Temperature optima

Contrary to common chemical reactions, enzyme-catalyzed reactions as well as growth of microorganisms show a so-called temperature optimum, which is a temperature-dependent maximum resulting from the overlapping of two counter effects with significantly different activation energies (cf. 2.5.4.2)  

For starch hydrolysis by microbial a-amylase, the following activation energies, which lie between the limits stated in section 2.5.4.2, were derived from e. g. the Arrhenius diagram (Fig. 2.35)  

As a consequence of the difference in activation energies, the rate of enzyme inactivation is substantially faster with increasing temperature than the rate of enzyme catalysis. Based on activation energies for the above example, the following relative rates are obtained (Table 2.14). Increasing 8 from 0 to 60 °C increases the hydrolysis rate by a factor of 5, while the rate of inactivation is accelerated by more than 10 powers of ten. 

The growth of microorganisms follows a similar temperature dependence and can also be depicted according to the Arrhenius equation (Fig. 2.36) by replacing the value k by the growth rate and assuming Ea is the reference value ja of the temperature for growth. 

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Handling Temperatures. Optimum temperature for pumping is in 37—48°C range. Piping should be stainless steel, aluminum, or galvanized iron. Valves and pumps should be bronze, cast-iron with bronze trim, or stainless steel. A pump of 3.15-L/s (50-gal/min) capacity unloads a tank car of warm glycerol in ca 4 h. 

Protein residues, eg, soft-boiled egg yolk, are difficult stains to handle. If the stains are not totally denatured, proteases can decompose them. There are commercial proteases with a high temperature optimum (60°C) that can remove most protein soils in a dishwasher (63). Patents on the use of Upases in ADDs have claimed that Upases can reduce the formation of spots and films on glasses (62,64—66) however, no commercial appUcation of Upases in ADDs has been implemented. 

The effect of temperature on the rate of ethanol production is markedly different for free and immobilised systems. Thus while a constant increase in rate is observed with free S. cerevisiae as temperature is increased from 25 to 42 °C, a maximum occurs at 30 °C with cells immobilised in sodium alginate. The lower temperature optimum for immobilised systems may result from diffusional limitations of ethanol within the support matrix. At higher temperatures, ethanol production exceeds its rate of diffusion so that accumulation occurs within the beads. The achievement of inhibitory levels then causes the declines observed in the ethanol production rate. 

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. 

Soluble reductases with a temperature optimum of 80°C have been described from (a) Pseudomonas putida that reduces chromate to insoluble Cr(lll) (Park et al. 2000) and (b) Archaeoglobus fulgidus that can reduce Fe(IIl)-EDTA (Vadas et al. 1999). 

Some effort has been directed toward understanding the growth requirements of the alga of interest, allelopathic alga (13), the optimum salinity (14), the distribution of allelopathic agents (15), and the temperature optimum (16). This paper reviews the approaches taken to separate the allelopathic agents from the other materials and the methods used to characterize the biological activity of aponin from Nannochloris sp. 

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. 

Hyperthermophile an organism having a growth temperature optimum of 80° C or higher. 

The temperature optimum for this proteinase may also be a result of evolutionary adaptation to the hot temperatures... 

Abiotic, for example by adsorption of reactants onto mineral surfaces, distinguished from biotic catalysis by the absence of a temperature optimum. 

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...

Other complexants have been used for PbSe deposition. Triethanolamine was used in one study [66]. While deposition occurred over a wide range of temperatures, optimum results (in terms of rate of deposition and film thickness) were obtained at a deposition temperature of 75°C. In another study, lead nitrate was dissolved in an excess of hydroxide and excess selenosulphate was also used as an additional complexant [67]. The pH was 10 (adjusted with acetic acid), and depo-... 

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). 

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