Phosphate buffer enzyme activity

PG activity was assayed from cells grown in a medium containing 1% glucose in one-litre self-induced anaerobic fermentation for 5 days by increase of reducing sugars. Enzyme activity increased from pH 3.0 to pH 5.0 (citrate buffer) and decreased drastically above 5.0 (phosphate buffer), but activity was not affected differentially by the two buffers used (data not shown). PG activity increased almost linearly from 20°C to 40°C but above this optimum, activity was lost rapidly and the enzyme was completely inactivated at 60°C and 70°C after 10 and 6 min, respectively (data not shown). No PL, PGL or PME were detected. 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

For in vitro studies there are a number of compounds available to block protein phosphatase activity. Phosphate buffers inactivate all of these enzymes. Several naturally occurring toxins are potent inhibitors of PPPs, e.g., okadaic acid or microcystin, and are frequently used tools. PPM and PTP family members are not affected by these toxins. Vanadate containing solutions are competitive inhibitors of PTPs, pervanadate is an irreversible inhibitor of PTPs. 

Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens 1AM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely... 

This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 C. The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. 

Extracellular Enzyme Activities. The protein, carrier of the polygalacturonase and pectinmethylesterase activities, was salted out from the H.annuus 1805 culture medium by adding ammonium sulphate to 70 % saturation. The precipitate was separated by centrifugation (30 min, 4500 xg), diluted with 0.1 M phosphate buffer (pH 7.0) and dialyzed for 12 hours against distilled water. After dilution to the required volume with the same buffer, both enzyme activities under study were determined in the solution. 

Material required For total protein extract and enzyme activities extraction buffer 50 mM potassium phosphate, pH 7.0,1 mM EDTA, 1% of PVP (100 mg/mL), for CAT, GR, and SOD and with 10 mM ascorbato for APX activity. Bio-Rad protein assay, centrifuge, spectrophotomer and quartz cuvette. 

Zoungrana et al. (1997) and Norde and Zoungrana (1998) investigated the influence of adsorption on the structure, structure stability and biological activity of a proteolytic enzyme, a -chymotrypsin. The enzyme was adsorbed from 0.01 M phosphate buffer at pH 7.0 and at 22°C onto solid surfaces of different hydrophobicities and morphologies. 

The pH optimum of an enzyme will often vary from one substrate to another and must be determined for each substrate. The buffer system used will often affect the overall activity of an enzyme and may alter its pH optimum. In general, the amino buffers such as glycylglycine and tricine, etc. (Table 8.2) result in a greater enzyme activity than do the simple inorganic buffers such as phosphate and carbonate. Buffers are most effective over a narrow pH range (approximately two units) which centres on their pK, value. Those buffers with pKa values similar to the known optimum pH of the enzyme should be tested for their effect on the activity of the enzyme over a limited pH range. 

The / -glucosidase activity was determined by measuring the release of p-nitrophenol from p-nitrophenyl-/i-D-glucopyranoside one unit of / -glucosidase activity (U) is defined as the amount of enzyme that releases 1 [mu] mol p-nitrophenol per minute. AU samples were assayed in potassium phosphate buffer (50 mM, pH 7.0) at 50 °C under conditions that activity was proportional to enzyme concentration. 

A. bronchisepticus was cultivated aerobically at 30 °C for 72 h in an inorganic medium (vide supra) in 1 liter of water (pH 7.2) containing 1 % of polypeptone and 0.5 % of phenylmalonic acid. The enzyme was formed intracellularly and induced only in the presence of phenylmalonic acid. All the procedures for the purification of the enzyme were performed below 5 °C. Potassium phosphate buffer of pH 7.0 with 0.1 mM EDTA and 5 mM of 2-mercaptoethanol was used thoughout the experiments. The enzyme activity was assayed by formation of pheylacetic acid from phenylmalonic acid. The summary of the purification procedure is shown in Table 2. The specific activity of the enzyme increased by 300-fold to 377 U/mg protein with a 15% yield from cell-free extract [9]. One unit was defined as the amount of enzyme which catalyzes the formation of 1 mmol of phenylacetic acid from phenylmalonic acid per min. 

Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and >galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and -galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and -galactosidase, respectively, were preincubatcd at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

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