Temperature optimum, enzyme

Hydroxybenzoate decarboxylase (EC 4.1.1.61) of anaerobe C. hydroxyben-zoicum was purified and characterized for the first time. ° It has an apparent molecular mass of 350 kDa and consists of six identical subunits of 57kDa. The temperature optimum for the decarboxylation is approximately 50°C, the optimum pH being 5.6-6.2. The activation energy for decarboxylation of 4-hydroxybenzoate is 65kJmor (20-37°C). The enzyme also catalyzes the decarboxylation of... 

Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 °C. It is shown on Figure 1 that pectinesterase was stable at pH 4 — 5. At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 — 8%. At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. 

An enantioselective nitrilase from Pseudomonas putida isolated from soil cultured with 2 mM phenylacetonitrile was purified and characterized. This enzyme is comprised of 9-10 identical subunits each of 43 kDa. It exhibits a pH optimum at 7.0 and a temperature optimum at 40 °C (Ty2 = 160 min) and requires a reducing environment for activity. This nitrilase was shown to have an unusually high tolerance for acetone as co-solvent, with >50% activity retained in the presence of 30% acetone. The kinetic profile of this nitrilase reveals KM= 13.4mM, cat/ M = 0-9s 1mM 1 for mandelonitrile, ZfM = 3.6mM, kclJKM 5.2 s him-1 for phenylacetonitrile, and KM = 5.3 mM, kC lt/KM = 2.5 s 1 him 1 for indole 3-acetonitrile. Preliminary analysis of this enzyme with 5 mM mandelonitrile revealed formation of (/t)-mandelic acid with 99.9% ee [59]. 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

The enzyme BdsC from the thermophilic strain B. subtilis WU-S2B has also been isolated [150], This enzyme has a subunit MW of 45kDa and a native molecular mass of 200 kDa. It has a temperature optimum of 50°C and a pH optimum of 8.0. Its stability at this temperature was 80% after incubation for 30 min. This enzyme demonstrated inhibition characteristics similar to other DBT monooxygenases, indicating involvement of metal ions and cysteine/SH groups in catalytic activity. 

Kinetics of Bound and Free Enzyme. The kinetics of the IME were obtained with the recirculating differential reactor system as described above. The appropriate flow rate, the temperature optimum, and pH optimum as described above were used to most accurately establish the kinetic parameters for this IME emgmie. Substrate solutions from 3 to 150 mM cellobiose in 10 mM sodium acetate were appropriate for this portion of the study. Results were analyzed with the ENZFTT software package (Elsevier Publishers) that permits precise Lineweaver-Burk regressions. 

The standard CGTase employed in the cyclodextrin industry is produced by Bacillus mascerans. This enzyme is reported to be stable only at temperatures around 50 C and loses activity rapidly above 50 C (19), A more thermostable CGTase compared to the B, mascerans CGTase is produced by Bacillus stearothermophilus with a temperature optimum of 1(PC (20). However, the Thermoanaerobaaer CGTase is far more thermostable with its optimum of 950C, and is to our knowledge, the most thermostable CGTase. 

The role of the enzymes is three-fold. Firstly there is the use of very thermostable df-amylases to pre-thin the gelatinised starch, reducing its viscosity so that it can be easily handled and further processed. This process is conceptually very similar to many other commercial uses of hydrolases, especially proteases and glycosidases. Pre-thinning takes place at 105°C and the thermostable df-amylase from B. licheniformis actually has a temperature optimum of almost 100°C. 

Figure 8.3 The effect of temperature of an enzyme reaction and the effect of the time-period of the activity measurements on the apparent temperature optimum (after Wiseman, 1975). The index numbers indicate the increase of temperature. It is important to note that in all cases the decrease of the rate of product formation is the consequence of partial inactivations only, i.e. the concentration of substrate must be enough to saturate the enzyme even at time...

It undergoes marked self-association and can be purified readily by chromatography on porous glass. The enzyme has a molecular weight of about 89 kDa, a pH optimum of 6.8-7.0, and a temperature optimum of 35°C. Its amino acid composition, its requirement for iron but not for molybdenum and FAD, and the catalytic properties of the enzyme, indicate that sulphydryl oxidase is a distinct enzyme from xanthine oxidase and thiol oxidase (EC 1.8.3.2). 

Apparent Temperature Optimum. A rise in temperature has a dual effect upon an enzyme-catalyzed reaction it increases the rate of the reaction, but it also increases the rate of thermal inactivation of the enzyme itself. Like the pH optimum, the temperature optimum may in certain instances be altered by environmental conditions, e.g., pH, type and strength of buffer, etc. The term temperature optimum, therefore, is useless unless the incubation time and other conditions are specified. A more enlightening term is apparent temperature optimum, which indicates that the optimum has been obtained under a... 

Several lactases suitable for industrial processing of whey or lactose are available. The enzyme prepared from the yeast Kluveromyces lactis has a pH optimum between 6 and 7 and a temperature optimum of about 35 °C. The lactase from K. fragilis has a pH optimum of 4.8 and a temperature optimum of about 50°C (MacBean 1979). 

A colleague has measured the enzymatic activity as a function of reaction temperature and obtained the data shown in this graph. He insists on labeling point A as the temperature optimum for the enzyme. Try, tactfully, to point out the fallacy of that interpretation. 

Chandrashekara and Swaminathan (1953b) examining crude extracts of ragi for the properties of the amylases contained therein. The pH optimum for enzyme activity was pH 4.6, the temperature optimum was at 60 °C, and no effect was observed for added salt. They reported that after heating to 70 °C for 15 min, a procedure claimed to destroy (3-amylase, the enzyme activity was reduced by half. 

This NAD-dependent enzyme was purified up to a specific activity of 1060 U/mg (diacetyl as substrate). The enzyme is stable at 57°C for 10 min, the temperature optimum is at 70°C. Besides diacetyl several other diketones were reduced. 

Alpha-amylase is most active at its pH optimum of 6.3 to 6.8.108,109 It is inactive at pH values below 4 and above 9. Enzymic starch conversion is terminated by raising the temperature until enzyme denaturation occurs or by the addition of enzyme poisons, such as the ions of copper, mercury or zinc. Inactivation can also be achieved by moving the pH outside the enzyme s active limits or by the addition of oxidizing agents, such as sodium hypochlorite, hydrogen peroxide or barium peroxide. 

Sucrose sucrose 1-fructosyl transferase appears to be a glycoprotein with a pH optimum of 5.0 and a molecular weight of 65 to 70 kDa (Scott, 1968). It has a lower temperature optimum than many plant enzymes and a relatively low Q10, allowing it to function effectively at lower temperatures. For example, the enzyme s activity decreases slowly (i.e., by only a factor of 2) between 28 and 8°C (Wagner and Wiemken, 1986). 

Fructan fructan fructosyl transferase has a molecular weight of approximately 70 kDa and can be separated into five species with pH values between 4.5 and 5.0. The enzyme has a pH optimum for fructosyl transfer activity between 5.5 and 7.0 and a temperature optimum in the 25 to 35°C range. Like 1-SST, 1-FFT has a low Q10 (i.e., 1.14 between 25 and 5°C), indicative of its ability to function at relatively low temperatures (Koops and Jonker, 1994). The rate of transfer of fructosyl groups increases with substrate concentration up to 100 mol nr3. 

Thermal Stability. The temperature optimum for arctic cod pepsin is approximately 32°C see Table III) and the enzyme is unstable when incubated at temperatures above 37°C see Figure 4) in contrast to porcine pepsin which has a temperature optimum of approximately 47°C and is unstable at temperatures above 50°C see Figure 4). Accordingly, there is a difference in thermal stability of approximately 13°C for the two enzymes. Similar differences in temperature optima were observed when Greenland cod and American smelt were compared with PP see Table III). These data are consistent with previous reports for intracellular enzymes and for crude preparations of pyloric caeca enzymes from other low-temperature-adapted organisms (42). 

Temperature has two counteracting effects on the activity of enzymes. At lower temperatures, there is a Ql0 of about 2, but at temperatures over 40°C, the activity quickly decreases because of denaturation of the protein part of the enzymes. The result of these factors is a bell-shaped activity curve with a distinct temperature optimum. 

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