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This trend in increased thermostability was observed for many other enzymes including proteases (Cowan, 1992), a-amylases and asparaginases (Daniel, 1986). It may therefore be assumed that a protein from a thermophilic micro-organism will be more thermostable then the homologous enzyme from a mesophilic source. This however, does not imply that all proteins from thermophiles will be more stable than all proteins from mesophiles (Cowan, 1992) because some proteins from mesophiles exhibit remarkable thermostability (e.g. myokinase; Daniel, (1986)).

Introduction Chapter I

1 11. T herm ostability

Thermostability is not an independant property, since it confers stability against dénaturation by organic solvents (Owasu and Cowan, 1989), detergents (Guagliardi et al., 1989) and proteolysis (Daniel e t a i , 1982).

The stabilisation o f a protein is due to many properties and is generally grouped as intrinsic and extrinsic stabilisation properties. These properties are associated with either the environment in which the protein exists or molecular forces within the protein.

1-11.1. intrinsic stabilisation

Intrinsic stabilisation in proteins is a result o f intramolecular electrostatic and hydrophobic interactions. Electrostatic interactions include ion pairs, hydrogen bonds, weak polar interaction and Van der Waals forces. Hydrophobic effects involve Van der Waals interactions and hydration effects o f non-polar groups (Privalov and Gill, 1988). These properties are specified by the amino acid sequence and thus reside in the secondary and tertiary structure o f the protein (Daniel, 1982).

Much o f the current understanding o f intrinsic protein stabilisation has arisen from amino acid homology comparisons, from site directed mutagenesis studies and from protein crystallographic analysis. The following are examples o f the “design rules” associated with protein stabilisation.

Mutations which lead to increases in cavity volume witliin the protein tend to be the most destabilising, whereas mutants in which the structure adjusts to minimise cavity volume tend to destabilise less (Ericksson et a i, 1992). Relaxation in the protein structure offsets the energy cost o f creating the cavity. The amount o f structural relaxation that occurs is therefore a major contributing factor in determining the change in protein stability (Ericksson et a i, 1993).

Flexibility indices (Vihinen, 1987), amide proton and hydrogen deuterium exchange rates and rates o f proteolysis (Wagner and Wuthrich, 1979; Daniel et a i,

Introduction C hapter I

counterparts at ambient temperatures but this stability becomes marginal at physiological temperatures (Daniel, 1986; Harris et a l, 1980; Vali et al., 1980). Furthermore, similar activity and catalytic mechanisms for enzymes o f mesophilic and thermophilic origin suggest similar conformational mobility at corresponding physiological temperatures. Lactate dehydrogenase from thermophiles were shown to be structurally more rigid than the mesophilic enzyme at mesophilic temperatures but some local sites o f flexibility were highly conserved (Vihinen, 1987). This suggested that only specific interactions were needed for an increase in stability and not a global amino acid composition change.

The comparison o f the amino acid sequence o f 3-phosphoglycerate kinase from

Saccharomyces cerevisiae and B. stearothermophilus showed significant amino acid

substitutions. In the thermophilic enzyme six lysine residues were substituted for arginine or glutamate and 2 0 alanine residues substituted by threonine and serine.

Alanine exhibits an higher affinity for helix formation than serine or threonine and glutamate is a known a-helix stabiliser (Davies et a l, 1993). These results suggest that keypoint amino acid substitutions can result in overall increased thermostability.

Further correlations between stability and aliphatic amino acid content have also been reported. The valine, alanine, isoleueine and leucine content o f thermostable enzymes are higher than in mesophilic counterparts in examples such as glucose phosphate isomerase (Muramatsu and Nosoh, 1971), malate synthase (Sundaram et a l,

1980) and glyceraldehyde-3-phosphate dehydrogenase (Hoching and Harris, 1976). This increase was presumed to increase hydrophobic interactions but studies on lactic acid dehydrogenase from thermophilic and mesophilic bacilli indicated an increase in the number o f polar amino acid residues at the active site. The increased thermostability was attributed to the presence o f more ionic and hydrogen bond interactions (Zuber, 1979).

Pyrococcus furiosus glutamate dehydrogenase exhibited a half-life at 100"C of twelve hours whereas a glutamate dehydrogenase o f Clostridium symbiosum displayed a half life o f twenty minutes at 52^C. One major contributing property for this difference m thermostability was the number o f surface residues that formed ion pair networks.

Introduction C hapter I

The thermophilic and mesophilic enzymes were seen to have a similar number of charged residues but the thermostable enzyme formed a greater number o f salt bridges o f which some were at crucial inter-subunit domains (Yip et al., 1995; Rice et at.,

1996). Structural data from this comparison indicated that salt-bridge networks may play an important role in maintaining the stability o f the thermophilic protein at high temperatures. Further comparisons o f glutamate dehydrogenase o f P. furiosus and

Thermotoga litoralis showed a decrease in the number o f sulphur containing amino acids with increased thermostability (Britton et a i, 1995). These findings indicate that there are several mechanisms involved in protein stabilisation and that their use varies considerably.

Various studies comparing proteins o f mesophilic and thermophilic origin (Craik

et al., 1985; Tanaka et at., 1971) show that glycine, serine, lysine and asparagine are generally substituted by alanine, threonine, arginine and glutamate respectively. These substitutions cause an increase in internal and decrease in external hydrophobicity and favour helix formation. Glutamate was found to promote helix formation and the glutamate and pro line contents increased with increasing thermostability properties. Furthermore, it was calculated that there was only a slight energy difference between thermostable andtherm olabile enzymes in the order o f 20 to 30 kJ.m of' (Kinney, 1980). This difference in free energy can be accounted for by only several additional hydrogen bonds or one to two salt bridges (Perutz, 1978). This demonstrates that an increase in stability does not require an overall structural change but only a few keypoint changes.

In summary, increased thermostability arises from a decrease in tertiary structure flexibility. This may be achieved via numerous mechanisms including disulphide bridges, ion-pairs and increased internal and decreased external hydrophobicity^. These stabilisation mechanisms vary in significance from protein to protein and although the majority o f stability is gained through intrinsic mechanisms many enzymes in thermophiles obtain added stabilisation via extrinsic stabilisation.

Introduction Chapter I

1-11.2. Extrinsic stabilisation.

Extrinsic stabilisation is gained by interaction with cell constituents such as substrates, metal ions, cofactors, solutes and membranes.

For example, alkaline phosphatase o f B. stearothermophilus lost activity when released from protoplasts. This phenomenon was attributed to cell membrane stabilisation (Welker, 1976). Chemical binding o f manganese, ATP and L-glutamate was shown to increase the stability o f glutamine synthases o f B. stearothermophilus and o f B. caldolyticus (M erkler et al., 1988). Calcium ions incorporation in proteases was also shown to increase with increased thermostability (Cowan et al., 1985). Subtilisin and thermolysin contained four calcium ions whereas caldolysin contained six calcium ions and was at least 30 times more thermostable.

Chang and Mahoney (1994) reported that bovine serum albumin (BSA) could increase the thermostability o f p-galactosidase o f Streptococcus thermophila

approximately ten fold. The relationship was found to be concentraton dependant. They reported that the stabilisation was probably due to hydrophobic interactions between the two proteins, which did not affect the catalytic activity.

Various novel low molecular weight organic compounds such as di-glycerol phosphate, 2-0 -p-mannosylglycerate and p-mannosylgl y cerate were found in

thermophiles and hyperthennophilies. The cellular concentration o f these compounds were found to increase with increasing growth temperatures (Martins & Santos, 1995; 1996; 1997; Ramos et a i, 1997). P-Mannosylglycerate was shown to protect mesophilic, thermophilic and hyperthermophilic proteins against freeze-drying and heat dénaturation (Ramos et al., 1997).

The result o f maintaining an osmotic equilibrium forces many halophiles to produce low molecular weight compounds. Two o f these compounds are 2-methyl-4- carboxy-l,4,5,6-tetrahydropyrimidines and hydroxyectoines and were reported to be powerful protein stabilising agents against heating, freezing and drying o f proteins (Lippert and Galinski, 1992).

Introduction C hapter I

1-12. Industrial applications and potential of thermophiles

Thermophilic bacteria and their enzymes possess several potentially beneficial properties which might be considered advantageous in high temperature industrial processes. Some o f these factors are outlined in Table 1-12.1.

Table 1-12.1. Advantages of high temperature industrial hioconversions.

Property Advantage in Process

Thermostability Tolerate high temperatures, long half-lives, increased resistance to denaturing agents such as organic solvents, high and low pH, detergents etc.

Little activity at low temperature, long shelf life Tolerate “harsh” purification methods; produces increased yields

Genes can be cloned into mesophiles A heating step simplifies purification High optimum teinperature

General robustness

Chemical reaction rates

Solubility Viscosity

Microbial contamination

Biological activity in raw materials

Operate at high temperatures

Diffusion and other chemical processes are accelerated

Higlier concentration of poorly insoluble compounds Viscosity decreases, mass transfer rates increase, mixing and pumping can be increased

Growth of most pathogens and most environmental mesophiles is prevented. Thermophilic treatment of sewage wastes will kill of pathogenic bacteria and viruses

Heating kills most interfering enzyme and microbial

activities v.

Distillation of volatile end products such as ethanol Table 1-12.1. taken from Kristjansson, (1989).

These advantages do not apply to all processes as there are significant drawbacks in carrying out some bioconversions at elevated temperatures, particularly in the use o f thermo-sensitive raw materials, cofactors and other chemicals. High temperatures also place greater stress on equipment and limit the materials that can be used. Gas solubility decreases with increasing temperatures, which may have limited benefit to processes such as those utilising anaerobic bacteria. For this and other reasons replacing existing

Introduction Chapter I

micro-organisms or enzymes for thermostable counterparts may not be a feasible option. This was outlined in a review by Cowan (1995) - “An analysis o f the operational properties o f some o f the existing industrial enzymes, suggests that in many cases, a thermostable alternative would be very unlikely to replace the enzyme in current use. This conclusion is based on several practical considerations; (i) Certain industrial biocatalytic processes are incompatible with high temperature operation (e.g., cheese-making); (ii) Some commercial trends favour low temperature operation (e.g., domestic detergents), (iii) Enzymes o f sufficient thermostability are already in use (e.g., saccharification o f starch); (iv) There is no obvious process advantage in increasing the reaction temperature (e.g., biosynthetic production o f fine chemicals such as amino acids and penicillins) and (v) Many o f the large industrial biocatalyst users are committed to existing low temperature operation through investment in specific plant and equipment. The advantages o f higher temperature operation would be insufficient to offset the costs o f replacing that equipment.

In summary, the conditions under which a hyperthermophilic enzyme would be a serious contender as a replacement for any "bulk" commercial enzyme might include many (if not all) o f the following:

(i) Available in similar (or equivalent) quantity with equally reliable supply.

(ii) Similar or lower price (per unit o f activity).

(iii) Significantly better performance in many respects (not just stability). (iv) No major alterations to existing plant and equipment (or investment

in new plant) required.”

Introduction C hapter I

Nevertheless, there are successful applications o f thermophilic micro-organisms and thermostable enzymes, o f which some are listed in Table 1-12.2.

Table 1-12.2. Examples of commercially available thermostable enzymes,

organisms and their applications

Enzyme Organism Application

DNA polymerase T. aquaticus Polymerase chain reaction in

molecular biology

T th ll 1\ restriction T. thermophila DNA sequencing

endonuclease

BstEll restriction B. stearothermophilus DNA sequencing

endonuclease

Taq\ restriction T. aquaticus DNA sequencing

endonuclease

?TcTaq (protease) Thermus. spp. DNA purification

Thermolysin B. thermoproteolyticus Synthesis o f aspartame

Acetate kinase B. stearothermophilus ATP regeneration system

Leucine dehydrogenase B. stearothermophilus Leucine aminopeptidase measurement

Alanine dehydrogenase B. stearothermophilus Synthesis and measurement o f alanine

B. stearothermophilus ‘'Spore strip” sterility indicators

B. stearothermophilus Penicillin measurement in milk

... L ... ... . . . . .

Introduction C hapter I

1-13. Aims

Relatively few examples o f thermostable nitrile metabolising enzymes have yet been reported. It was therefore the aim o f this project to identify, isolate and characterise a nitrile degrading system from a thermophilic organism, on the assumption that such an enzyme would be more thermostable than those derived from mesophilic organisms. Specific aims included microbial identification and growth characterisation, development o f a protein purification strategy, investigation o f enzyme(s) characteristics and cloning studies on the gene encoding the nitrile hydrolysing enzyme.

These results could potentially answer some fundamentally important questions including;

• How closely related are thermophilic bacterial nitrile metabolising enzymes to their mesophilic counterparts?

• Are nitrile hydrolysing enzymes from mesophiles and thermophiles structurally and functionally similar?

• How does enzyme thermostability affect nitrile hydrolysis? Is there a compromise between stability, specific activity and substrate specificity?

• Do thermostable nitrile hydratases have suitable functional properties for industrial application?

M aterials and M eth od s Chapter II

Chapter II