Stable Enzymes

The enzymes with the long half-lives listed in Table 1 include the glycolytic enzymes aldolase, glyceraldehyde-phosphate dehydroge- 

A summary of the properties of both types of stable enzymes is given in Table 2. 

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Enzyme electrodes for other substrates of analytical significance have been developed. Representative examples are listed in Table 6-1. Further advances in enzyme technology, and particularly the isolation of new and more stable enzymes, should enhance the development of new biocatalytic sensors. New opportunities (particularly assays of new environments or monitoring of hydrophobic analytes) derive from the finding that enzymes can maintain then biocatalytic activity in organic solvents (31,32). 

Aqueous solutions are not suitable solvents for esterifications and transesterifications, and these reactions are carried out in organic solvents of low polarity [9-12]. However, enzymes are surrounded by a hydration shell or bound water that is required for the retention of structure and catalytic activity [13]. Polar hydrophilic solvents such as DMF, DMSO, acetone, and alcohols (log P<0, where P is the partition coefficient between octanol and water) are incompatible and lead to rapid denaturation. Common solvents for esterifications and transesterifications include alkanes (hexane/log P=3.5), aromatics (toluene/2.5, benzene/2), haloalkanes (CHCI3/2, CH2CI2/I.4), and ethers (diisopropyl ether/1.9, terf-butylmethyl ether/ 0.94, diethyl ether/0.85). Exceptionally stable enzymes such as Candida antarctica lipase B (CAL-B) have been used in more polar solvents (tetrahydrofuran/0.49, acetonitrile/—0.33). Room-temperature ionic liquids [14—17] and supercritical fluids [18] are also good media for a wide range of biotransformations. 

The entrapment method is based on confining the enzyme within the lattice of a polymeric matrix. Polyacrylamide gels have successfully yielded stable enzyme films with a high retention of activity... 

While PDF was originally proposed to be a zinc-metalloprotease [51], it is now generally accepted that Fe is the physiologically relevant metal ion occupying the active site in vivo [52], The native forms of most PDF enzymes are highly unstable due to propensity to oxidation, rendering them difficult to purify [53, 54], However, the Fe can be suitably replaced by either or Co, both of which provide a stable enzyme and main-... 

Archaea or Archaebacteria, which live in sulphurous waters around undersea volcanic vents. An extraordinarily stable enzyme which functions even at 135 °C and survives at pH 3.2-12.7 has been identified [142]. This enzyme has been termed STABLE (stalk-associated archaebacterial endoprotease). It is suggested that such exceptional stability may be attributable to unusually large Mr and tight folding of the protein chain. Suggested uses include washing powders and detergents, as well as industrial catalysts. It is even proposed that such remarkable properties may have contributed to the early evolution of life on earth [142]. 

This is a smaller, stable enzyme that has been highly purified. It is immunologically distinct from the other polymerases, indicating that it is not merely a subunit of the larger polymerases. Polymerase p is undoubtedly a repair enzyme. 

Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T. and Kivirikko, K.I. (1997) Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris. formation of a stable enzyme tetramer requires coexpression... 

Designing more stable enzymes e.g. by genetically engineering... 

Designing more stable enzyme proteins, e.g. via genetically replacing oxidation labile amino acids with more stable ones, has been followed for several other... 

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... 

The general rules that should therefore be observed include the use of a blanket of nitrogen whenever possible and evaporation of solvents at the lowest feasible temperatures, which must not exceed 50°C. The addition of an antioxidant such as butylated hydroxytoluene (2,6-di-/-butyl-4-methylphenol) to the extraction solvents (0.1 g 1 ) might be necessary to prevent deterioration of unsaturated lipids but it is essential for storage of lipid extracts at about 0.1% of the weight of lipid. Inactivation of lipolytic enzymes may usually be achieved by addition of an alcohol such as methanol or, in some cases, isopropanol. The latter is recommended for some more stable enzymes sometimes found in plant tissues. Alternatively the plant may be briefly immersed in boiling water. 

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). 

We should also remember that not all of the states that we see when freezing the enzyme (Section 7.4) are necessarily part of the mechanism. The most stable enzyme molecule is a dead one, so we must be aware that some of the spectroscopic signals represent damaged molecules. In the [NiFe] hydrogenases, the NiA and NiB states probably are not involved in the catalytic cycle, because they react slowly, if at all, with H2. In the mechanism shown in Fig. 8.3, it is assumed that the relevant active states are NiSR, NiA and NiR. 

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. 

If a stable enzyme form isomerizes and these isomerization steps are a part of the reaction pathway, the term Iso is provided. For a Uni Uni mechanism in which an isomerization step occurs [e.g., the reaction sequence E -I- A EA EA FP FP F -I- P and F E] is designated an Iso Uni Uni scheme. If more than one isomerization event occurs, then the prefixes di-, tri-, etc, are utilized with Iso. Note that this aspect of the nomenclature does not refer to isomerization steps which may or may not occur between central complexes. 

Chance Bi Bi mechanism. (This expression also applies to isomerization mechanisms for the ordered and Theorell-Chance schemes in which a stable enzyme form undergoes isomerization.) The points of intersection for each different mechanism can be used to exclude certain possibilities, depending on the quality of the rate data. 

However, the procedure has some drawbacks to overcome. First, it requires an elevated temperature (70 °C) for the activation of the racemization catalyst Such a high temperature is unacceptable for thermally less-stable enzymes. Second, the racemization proceeds through a mechanism including the release of ketone as a byproduct and thus the lowering of yield is inevitable. Third, PCPA used in an... 

Not much is known about the mechanisms that ensure efficient discrimination between ER proteins, which are in the process of folding and will finally form stable enzymes, and those, which fail to acquire their mature structure and have to be degraded. For example, small peptide stretches, which are buried in the mature but exposed in a native conformation of a protein, have... 

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. 

The deactivation process of enzymes can be classified generally into first-order and non-first-order processes. The first-order model is often sufficient for describing the deactivation, especially in case of highly stable enzymes in microemulsions, like lipases. For a lot of enzymes the activity decay in microemulsions has to be fitted to the deactivation model which involves an active intermediate in order to describe the deactivation processes qualitatively [34,100-102] ... 

Comparing the published synthesis reactions in reverse micelles, lipases seem to be the most stable enzymes in the microemulsion as reaction media and... 

Spray-dried cells have a stable enzyme activity enabling easy storage and transport of the dehalogenase biocatalyst. 

A traditional in vivo approach has been to select stable enzymes from organisms that grow in extreme conditions—engineering by nature. This highlights the importance of extensive screening for enzymes (Cheetham, 1987), and has provided insight into mechanisms of stabilization. 

Furthermore, it can be shown that, in the limiting cases of first-order kinetics [Equation (11.35) also holds for this case] and zero-order kinetics, the equal and optimal sizes are exactly the same. As shown, the optimal holding times can be calculated very simply by means of Equation (11.40) and the sum of these can thus be used as a good approximation for the total holding time of equal-sized CSTRs. This makes Equation (11.31) an even more valuable tool for design equations. The restrictions are imposed by the assumption that the biocatalytic activity is constant in the reactors. Especially in the case of soluble enzymes, for which ordinary Michaelis-Menten kinetics in particular apply, special measures have to be taken. Continuous supply of relatively stable enzyme to the first tank in the series is a possibility, though in general expensive. A more attractive alternative is the application of a series of membrane reactors. 

C), there is a broad range of temperature-induced decreases in enzyme activity. Catalase is seen as the most thermally stable enzyme in this group, while creatine phosphokinase (or CPK) is the most thermally sensitive enzyme. 

An NADP(H)-dependent ADH of Lactobacillus brevis (LBADH) was identified as a suitable catalyst accepting a broad range of diketo esters A as substrate [8]. This stable enzyme is easily available in the form of a crude cell extract (recLBADH) from a recombinant E. coli strain [9]. The reaction with diketo esters la-lc was performed on a preparative scale, using substrate-coupled regeneration of NADPH (Scheme 2.2.7.2). 

The occurrence of a peroxidase, lactoperoxidase (LPO), in milk was recognized as early as 1881. It is one of the most heat-stable enzymes in milk its destruction was used as an index of flash pasteurization (now very rarely used) and is now used as an index of super-HTST pasteurization. 

Coenzyme and substrate analogs. The structures of enzyme NAD1 substrate complexes (Fig. 15-3) may be studied by X-ray crystallography under certain conditions or can be inferred from those of various stable enzyme-inhibitor complexes or from enzyme reconstituted with NAD+ that has been covalently... 

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer of Mr2 X 47 316. It catalyzes the aminoacylation of tRNA1 in a two-step reaction. Tyrosine is first activated (equation 15.1) to form a very stable enzyme-bound tyrosyl adenylate complex. Tyrosine is then transferred to tRNA (equation 15.2).6... 

Methods for examining stable enzyme-substrate complexes 39... 

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