Enzymes thermostability

Tanner, J J., R.M. Hecht, and K.L. Krause. 1996. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceralde-hyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry 35 2597-2609. 

The kit includes necessary enzymes (thermostable a-amylase and amyloglucosidase), some reagents (buffer concentrate and glucose oxidase/peroxidase [GOPOD] reagent) and standards (glucose solution and com starch) to carry out 100 starch determinations. The other reagents required for this procedure may be obtained from any chemical supplier. 

The by-products, such as isomaltose, have to be removed chromatographically, which is energy- and cost-intensive. Protein engineering focuses on increasing the glucose yield (affecting enzyme selectivity) and enzyme thermostability. 

T. J. Ahern, J. I. Casal, G. A. Petsko, and A. M. Klibanov, Control of oligomeric enzyme thermostability by protein engineering, Proc. Natl. Acad. Sci. USA 1987, 84, 675-679. 

The incubation time t, although theoretically as short as possible, is typically between 15 and 45 min. Such a reaction period is based largely on the following factors the metabolite formation rates, the linearity of metabolite formation rates, the responsible enzyme thermostabilities, and substrate and enzyme concentrations. For CYP-mediated reactions the rates of metabolite formation are often moderate, and the CYP enzymes are often thermostable, resulting in... 

The serine protease from Thermus caldophilus strain GK-24 [292] gave maximum activity at 90 °C in 20 min assays and had a broad pH optimum with casein as substrate. The enzyme showed hydrolytic activity on some small peptide substrates (e.g. CBZ-L-leu-L-tyr-NHj) and also possessed esterase activity. Hydrolysis of synthetic chromogenic peptides and esters is also a property of the recently described serine protease, caldolase, from Thermus strain ToK3 [293]. This enzyme contained 10% carbohydrate and four disulphide bonds, but neither calcium nor zinc were detected in the purified enzyme. Thermostability of the enzyme was high in 0.4 M NaCl, but in low ionic strength buffer rapid thermal denaturation occurred at 75 °C. Work has also been undertaken in this laboratory... 

Low Temperature Process. The low temperature process was developed when B. licheniformis and B. stearothermophilus a-amylases became commercially available in the 1970s. These enzymes ate more thermostable, more acidutic, and requite less calcium for stabiUty than the B. subtilis enzyme used in the EHE process. Consequendy, the high temperature EHE heat treatment step was no longer requited to attain efficient Hquefaction. 

Dual-Enzyme Processes. In some cases, especially in symp production in Europe, a Hquefaction process is used that incorporates both a thermostable enzyme and a high temperature heat treatment. This type of process provides better hydrolyzate tilterabiHty than that attained in an acid Hquefaction process (9). Consequendy, dual-enzyme processes were developed that utilized multiple additions of either B. licheniformis or B. stearothermophilus a-amylase and a heat treatment step (see Eig. 1). 

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. 

In the alcohol industry, grain or potato raw materials are milled and water added to form a slurry or mash which is heated either batchwise or continuously. Traditionally, the mash is heated to 150°C by the injection of Uve steam. To reduce viscosity, a-amylases are added both during beating to 150°C and during cooling. Thermostable a-amylases from Bacillus licheniformis are the most commonly used enzymes for these processes (68). 

The interest and success of the enzyme-catalyzed reactions in this kind of media is due to several advantages such as (i) solubilization of hydrophobic substrates (ii) ease of recovery of some products (iii) catalysis of reactions that are unfavorable in water (e.g. reversal of hydrolysis reactions in favor of synthesis) (iv) ease of recovery of insoluble biocatalysts (v) increased biocatalyst thermostability (vi) suppression of water-induced side reactions. Furthermore, as already said, enzyme selectivity can be markedly influenced, and even reversed, by the solvent. 

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]. 

The research group of Backvall employed the Shvo s ruthenium complex (1) [21] for the racemization. This complex is activated by heat. For the KR they used p-chlorophenyl acetate as the acyl donor in combination with thermostable enzymes, such as CALB [20] (Figure 4.7). This was the first practical chemoenzymatic DKR affording acetylated sec-alcohols in high yields and excellent enantioselectivities. In the best case 100% conversion (92% isolated yield) with 99% ee was obtained. This method was subsequently applied to a variety of different substrates and it is employed (with a different ruthenium complex) by the Dutch company DSM for the large-scale production of (R)-phenylethanol [22]. 

The thermostable enzyme PAMO was the first BVMO identified to oxidize enolizable diketones in acceptable stereoselectivity (82% ee). The J -acetate obtained was hydrolyzed to J -hydroxyphenylacetone as an interesting intermediate for various pharmaceutical compounds (Scheme 9.23) [179]. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

In addition to proteases, other inhibitors reduce the activity of amylase and other digestive enzymes (Ishimoto et al, 1999). Many varieties of beans produce a glycoprotein that complexes with and inhibits a-amylase (Mirkov et al, 1995). The amylase inhibitors are non-competitive and thermostable (Gallaher and Schneeman, 1986) and, unlike protease inhibitors, do not elicit heightened secretion of amylase (Toskes, 1986). Although over-expression... 

CARMONA A (1996) Tannins thermostable pigments which complex dietary proteins and inhibit digestive enzymes. Latinoam Nutr. 44 31S-35S. 

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