Temperature optima and stabilities

Figure 3(B). Comparison of temperature optima for stabilities of glucose isomerase, amylase, and d-galactosidase. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

Because this biocatalyst is of such industrial significance, efforts to redesign it with altered properties could have a profound economic effect on the cost of HFCS. With the advances in molecular biology and prediction of protein structure-function relationships, these studies have been under way for a number of years and include thermal stabilization, alteration of pH and temperature optima, and modifications to substrate specificity.284 286,287... 

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). 

A much more ambitious database that builds on the IUBMB classification is BRENDA, maintained by the Institute of Biochemistry at the University of Cologne. In addition to the data provided by the ENZYME database, the BRENDA curators have extracted a large body of information from the enzyme literature and incorporated it into the database. The database format strives to be readable by both humans and machines. The categories of data stored in BRENDA comprise the EC-number, systematic and recommended names, synonyms, CAS-registry numbers, the reaction catalyzed, a list of known substrates and products, the natural substrates, specific activities, KM values, pH and temperature optima, cofactor and ion requirements, inhibitors, sources, localization, purification schemes, molecular weight, subunit structure, posttranslational modifications, enzyme stability, database links, and last but not least an extensive bibliography. Currently, BRENDA holds entries for approximately 3500 different enzymes. 

Four chimeric enzymes showed marked differences in their temperature optima (Fig. 5A). The chimeric enzymes were optimally active at 45-50°C, showing an intermediate temperature optimum between C. gi7v s andA. tumefaciens enzymes. CHSTY showed the temperature optima of 45 C with complete loss of activity at 65 °C. CHBSA showed the temperature optima of 45 C with no activity at 70°C. On the other hand, CHAGE was optimally active at 50 C and exhibited 52% of its maximum activity at 60 0. CHBSM exhibited maximum activity at 50°C and 61% of its maximum activity at 60 C. The temperatures at which 50% loss of the enzyme activities occurred were 41, 47, 50, 55 and 57 and 67°C for C. gilvus, CHSTY, CHBSA, CHAGE, CHBSM and A. Tumefaciens enzymes, respectively (Fig. 5B). Thus heat stability of chimeric enzymes was increased by 6-16 C as compared to C. gilvus enzyme though none of them exceeded that of A. Tumefaciens enzyme. 

However, there is evidence that some enzymes may have genuine temperature optima. That is, at some point in the temperature profile, an enzyme may actually become less active as the temperature is raised, but this is not caused by denaturation. Although it is not clear yet if this phenomenon is widespread among enzymes, where it is present this genuine temperature optimum will be an important and diagnostic characteristic of an individual enzyme, alongside pH optimum, stability, and kinetic properties. 

Properties. In general, the free and the immobilized enzyme catalyse the same reaction, but depending on the supporting material and nature of the binding, there may be differences in pH and temperature optima (the latter is usually increased), value and specific and maximal activities (the latter is usually decreased) (see Enzyme kinetics). Chief reasons for these alterations are the decreased flexibility and mobility of the coupled enzyme, and steric factors which interfere with access of the substrate to, and diffusion of product from, the active center. These changes are usually more than compensated by increased stability of the enzyme. They can be avoided or reduced by attaching the enzyme to the support by a side chain, or spacer, which allows greater mobility and unhindered contact with substrates. 

The existence of upper and lower temperature limits for stabilizers of various types is confirmed by the data of Table 17. The temperature limits of the optima are evidently determined by the values of the total activation energy of the reactions in the polymer-stabilizer system, as well as the selective action of the stabilizer and its conversion products with respect to the decomposition products of the pol3rmer. On the basis of the principle of the action of inhibitors of chain reactions initiated by radiations, we might assume that limits of optimum irradiation intensities exist for them, just as for thermal stabilizers. When the intensity of the influence is lower than the optimum value, the additives remain inactive when the intensity of the influence is higher than the... 

The specific activity of dextranase from Penicillium funiculosum immobilized on agarose cyclic imidocarbonate was as high as 30% of that of the free enzyme. The pH and temperature optima of the enzyme were unchanged by immobilization, whereas the pH and temperature stabilities were enhanced. The immobilized dextranase was more active towards low-molecular-weight dextrans, and the Michaelis constants were two to five times greater than those for the free enzyme acting on substrates of the same chain length the action patterns of the free and immobilized enzymes are virtually identical. Columns of the immobilized enzyme were suitable for repeated use. 

Immobilization of Brevibacterium fuscum dextranase on agarose cyclic imido-carbonate reduced its specific activity by 70% but increased its pH stability, whereas its action pattern, thermal stability, and pH and temperature optima were unaffected/ ... 

Serine hydroxymethylase was purified to homogeneity from mung beans (Vigna radiata) (Rao and Appaji Rao, 1982). It required one of the substrates to be present for stability as well as a sulfhydryl reducing agent. Activity was optimal at pH 8.5 and the enzyme had two temperature optima, 35 and 55°C. The substrate dependence was biphasic vnth respect to serine with values of... 

An enzyme similar to the 3 -nucleotidase of mung bean has been isolated from germinating wheat seedlings and purified 800-fold (90). The preparation possessed DNase, RNase, and 3 -nucleotidase activities. These three activities were similar in pH optima, requirements for Zn2+ and sulfhydryl compounds, stability to storage, temperature inactivation... 

Although the enzymic activity of RNase-S is very similar to RNase-A, it is not identical. An extensive comparative study has been reported by Takahashi et al. (91). These authors varied the substrate, the temperature, and the pH. The pH optima are the same with RNA but different with C>p or CpA as substrates. The pH profiles vary with temperature. The effect of the lower thermal stability of RNase-S is evident above 30°. 

Enzymes are protein catalysts of remarkable efficiency and specificity. Lipid, carbohydrate, nucleotide, or metal-containing prosthetic groups may be attached to these enzymes and serve as essential components of their catalyses by enhancing specificity and/or stability (8—13). Each enzyme has a specific temperature and pH range where it functions to its optimal capacity the optima for these proteins usually He between 37—47°C, and pH optima range from acidic, ie, 1.0 in the case of gastric pepsin, to alkaline, ie, 10.5 in the case of alkaline phosphatase. However, enzymes from extremely thermotolerant bacteria have become available these can function at or near the boiling point of water, and therapeutic use of these ultrastable proteins can be anticipated. 

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycete Phellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting a-D-mannosidic bonds a-D-mannosidase, exo-D-mannanase, and e/ido-D-mannanase, which were separated. Some properties of the D-mannanase complex of the crude enzyme preparation, and of a partially purified a-D-mannosidase, were examined. The D-mannanase complex exhibited two pH optima, its temperature optimum being 45 C. The pH optimum of purified a-D-mannosidase was at pH 5.0, the temperature optimum was at 60 °C the enzyme had a relatively high heat stability. The of a-D-mannosidase for 4-nitrophenyl a-D-mannopyranoside was 1.5xl0 M. Pure a-D-mannosidase did not split D-mannan. 

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