Heat-stable enzymes

Thermostable enzymes, heat stable enzymes a small number of enzymes, mostly hydrolases, which show their highest activity between 60 and 80 °C, and which are stable and catalytically active at up to -100 °C. T.e. usually have a compact structure, stabilized by many disulfide bonds and/or extensive hydro-phobic regions, and a low a-helix content. Examples... 

Co-enzymes.—Heat-stable organic compomids naturally associated with complex enzyme systems, and necessary for zymolysis. They act as carriers of hydrogen, phosphate, or other intermediate reactants in the system. 

A slurry of the starch is cooked in the presence of a heat-stable bacterial endo-a-amylase. The enzyme hydrolyzes the a-l,4-glycosidic bonds in pregelatinized starch, the viscosity of the gel rapidly decreases, and maltodextrins are produced. The process may be terrninated at this point. The solution is purified and dried, and the maltodextrins are utilized as blandtasting functional ingredients in dry soup mixes, infant foods, sauces and gravy mixes, etc. 

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. 

In this chapter we describe the use of pea seeds to express the bacterial enzyme a-amylase. Bacterial exoenzymes like the heat stable a-amylase from Bacillus licheni-formis are important for starch hydrolysis in the food industry. The enzymatic properties of a-amylase are well understood [13,14], it is one of the most thermostable enzymes in nature and it is the most commonly used enzyme in biotechnological processes. Although fermentation in bacteria allows highly efficient enzyme production, plant-based synthesis allows in situ enzymatic activity to degrade endogenous reserve starch, as shown in experiments with non-crop plants performed under greenhouse conditions [12,15]. Finally, the quantitative and sensitive detection of a-amylase activ-... 

The purple colonies of T. roseopersicina are lifted on a filter paper, transferred onto a stack of filter papers soaked with oxidised redox dye (benzyl viologen) under air. Following heat treatment, the cells containing heat stable, active enzyme turn blue under hydrogen atmosphere,those containing defected hydrogenase remain purple [Fodor et al., 2001],... 

C5a is inactivated by the myeloperoxidase-H202 system, which oxidises a methionine residue (Met 70) on the molecule group A streptococcal endo-proteinases also abolish chemotactic activity of C5a and related compounds. Neutrophil lysosomal enzymes (e.g. elastase and cathepsin G) also destroy C5a chemotactic activity, but as these proteases are inhibited by the serum antiproteinases, a -antiproteinase and a2-macroglobulin, the physiological role of neutrophilic proteases in the inactivation of C5a is questionable. Two chemotactic factor inactivators have been found in human serum an a-globulin that specifically and irreversibly inactivates C5-derived chemotactic factors, and a / -globulin that inactivates bacterial chemotactic factors. These activities are heat labile (destroyed by treatment at 56 °C for 30 min) and are distinct from those attributable to anaphylatoxin inactivator. An apparently specific inhibitor of C5-derived chemotactic activity has also been described in human synovial fluid and peritoneal fluid. This factor (molecular mass of 40 kDa) is heat stable and acts directly on C5a. 

Enzymes from these organisms find numerous applications beyond PCR. Certain ones can modify plant fibers or break down proteins or fats. Heat-stable enzymes with these digestive properties are very attractive to the food processing industry. Properly controlled, their action can make prepared foods more palatable, and they can be employed where ordinary enzymes fail. In food processing, all operations must be carried out under sterile conditions. Frequently, the easiest way to maintain sterility is to keep the... 

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

ATP and magnesium were required for the activation of acetate. Acetylations were inhibited by mercuric chloride suggesting an SH group was involved in the reaction either on the enzyme or, like lipoic acid, as a cofactor. Experiments from Lipmann s laboratory then demonstrated that a relatively heat-stable coenzyme was needed—a coenzyme for acetylation—coenzyme A (1945). The thiol-dependence appeared to be associated with the coenzyme. There was also a strong correlation between active coenzyme preparations and the presence in them of pantothenic acid—a widely distributed molecule which was a growth factor for some microorganisms and which, by 1942-1943, had been shown to be required for the oxidation of pyruvate. 

Selenoprotein A is remarkably heat stable, as seen by the loss of only 20% of activity on boiling at pH 8.0 for lOmin (Thrner and Stadtman 1973). Although selenoprotein A contains one tyrosine and no tryptophan residues, it contains six phenylalanine residues and thus has an unusual absorbance spectrum (Cone et al. 1977). Upon reduction, a unique absorption peak emerges at 238 nm, presumably due to the ionized selenol of selenocysteine, which is not present in the oxidized enzyme. The activity of selenoprotein A was initially measured as its ability to complement fractions B and C for production of acetate from glycine, in the presence of reducing equivalents (e.g., dithiothreitol). Numerous purification schemes were adopted for isolation of selenoprotein A, all of which employed the use of an anion exchange column to exploit the strongly acidic character of the protein. 

APS reductase from different sources shows considerable differences in stability of activity. The enzyme from Desulfovibrio desulfuricans was stable, but that from T. thioparus was labile and could not be purihed (Peck et al. 1965). In contrast, APS reductase from T. denitrificans was purihed to homogeneity and was remarkable heat-stable (Bowen et al. 1966 Taylor 1989). 

PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonu-cleoside triphosphates. We have already looked at the chemistry of DNA replication (see Section 14.2.2), and this process is exactly the same, though it is carried out in the laboratory and has been automated. 

Serum alkaline phosphatase elevations have been reported following administration of salt-poor albumin (B5). Placenta is very rich in a heat-stable alkaline phosphatase, and albumin prepared from placental blood has a high activity of this enzyme. In one cirrhotic patient who received 1-6 units per day of albumin obtained from pooled human blood and/or human placenta, the alkaline phosphatase before infusion was 5 Bodansky units and by the thirteenth day of administration had reached a value of 160 units. The physician administering the albumin at first thought the patient was having a severe toxic liver reaction and stopped the therapy. The alkaline phosphatase then started to go down and within 10 days returned to normal levels. Analysis of the albumin indicated that it contained 470 units of alkaline phosphatase activity and was probably responsible for the observed elevations in the serum enzyme activity. Albumin prepared from venous blood did not cause an alkaline phosphatase elevation, but placenta-albumin caused elevations with a half-life of about 8 days (Ml). 

Ethambutol is a water-soluble, heat-stable compound that acts by inhibition of arabinosyl transferase enzymes that are involved in cell wall biosynthesis. Nearly all strains of M tuberculosis and M. kansasii and most strains of Mycobacterium avium-intracellulare are sensitive to ethambutol. Drug resistance relates to point mutations in the gene (EmbB) that encodes the arabinosyl transferases that are involved in mycobacterial cell wall synthesis. 

In spite of a rich life, no signaling or defensive secondary metabolite has ever been reported from organisms thriving on the hydrothermal vents. Lack of competition in these areas, where the hostile environment provides protection from invaders, has not stimulated the formation of defensive metabolites. Defensive heat-stable enzymes and cell walls were raised against the hostile environment. 

To increase the thermostability of existing enzymes by genetic engineering techniques is difficult, since molecular determinants that increase thermostability are hard to predict. Comparisons of heat labile and heat stable stmctures do however indicate that... 

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