Thermostability of enzymes

Thermostability of enzymes increases in apolar organic solvents increasing the stability and rigidity of the molecules. This effect is probably due to low-water activity [130]. 

Martinek, K., Klibanov, A.M. Goldmacher, V.S. and Berezin, I.V. (1977) The principles of enzyme stabilization. I. Increase in thermostability of enzymes covalently bound to a complementaiy surface of a polymer support in a multipoint fashion. Biochim. Biophys. Acta, 485, 1-12. 

Peptides that form a helices that associate as coiled coils, or as three- or four-helix tetrameric bundles or amphipathic helices that associate with lipid bilayers have been made. More difficult has been the design of proteins that form (3 sheets. Many efforts are being made to xmderstand protein stability " by systematic substitutions of one residue for another. Addition of new disulfide linkages at positions selected by study of three-dimensional structures sometimes stabilizes enzymes. On the other hand elimination of unnecessary cysteine residues can enhance stability by preventing p elimination and replacement of asparagine by threonine can improve the thermostability of enzymes by preventing deami-dation. ... 

Cabral JMS and Kennedy JF (1993) Immobilisation techniques for altering thermal stability of enzymes. In Gupta MN (ed.) Thermostability of Enzymes, pp. 163-179. Berlin Springer. 

Cabral JMS, Keimedy JF (1993) In Gupty MN (ed) Thermostability of Enzymes. Springer, Berlin, p 163... 

Martinek, K., A. M. Klibanov, V. S. Goldmacher, and 1. V. Berezin. 1977. The Principles of Enzyme Stabilisation. 1. Increase in Thermostability of Enzymes Covalently Bound to a Complementary Surface of a Polymer Support in a Multi-Point Fashion. Biochim Biophys Acta 485 1-12. 

Another line of approach to the elucidation of increased thermostability of enzymes is the analysis of significant priorities in amino acid replacements, resulting from a comparison of pairs of homologous proteins from thermophiles and mesophiles [21] (Figure 8). The left-hand part of the figure shows a general scheme derived from a statistical treatment of several pairs of thermostable and thermolabile enzymes. 

Table 1. Thermostabilities of enzymes from extreme thermophiles. Enzymes are listed under class headings in order of discussion in the text a ... 

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. 

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]. 

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. 

Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm ) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response. 

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

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. 

For design of a simple manufacturing process, the thermostability of the NP enzymes is a very useful feature. Although heat treatment can be used as part of a purification protocol to isolate the enzymes from contaminating materials, the high temperature of operation itself excludes undesired enzyme-catalysed side reactions. For example, in the synthesis of 9-p-D-arabinofuranosyladenine from Ara-U and adenine, using a wet cell paste of Enterobacter aerogenes, adenine and Ara-U mainly underwent deamination at lower temperatures to form hypoxanthine and uracil respectively. At elevated temperature, deamination was completely eliminated and the rate of transarabinosylation increased. 

HPMA copolymers are water-soluble biocompatible polymers, widely used in anticancer drug delivery (reviewed in Reference [22]). HPMA copolymers containing reactive groups at side-chain termini were previously used for the modification of trypsin [23], chymotrypsin [23,24], and acetylcholinesterase [25]. The modification dramatically increased the acetylcholinesterase survival in the blood stream of mice and the thermostability of modified enzymes when compared to the native proteins. However, the modification involved multipoint attachment of the copolymers to the substrates, which may cause crosslinking. To modify proteins or biomedical surfaces by one point attachment, semitelechelic polymers should be used. 

There have been a number of reports of the use of enzymes in the extraction of oils from sources such as fish, rape seed, yeast, palms, and soya beans. Celluloses and pectinases are used in pdm oil extraction. In soya bean and fish, much oil has been found to be associated with protein, so that addition of proteases increases the yield of oil and protein. Use of thermostable proteases is preferred, but m general the use of enzymes is limited by the minimal water contents of the various process streams. Trichoderma uride and A niger celluloses, hemicellulases and proteases have been used to extract hydrocarbons from Euphorbia plants 39 40) and similar enzymes used to extract sapogenins from Helleborus 41). 

Activity Assays. The standard activity assay mixture of 3 ml contained about 0.1 U/ml lignin peroxidase, 0.4 mM veratryl alcohol (Fluka, purum >97%) and 0.1M sodium tartrate, pH 3.0. The reaction was started by adding 15 fil of 54 mM H2O2 to make a final concentration of 0.28 mM in the reaction. The production of veratraldehyde was followed by recording the change of absorbance for 12 seconds at 310 nm in a cuvette which was thermostated to 37°C. The reaction was started 24 seconds before the recording. One unit of lignin peroxidase is defined as the amount of enzyme required to oxidize one imol of veratryl alcohol to veratraldehyde in one minute. 

Thermostability of Thermoanaerohacter sp. CGTase. The addition of 40ppm Ca+ + to the CGTase preparation during incubation at high temperatures in the absence or presence of starch substrate provided no enhancement of the thermostability of the enzyme. A comparison of the thermostable CGTase was made to other thermostable enzymes used in starch liquefaction including Termamyl Bacillus licheniformis) and Bacillus stearothermophilus alpha-amylase. 

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