Denaturation characteristics

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Table 11.2. Denaturation Characteristics of Some Milk Proteins (Heated at 21.4°K/mln In 0.7 M Phosphate Buffer at pH 6).

Heat denaturation characteristics of M-/3-L were compared with those of endogenous / -lactoglobulin (see Figure 4). Values for the latter... 

Ahvenainen, R. 1996. New approaches in improving the shelf life of minimally processed fruits and vegetables. Trends in Food Science and Technology 7 179-186. Aktas, N. and Kaya, M. 2001a. Influence of weak organic acids and salts on the denaturation characteristics of intramuscular connective tissue. A differential scanning calorimetry study. Meat Science 58 413-419. 

Likewise, the strucmre of subtilisin (pH 3.0) suspended in varying ratios of acetonitrile and water demonstrated a-helical content similar to that in the lyophilized powder (Griebenow and Klibanov, 1996). Furthermore, the rate of transesteriflcation reactions of subtilisin (pH 7.8) suspended in DMSO/acetonitrile, formamide/acetonitrile or formamide/dioxane were increased approximately 100-fold over aqueous conditions (Almarsson and Klibanov, 1996). Similar results were obtained for subtilisin (pH 7.8) in a tetrahydrofuran/1-propanol mixture (Affleck et al., 1992). These results can be attributed to the increased structural rigidity of the active conformation of the protein in the solid, and the denaturing characteristics of the solvent at the solvent-particulate interface. Preservation of this molecular memory or molecular imprint of the protein can also be used to stabilize structure and activity (Mishra et al., 1996 Rich and Dordick, 1997 Santos et al., 2001). Subtilisin was lyophilized from crown ethers, resulting in more native like structure, by FTIR, and increased enzyme activity in THF, acetonitrile and dioxane (Santos et al.,2001). 

A DuPont model 990 thermal analyzer, modified to improve sensitivity and signal-to-noise ratio (Donovan, 1979), was used. Operating procedures and methods for determining denaturation characteristics were as described (Donovan and Beardslee, 1975 Donovan and Ross, 1973,1975). In a typical DSC experiment, components of the reaction mixture were combined and allowed to react in a small tube. An aliquot was weighed into a tared sample pan, and the pan was hermetically sealed. Weights of solvent were matched in the sample and reference pans. Sample and reference were placed in the DSC cell and heated. The heating rate was 10°C/min unless noted. 

Guaiacol has a very characteristic odour and burning taste its medicinal properties are identical with those of creosote. Used in the preparation of vanillin and paparvarin and for denaturing alcohol. 

Casein. Milk contains proteins and essential amino acids lacking in many other foods. Casein is the principal protein in the skimmed milk (nonfat) portion of milk (3—4% of the weight). After it is removed from the Hquid portion of milk, whey remains. Whey can be denatured by heat treatment of 85°C for 15 minutes. Various protein fractions are identified as a-, P-, and y-casein, and 5-lactoglobulin and blood—semm albumin, each having specific characteristics for various uses. Table 21 gives the concentration and composition of milk proteins. 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

Chemical Designations - Synonyms-. Alcohol Cologne spirit Denatured alcohol Ethanol Fermentation alcohol Grain alcohol Spirit Spirits of wine Chemical Formula CjHjOH. Observable Characteristics - Physical State (as normally shipped) Liquid Color Colorless Odor. Mild, pleasant Like wine or whiskey. (Denatured alcohol may be unpleasant). 

Like most chemical reactions, the rates of enzyme-catalyzed reactions generally increase with increasing temperature. However, at temperatures above 50° to 60°C, enzymes typically show a decline in activity (Figure 14.12). Two effects are operating here (a) the characteristic increase in reaction rate with temperature, and (b) thermal denaturation of protein structure at higher tem-... 

Filter aids are widely used in die fermentation industry to improve the efficiency of filtration. It is a pre-coated filter medium to prevent blockage or blinding of the filter by solids, which would otherwise wedge diemselves into the pores of the cloth. Filter aid can be added to the fermentation broth to increase the porosity of the cake as it formed. This is only recommended when fermentation product is extracellular. Filter aid adds to the cost of filtration. The minimum quantity needed to achieve the desired result must be established experimentally. Fermentation broths can be pretreated to improve filtration characteristics. Heating to denature proteins enhances the filterability of mycelial broths such as in penicillin production. Alternatively, electrolytes may be added to promote coagulation of colloids into larger, denser particles, which are easier to filter. The filtration process is affected by the viscosity and composition of the broth, and the cell cake.5... 

Its solubility characteristics in aqueous systems are such that retention of toxicity to insects by dissolved crystal protein is always suspect, and loss of activity on dissolution owing to denaturation is often observed. The protein is soluble only in relatively strong aqueous alkali. Thus, it has been variously reported to be soluble in 0.01N- to 0.05N sodium hydroxide (1) and alkali at pH 10.5 in the presence of thioglycollate (35) we have also observed its solubility in alkali at pH 9.5 in the presence of urea and potassium boro hydride. One difference between the characteristic proteins produced by various strains of crystalliferous bacilli is observed in the degree of alka-... 

There is a continuing interest to improve and extend the fimctional properties range of dairy proteins to provide both health benefits and their characteristic physical behaviors under different temperature, moisture, and pH conditions so that they may be included in foods that ordinarily do not contain them. One such research area is the extrusion texturization of whey proteins, which have resulted in dairy proteins with new characteristics imparted by a controlled texturization process, depending on the application desired (Hale et al., 2002 Manoi and Rizvi, 2008 Onwulata, 2009 Onwulata et al., 1998). Protein texturization is a two-step process that involves, first, the unfolding of the globular structure (denaturation) and, second, the alignments of the partially unfolded structures in the direction of mass flow in the extruder. The surface characteristics are imparted at the extruder die as the molten mass exits (Onwulata et al., 2003a). 

Extrusion is an effective means of denaturing whey proteins to create texturized products. TWP may be used as an ingredient to improve the characteristics of many foods. The production of snack foods wifh... 

In soil, the chances that any enzyme will retain its activity are very slim indeed, because inactivation can occur by denaturation, microbial degradation, and sorption (61,62), although it is possible that sorption may protect an enzyme from microbial degradation or chemical hydrolysis and retain its activity. The nature of most enzymes, particularly size and charge characteristics, is such that they would have very low mobility in soils, so that if a secreted enzyme is to have any effect, it must operate close to the point of secretion and its substrate must be able to diffuse to the enzyme. Secretory acid phosphatase was found to be produced in response to P-deficiency stress by epidermal cells of the main tap roots of white lupin and in the cell walls and intercellular spaces of lateral roots (63). Such apoplastic phosphatase is safe from soil but can be effective only when presented with soluble organophosphates, which are often present in the soil. solution (64). However, because the phosphatase activity in the rhizo-sphere originates from a number of sources (65), mostly microbial, and is much higher in the rhizosphere than in bulk soil (66), it seems curious that plants would have a need to secrete phosphatase at all. 

Fig. 6. Spectral monitoring of the thermal denaturation of the highly helical, Ala-rich peptide Ac-(AAAAK)3AAAA-YNH2 in D20 from 5 to 60°C, as followed by changes in the amide V IR (left) and VCD (right). IR show a clear shift to higher wavenumber from the dominant a-helical peak (here at an unusually low value, 1637 cm-1, due to full solvation of the helix) to a typical random coil value ( 1645 cm-1). VCD loses the (—,+,—) low-temperature helical pattern to yield a broad negative couplet, characteristic of a disordered coil, at high temperature. Spectra were normalized to A = 1.0 by 45°C.

Thermally denatured proteins have been studied for a variety of systems using FTIR and VCD. The resulting high-temperature spectra often reflect the characteristics seen earlier for random coil peptides as well as that seen for the unstructured casein. Particularly the amide I IR bands show a frequency shift to center on a broadened band at 1645-50 cm-1. The amide I VCD loses its distinctive character (Fig. 11) and tends toward... 

Fig. 11. Amide F thermal denaturation spectra for ribonuclease A as followed by FTIR (left) and VCD (right), which show the IR peak shifting from the dominant /3-sheet frequency (skewed with a maximum at 1635 cm-1) to the random coil frequency ( 1645-1650 cm-1) and the VCD shape changing from the W-pattern characteristic of an a + p structure to a broadened negative couplet typical of a more disordered coil form. The process clearly indicates loss of one form and gain of another while encompassing recognition of an intermediate form. (This is seen here most easily as the decay and growth back of the 1630 cm-1 VCD feature, but is more obvious after factor analysis of the data set, Fig. 15).

Gudiksen, K.L., Gitlin, I., Whitesides, G.M. (2006b). Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS. Proc. Natl. Acad. Sci. USA. 103, 7968-7972. 

The molecular folding of the backbone chain of the enzyme, as well as the distribution and content of amino acids [71] plays a decisive role in determining the characteristic specificity of an enzyme with regard to its reactions. This folding is markedly affected by temperature, for example. As the temperature rises, the chain gradually unfolds until a point is reached at which the enzyme becomes denatured and the catalytic activity is lost. 

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