Enzymes thermal stability

Enzyme thermal stability in the PE presence. This technique allows us to know the melting temperature of a macromolecule (Tj ), the temperature where 50 % is in native form and 50 % is in its denatured form. Also, the enthalpy and entropic changes of the denaturalization process can be calculated in this way. The information given by this technique is useful in the sense that it allows us to determine whether the PE presence increases o decreases the thermal stability of an enzyme [38]. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Several reports have indicated that enzymes are more thermostable in organic solvents than in water. The high thermal stability of enzymes in organic solvents, especially in hydrophobic ones and at low water content, was attributed to increased conformational rigidity and to the absence of nearly all the covalent reactions causing irreversible thermoinactivation in water [23]. 

Enhanced thermal stability enlarges the areas of application of protein films. In particular it might be possible to improve the yield of reactors in biotechnological processes based on enzymatic catalysis, by increasing the temperature of the reaction and using enzymes deposited by the LB technique. Nevertheless, a major technical difficulty is that enzyme films must be deposited on suitable supports, such as small spheres, in order to increase the number of enzyme molecules involved in the process, thus providing a better performance of the reactor. An increased surface-to-volume ratio in the case of spheres will increase the number of enzyme molecules in a fixed reactor volume. Moreover, since the major part of known enzymatic reactions is carried out in liquid phase, protein molecules must be attached chemically to the sphere surface in order to prevent their detachment during operation. 

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

Kumar, C.V. and Chaudhari A. (2003) Unusual thermal stabilities of some proteins and enzymes bound in the galleries of layered alpha-Zr(IV) phosphate/phosphonates. Microporous and Mesoporous Materials, 57,181-190. 

Tricalcium phosphate was also used as an enzyme embedding matrix. Das and coworkers [229] demonstrated that acid phosphatase and amylase immobilized on Ca3(P04)2 retained their activities with increased thermal stability. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

Removal of calcium from HRP C has a significant effect not only on enzyme activity and thermal stability, but also on the environment of the heme group. The calcium-depleted enzyme has optical, EPR, and H NMR spectra that are different from those of the native enzyme (211). Temperature dependence studies indicate that the heme iron exists as a thermal admixture of high- and low-spin states. Kinetic measurements at pH 7 show that ki, the rate constant for compound I formation, is only reduced marginally from 1.6 0.1 x 10 to 1.4 x lO M s , whereas k, the rate constant for compound II reduction, is reduced from 8.1 1.6 x 10 to 3.6 x lO M s (reducing substrate p-aminobenzoic acid), 44% of its initial value (211). There can be little doubt that this is the main reason for the loss of enzyme activity on calcium removal. 

Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

As described in the previous section, this strain produced significant amounts of three extracellular P-mannanases and a cell-associated P-mannosidase. The three P-mannanases differed in several enzymatic properties including optimum pH for enzyme action, optimum temperature, pH stability, thermal stability, isoelectric point and molecular weight. To elucidate the genetic basis for production of multiple forms,... 

In an attempt to dissect the overall endothermic envelope observed for the intact enzyme and the core fragment, both the core and the intact enzyme were studied by DSC at increasing pH values. In the case of two or more domains of a protein molecule that unfold independently of each other, but have thermal stabilities similar enough that the two independent transitions overlap at a given pH, it may be that the stabilities of the two domains are sufficiently different functions of pH that a change in pH will move the two peaks to different positions on the temperature scale. In an alternate... 

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