growth temperature for the organism. The purpose of this chapter is to cover practical aspects of enzyme assay procedures that are specific to high temperatures. Since by far the commonest routine assessment of enzyme stability is activity loss, and because it is always unwise to measure enzyme activity without being confident of its stability during the assay, we include an outline of procedures for measuring enzyme activity loss/stability at high temperatures

A new intrinsic thermal parameter for enzymes reveals true temperature optima

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, Teq, arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source (“evolved”) temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on Teq with respect to enzyme temperature/activity effects to be more important than on thermal stability. Teq is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution

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

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

Effect of Water Content on the Thermal Inactivation Kinetics of Horseradish Peroxidase Freeze-Dried from Alkaline pH

The thermal inactivation of horseradish peroxidase freeze-dried from solutions of different pH (8, 10 and 11.5, measured at 25 C) and equilibrated to different water contents was studied in the temperature range from 110 to 150 C. The water contents studied (0.0, 1.4, 16.2 and 25.6 g water per 100 g of dry enzyme) corresponded to water activities of 0.0, 0.11, 0.76 and 0.88 at 4 C. The kinetics were well described by a double exponential model. The enzyme was generally more stable the lower the pH of the original solution, and for all pH values, the maximum stability was obtained at 1.4 g water/100 g dry enzyme. Values of z were generally independent of water content and of the pH of the original solution, and in the range of 15–25 °C, usually found in neutral conditions, with the exception of the enzyme freeze dried from pH 11.5 and equilibrated with phosphorus pentoxide, where a z-value of the stable fraction close to 10 C was found

Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1

A cyclodextrin glucanotransferase (CGTase) was successively purified by ammonium sulphate precipitation, and affinity chromatography on a-CD (epoxy)-Sepharose 6B column. The specific activity of the CGTase was increased approximately 2200-fold, from 8.43 U/mg protein to 18,866 U/mg protein. SDS-PAGE showed that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 75 kDa. The molecular weight of the enzyme that was estimated by gel filtration under native condition was 79 kDa. This has indicated that Bacillus sp. G1 CGTase is a monomeric protein. The isoelectric point (pI) of the enzyme was about 8.8. Characterization of the enzyme exhibited optimum pH and temperature of 6.0 and 60 8C, respectively. The enzyme was stable from pH 7.0 to 9.0 and retained its high activity up to 60 8C. However, in the presence of 20 mM Ca2+, the purified CGTase is able to prolong its thermal stability up to 70 8C. CGTase was strongly inhibited by ZnSO4, CuSO4, CoCl2, FeSO4, FeCl3 and EDTA. Km and Vmax for the purified enzyme were 0.15 mg/ml and 60.39 mg bcyclodextrin/( ml min), respectively, with soluble starch as substrate. In cyclodextrin production, tapioca starch was found to be the best substrate used to produce CDs. The enzyme produced g- and b-CD in the ratio of 0.11:0.89 after 24 h incubation at 60 8C, without the presence of any selective agents

Activity of glucose oxidase functionalized onto magnetic nanoparticles

BACKGROUND: Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. METHODS: Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. RESULTS: The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles CONCLUSION: Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological application

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

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