39

Extremozymes David W Hough* and Michael J Danson

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.
Addresses Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK *e-mail: D.W.Hough@bath.ac.uk Current Opinion in Chemical Biology 1999, 3:3946 http://biomednet.com/elecref/1367593100300039 Elsevier Science Ltd ISSN 1367-5931

of enzymes (extremozymes) with extreme stability, and the application of these enzymes as biocatalysts is attractive because they are stable and active under conditions that were previously regarded as incompatible with biological materials. Furthermore, it is clear that some extremophiles, particularly those from the Archaea, have novel metabolic pathways and so might serve as a source of enzymes with novel activities and applications. Recent work suggests that the diversity of organisms in extreme environments is far greater than was initially suspected. The majority of extremophiles have not yet been isolated in pure culture, however, and thus it is difficult to determine the stability characteristics and precise substrate specificity and enantioselectivity of their enzymes. In this review, we have concentrated on papers appearing in 1997 and 1998 and discuss recent advances in the isolation and identification of extremozymes, progress towards understanding the stability of extremozymes at a molecular level and enzyme engineering to create novel biocatalysts with enhanced stability and altered specificity.

Introduction
The use of enzymes as biotransformation catalysts is well established and has been the subject of numerous texts and reviews. The majority of enzymes used to date, however, have been obtained from mesophilic organisms and, despite their many advantages, the application of these enzymes is restricted because of their limited stability to extremes of temperature, pH, ionic strength, etcetera. On the other hand, extremophiles are microorganisms that are found in environments of extreme temperature (2 to 15C, 60110C), ionic strength (25 M NaCl) or pH (<4, >9). The majority of extremophiles are members of the Archaea, one of the three phylogenetic domains of life, defined by comparison of 16S rRNA gene sequences, although extremophilic members of the bacterial domain are also known [1]. Extremophiles are a source
Table 1 Extremophiles and their environments. Phenotype Thermophilic Hyperthermophilic Environment 5580C 80113C

Extremophiles and extremozymes

It is now recognised that extreme environments, once thought to be too hostile to permit the survival of living organisms, are the natural habitat of certain microorganisms known as extremophiles. The discovery of extremophiles, particularly those that inhabit high temperature environments (thermophiles), stimulated attempts to define the most extreme conditions that remain compatible with the existence of life [2]. An overview of the range of extreme environments in which extremophiles have been detected is presented in Table 1. The majority of extremophiles identified to date are members of the Archaea [3]. Although mesophilic Archaea have been identified, the majority of archaeal

Typical genera Methanobacterium, Thermoplasma, Thermus*, some Bacillus* species Aquifex*, Archaeoglobus, Hydrogenobacter*, Methanothermus, Pyrococcus, Pyrodictium, Pyrolobus, Sulfolobus, Themococcus, Thermoproteus, Thermotoga* Alteromonas*, Psychrobacter* Haloarcula, Halobacterium, Haloferax, Halorubrum Acidianus, Desulfurolobus, Sulfolobus, Thiobacillus* Natronobacterium, Natronococcus, some Bacillus* species

Psychrophilic Halophilic Acidophilic Alkaliphilic

2 to 20C 25M NaCl pH<4 pH>9

*Genera of the domain Bacteria; all others are Archaea.

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Figure 1 The universal phylogenetic tree, constructed from rRNA sequence comparisons. Branches representing the three domains (Archaea, Bacteria, Eukarya) are indicated. Thermophilic and hyperthermophilic species are underlined and halophilic species are shaded. Reproduced with permission from [20].

Microsporidia

Animals Ciliates Slime moulds

Plants Fungi

Euglena

Diplomonads (Lambila)

Eukarya
Green nonsulphur bacteria (chlorflexus)

Bacteria

Gram-positives Thermotoga

Purple bacteria Flavobacteria Cyanobacteria

Green sulphur bacteria

Aquifex Hydrogenobacter

Desulfuro- Sulfolobus coccus Thermofilum PyroThermoproteus dictium Thermococcus Methanothermus Methanobacterium Archaeoglobus Halococcus Halobacterium Methanopyrus Methanoplanus Methanococcus Methanospirillum species Methanosarcina

Archaea

species have been detected in extreme environments. Extremophilic bacteria have also been identified, and it is interesting to note that they are amongst the most primitive bacteria, as suggested by their location close to the root of the universal phylogenetic tree (Figure 1). Recent developments in the isolation and sequencing of environmental DNA samples have indicated that only a small fraction, probably between 0.11%, of organisms from the environment can be obtained in pure culture by present methods [4], a statement that seems to apply to both moderate (i.e. mesophilic) [5] and extremophilic [6,7,8] environments. It is generally true that the enzymes of an organism are adapted to function optimally at or near its growth conditions, and so the range of extremes at which life is found defines the range of conditions at which enzyme activity might be detected. The greater the diversity of organisms detected in the environment, the greater the potential range of biocatalysts available for applications such as biotransformations. The classical route to microbial enzyme production, involving isolation of the organism as a pure culture followed by purification of the enzyme either from its natural host or by cloning and expression of the corresponding gene, has been used to obtain extremozymes [9,10]. Alternative approaches have been applied in situations where pure cultures are not available, however. These include screening of expression libraries prepared from environmental DNA [11] or cloning of PCR-amplified gene fragments to produce hybrid genes for expression in a mesophilic host [12].

Possibly the most significant recent advances in molecular microbiology have resulted from rapid progress in the field of genomics, the determination and analysis of the entire DNA sequence of an organism. The number of microbial genome sequences available has increased rapidly to sixteen, including five from extreme thermophiles [1317] with at least two more reported to be near completion [18,19]. Analysis of these genome sequences, involving comparison with gene sequences of proteins of known function from other organisms, has resulted in the identification of approximately half of their open reading frames. Since the genome data include the entire complement of enzymes from each organism, there is now a large database for the selection of extremozymes for biotransformations and other applications; however, although the putative function of a gene product can be identified by sequence comparisons, this needs to be confirmed by analysis of the expressed protein product. Also, characterisation of the expression product is essential to provide additional information such as substrate specificity, enantioselectivity and stability.

Structural basis of enzyme stability

High temperature

Many attempts have been made to understand the stability of extremozymes in terms of their three-dimensional (3D) structure. This approach requires high-resolution structural data for homologous enzymes from both mesophiles and extremophiles so that differences, which might result in enhanced stability of the extremozyme, can be identified

Extremozymes Hough and Danson

41

by structural comparison [20]. Most work in this area has focused on thermophilic and hyperthermophilic enzymes, where a number of studies of a series of glutamate dehydrogenase [21] and citrate synthase [22] structures have indicated the importance of ionic interactions, a suggestion first made by Perutz and Raidt [23] in 1975. In both these families of enzymes, the hyperthermophilic members show an increase in the number and extent of ion-pair networks, and in the hyperthermophilic citrate synthase, these networks involve intersubunit interactions. Also, in the case of citrate synthase there is an increase in compactness associated with increasing thermostability, together with additional inter-subunit interactions involving amino acid residues near the carboxyl terminus [22]. The association between an increase in ion-pair networks and hyperthermostability is not universal, however. Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has fewer intersubunit ion pairs and an increased number of hydrophobic interactions when compared with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus [24]. This observation suggests that different mechanisms of thermostabilisation might be utilised in bacterial and archaeal enzymes. A novel feature detected in the structure of a thermostable -glycosidase from Sulfolobus solfataricus is the existence of solvent-filled hydrophilic cavities in the core of the protein in addition to surface ion-pair networks [25]. It has been suggested that the surface ion-pair and buried solvent networks provide a degree of resilience and resistance to thermal denaturation. This is in contrast to the widely held view that increasing thermostability is associated with an increase in structural rigidity. It is clear that there are a number of features that have been identified as possible contributors to increased protein thermostability; however, there are substantial differences between the conclusions reached with different proteins and there appears to be no universal rule for the structural basis of stability. Furthermore, most comparative studies are qualitative in nature and at present we have little or no information on the quantitative significance of the various structural features that might contribute to the observed stability characteristics of thermozymes.
Low temperature

appears to be much more accessible to substrates than in mesophilic and thermophilic citrate synthases. Psychrophilic enzymes must also possess structural adaptations as protection against cold denaturation, which is caused in part by a diminished hydrophobic interaction at low temperatures. The cold-active citrate synthase contains more intramolecular ion pairs than the hyperthermophilic enzyme from P. furiosus, and it has been suggested that these may serve to prevent cold-induced unfolding.
High ionic strength

Extremozymes from halophilic organisms have adapted to remain stable and active at high ionic strength, an essential requirement since the intracellular environment in extreme halophiles is isotonic with the growth medium [29]. Amino acid sequence comparisons indicate that halophilic proteins are highly acidic compared with their mesophilic counterparts, and 3D structures obtained by X-ray crystallography [30,31] or homology modelling [32] show that these enzymes have an excess of negatively charged amino acids on their surfaces. The role of surface negative charge on halophilic enzymes is to bind hydrated ions, so retaining a surface hydration layer, and to reduce their surface hydrophobicity, thus reducing the tendency to aggregate at high salt concentrations. The recent report of the 3D structure of a formyltransferase [33] from the hyperthermophile Methanopyrus kandleri, which grows at 98C and has an internal phosphate concentration of 1.5 M, shows that this enzyme also has halophilic adaptations, having a surface with low hydrophobicity and high acidity. In addition, it has ionic networks connecting secondary structural elements, a feature that might relate to its thermostability characteristics.
Extreme pH

Both acidophilic and alkaliphilic extremophiles are known and, although their internal pH values are close to neutrality, it is clear that their extracellular enzymes must be stable and active at the appropriate pH extreme. Indeed, these enzymes were amongst the first extremozymes to be exploited because of their enhanced stability. At present, there are no structural data available for these enzymes, and so the basis of their stability remains a mystery.

Engineering or evolution?
Engineering

In contrast to hyperthermophilic enzymes, enzymes from psychrophiles are cold-active and undergo thermal inactivation at moderate temperatures. It has been suggested that these enzymes are more flexible than their mesophilic counterparts in order to remain active at low temperatures [26], and two recently published crystallographic structures support this conclusion [27,28]. Analysis of the structure of a cold-active citrate synthase [27] identified extended, charged surface loops that might confer additional flexibility in the active-site region. Although the overall mechanism of catalysis is conserved, the active site

If the goal of understanding the structural basis of the stability and activity of extremozymes can be achieved, this will provide a rational basis for engineering stability into mesophilic enzymes by site-directed mutagenesis. Although our understanding in this area is far from complete, the importance of ionic interactions in relation to thermostability, first suggested by Perutz and Raidt [23], has been demonstrated by several recent structural studies [20]. Experimental verification of the importance of ion pairs has been obtained by site-directed mutagenesis of pigeon liver malic enzyme to produce a double mutant containing an additional glutamate and lysine residue, and hence a putative additional ion pair [34]. This

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resulted in an enzyme with a 10C increase in melting temperature and improved stability to urea-induced unfolding, although this was achieved at the expense of reduced enzymatic activity. It has proved much more difficult to produce mutants with enhanced stability while retaining full catalytic activity, although the removal of stabilising features generally results in the expected decrease in enzyme stability [35]. Indeed, it has been suggested that there is an inverse relationship between the effects of mutation on stability and catalysis [36], and recent work on 3-isopropylmalate dehydrogenase from themophilic and mesophilic bacteria demonstrates a close relationship between conformational flexibility and catalytic function [37]. Interestingly, Giver et al. [38] have shown that thermostability and activity may be at least partially independent, although mutations that enhance both properties are rare. Since the structural basis of the stability of extremozymes is not fully understood, an alternative protein engineering approach is to design mutants by comparison of the amino acid sequences of homologous enzymes with differing stability. Using this approach, Van den Burg et al. [39] introduced a number of mutations into a moderately stable thermolysin-like protease from Bacillus stearothermophilus. They produced an eightfold mutant that was 340 times more stable at 100C than the wild type protein, with a half-life of 170 min and the ability to degrade proteaseresistant substrates at this temperature. The mutations were of the type generally considered as rigidifying mutations that are thought to reduce the entropy of the unfolded state; they included glycine to alanine and alanine to proline substitutions and the introduction of a disulphide bridge. In this case, rational design has produced an enzyme with increased thermal stability without compromising its catalytic activity. Again, this approach requires information on amino acid sequences, and preferably 3D structures, of families of enzymes that differ in their stability to temperature or other extreme conditions.
Evolution

gene. The T. thermophilus host system is available, and other thermophilic hostvector systems are under development [41]. In principle, a similar approach could be used to select mutant enzymes with improved activity under high salt or low temperature conditions. Suitable shuttle vectors are available for the halophilic host Haloferax volcanii [32], but expression vectors for use with a psychrophilic host have not yet been reported.
Directed evolution

Directed evolution is a method that involves sequential stages of random mutagenesis, recombination and screening for mutants with desired characteristics, and does not depend on in vivo selection of mutants in an appropriate extremophilic host. In a recent review of the directed evolution approach, Kuchner and Arnold [42] present data on the generation of a p-nitrobenzoyl esterase with enhanced thermostability. Five cycles of sequential mutagenesis by error-prone PCR, with selection on the basis of catalytic activity and thermostability, produced an active enzyme with a melting temperature that was increased by 14C above that for wild type. A similar approach has been used [43] with subtilisin BPN, an alkaline protease from the mesophilic bacterium Bacillus amyloliquifacieus. A mutant enzyme was obtained that has improved activity at low temperature but without loss of stability at higher temperatures. Although mutagenic rates in error-prone PCR can be controlled by the conditions used, the method has significant bias and not all amino acid substitutions are accessible by this approach [44].
DNA shuffling

A radically different approach to increasing the stability of an enzyme is to adopt an evolutionary strategy. In this case, introduction of mutations into a target gene on a random basis is followed by selection of mutants with the desired properties, which could include enhanced stability, altered catalytic specificity or any other change in property than can be used as a basis for a selective screen. Improved thermostability of a mesophilic 3-isopropylmalate dehydrogenase from B. subtilis has been achieved by Akanuma et al. [40]. They used a stepwise process of random in vitro mutagenesis followed by in vivo selection in a thermophilic host, Thermus thermophilus, to produce a triple mutant enzyme with enhanced thermostability and a significantly higher specific activity than the wild type; however, this approach is limited by its requirement for an extremophilic expression host with a deletion of the corresponding host

The random recombination of fragments from closely related gene sequences (DNA shuffling) creates novel genes and provides an alternative basis for directed enzyme evolution. This technique has been applied to mutants produced by error-prone PCR to yield enzymes with enhanced activity towards non-natural substrates [45]. As an alternative, DNA shuffling has been carried out between naturally occurring homologous genes, which provides functional diversity [46]. In this case, a library of chimaeric proteins comprising segments of the enzyme cephalosporinase originating from different species was produced and assayed for improved monolactamase activity. This process was much more efficient than shuffling DNA fragments of genes derived by mutagenesis from a single sequence. Clearly the DNA shuffling approach could also be applied to generate variants of extremozymes with improved stability or activity towards a particular substrate. DNA shuffling has also been used for functional evolution of the arsenic resistance operon (ars) of Escherichia coli [47], resulting in a 40-fold increase in resistance to the presence of arsenate in the growth medium. The resistant mutant was found to have 13 mutations in the ars operon and ten in the arsenite membrane pump, while other mutants resulted in increased levels of expression of the arsenate reductase gene. These results demonstrate that DNA shuffling can improve the function of metabolic

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pathways as a result of complex, unexpected effects and are of considerable significance for the development of in vivo biotransformation processes.

Novel enzymes
Attempts to enhance the stability of mesophilic enzymes or to manipulate the specificity of extremozymes by directed evolution depend on the availability of the appropriate cloned genes and expression systems. Furthermore, detailed structural information is an essential prerequisite for protein engineering. An alternative route to the discovery of enzymes with appropriate specificity and stability characteristics for a particular biotransformation process is via screening of either established cultures or expression libraries prepared from environmental DNA samples. This approach was followed by Ito et al. [48] to identify enzymes with potential for application in the detergent industry. Since laundry and dishwashing detergents operate under alkaline conditions, Ito et al. isolated alkaliphilic Bacillus strains that produce extracellular enzymes including cellulase, protease, -amylase and starch debranching enzymes. Similarly, an intracellular nitrile hydratase has been identified in a Bacillus strain growing optimally at 65C [49]. Unlike mesophilic nitrile hydratases, the enzyme was stable up to 60C in aqueous solution in the absence of substrates. Screening of expression libraries of the hyperthermophile P. furiosus has identified recombinants producing an extracellular -amylase with thermal activity and stability properties that are a significant improvement on the commercially available enzyme from Bacillus licheniformis [50,51]. Unlike -amylases, which degrade starch by hydrolysis of -1,4 linkages, amylopullulanases degrade starch and related polysaccharides by hydrolysis of both -1,6 and -1,4 glucosidic bonds. An amylopullulanase clone has also been isolated [52] and the enzyme shown to have high resistance to chemical denaturants and surfactants. A similar approach has yielded a thermostable esterase [53] that extends the range of microbial esterases available for biotransformation applications. On occasions, the use of an extremozyme allows the generation of novel products that are not produced by the mesophilic enzyme. Fischer et al. [54] identified a -glucosidase from P. furiosus that could be used as a catalyst for glucoconjugate synthesis at high temperature. The enzyme accepted a wide range of alcohols and organosilicon alcohols with cellobiose as the glucose donor, producing a series of novel glucoconjugates. Novel applications of extremophiles as biotransformation catalysts are not restricted to situations where a specific extremozyme has been identified and isolated. There are a number of reports of in vivo biotransformation or biodegradation processes mediated by growing cultures of extremophiles, particularly by moderately thermophilic anaerobes. Mixed cultures of anaerobes maintained at 75C were able to transform synthetic pyrethroid insecticides to noninsecticidal products [55], potentially a useful step in environmental bioremediation. Dehalogenation of

3-chlorobenzoate was also achieved in mixed cultures of anaerobic thermophiles [56]. Biodegradation using a defined bacterial strain has advantages over the use of mixed cultures, and degradation of halophenols such as 2-chlorophenol or 2-bromophenol to corresponding halocatechols was obtained using a strain of thermophilic Bacillus [57]. Clearly there is potential for further development in this area through the identification of isolates capable of other biodegradation reactions and of isolates able to carry out in vivo biotransformation to yield a required end product. Also, once the enzymology of these bioconversions is understood, it might be possible to carry out similar reactions in a cell-free environment using purified enzymes.

Enzyme production
The viability of processes based on the use of extremozymes is dependent on the availability of enzyme in sufficient quantity for practical application. This immediately raises problems if the enzyme is isolated directly from the source organism or for in situ reactions involving whole cells. The conditions required for the growth of extremophiles include temperatures above 80C (hyperthermophiles), often in an anaerobic environment, and media of extreme pH (acidophiles and alkaliphiles) or containing up to 5 M NaCl (halophiles). These conditions are incompatible with standard industrial fermentation and the downstream processing plant. Most studies on extremozymes have focused on thermophilic enzymes and, similarly, most work on large-scale cultivation methods is related to the problems of growing thermophilic and hyperthermophilic microorganisms. For example, three different approaches to the growth of these organisms were compared [58] and it was shown that hyperthermophiles and thermoacidophiles could be grown at high cell density, leading to improved productivity. Because of the problems presented by large-scale culture of extremophiles and subsequent purification of extremozymes, most applications rely on the expression of the corresponding gene in a mesophilic host. This approach avoids problems arising from the need to grow extremophiles, but it introduces further complications resulting from the expression of genes encoding extremozymes in a non-native, mesophilic environment. It is now generally accepted that genes from thermophiles and hyperthermophiles can be expressed successfully in a mesophilic host such as E. coli, although difficulties might be encountered when the expressed enzyme requires specific cofactors or metal ions that the host does not utilise. Expression normally yields a soluble product that has similar activity to the native material when assayed at an appropriate temperature [9]. Furthermore, heat precipitation of host cell protein provides a simple initial stage in purification of the expression product. On occasions, the expressed product is assembled in a slightly different oligomeric state from the native material [59], although this has little impact on the stability or catalytic properties of the enzyme. Similarly, successful expression of genes

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encoding cold-active enzymes from psychrophilic bacteria has been obtained [60,61], although it was found that the mesophilic expression host (E. coli) must be grown at temperatures below its growth optimum in order to avoid thermal denaturation of the expression product. Expression of halophilic enzymes in a mesophilic host is a much more complex problem than expression of thermophilic enzymes. The intracellular environment of an extreme halophile such as Haloferax volcanii is isotonic with the growth medium. The cells accumulate KCl at concentrations up to 5 M, and this is the environment in which halophilic enzymes are synthesised and in which they fold into their native conformation. Heterologous expression of genes from halophiles in a mesophilic host such as E. coli takes place in a low ionic strength environment under conditions that would result in the inactivation of halophilic enzymes. In some cases, heterologous expression yields a soluble, inactive product [62], whereas in other cases the expression product is insoluble [63]. Unfolding or solubilisation in the presence of a denaturant, such as guanidine hydrochloride or urea, followed by renaturation in the presence of NaCl, normally results in an active product with properties similar to the native material. In the recent report of expression in E. coli of seryl-tRNA synthetase from Haloarcula marismortui [64], however, the soluble expression product could not be reactivated. Although there are no reports in the literature to date, similar difficulties might be expected if genes encoding extracellular extremozymes stable at high or low pH are expressed in a mesophilic host. Although the internal pH of acidophilic and alkaliphilic microorganisms is close to neutrality, their extracellular enzymes are secreted into a low or high pH medium. Expression of such enzymes in a mesophilic host such as E. coli would not allow the proteins to fold in a native-like environment and therefore problems are likely to arise as a result of insolubility or inactivity.

far from complete. Therefore, we do not yet have the theoretical basis for engineering enhanced stability into a mesophilic enzyme of choice, nor are we able to alter the specificity and catalytic activity of extremozymes in a predictable fashion. For this reason, the directed evolution approach, involving random mutagenesis and DNA shuffling followed by selection for desirable characteristics, has so far proved much more successful than traditional protein engineering. This situation seems likely to continue well into the future, particularly as the mutations generated and selected in directed evolution experiments are not limited to those found in naturally occurring, stable enzymes.

Acknowledgements
The authors would like to thank the Biotechnology and Biological Sciences Research Council for their support, and also NATO, The British Council and The Royal Society for grants for collaboration and travel.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest

1. Brown JR, Doolittle WF: Archaea and the prokaryote-to-eukaryote transition. Microbiol Mol Biol Rev 1997, 61:456-502. An excellent review on the universal tree of life from different biochemical perspectives. The authors review the concept of Archaea, Bacteria and Eukarya, and then present and critically assess phylogenetic trees using a wide variety of protein sequences. 2. Blochl E, Rachel R, Burggraf S, Hafenbrandl D, Jannasch HW, Stetter KO: Pyrolobus fumarii, gen. and sp. nov. represents a novel group of Archaea extending the upper temperature limit for life to 113C. Extemophiles 1997, 1:14-21. This paper describes the most extreme hyperthermophile isolated to date. This organism has novel metabolic properties and can survive autoclaving for one hour at 121C. 3. Woese CR, Kandler O, Wheelis ML: Towards a natural system of organisms-proposal for the domains Archaea, Bacteria and Eukarya. Proc Natl Acad Sci USA 1990, 87:4576-4579. Hugenholtz P, Pace NR: Identifying microbial diversity in the natural environment: a molecular phylogenetic approach. Trends Biotechnol 1996, 14:190-197. Bintrim SB, Donohue TJ, Handelsman J, Roberts GP, Goodman RM: Molecular phylogeny of Archaea from soil. Proc Natl Acad Sci USA 1997, 94:277-282. Barns SM, Delwiche CF, Palmer JD, Pace NR: Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc Natl Acad Sci USA 1996, 93:9188-9193. Harmsen HJM, Prieur D, Jeanthon C: Distribution of microorganisms in deep sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture of thermophilic subpopulations. Appl Env Microbiol 1997, 63:2876-2883.

4.

5.

Conclusions and future prospects

The potential conflict between the harsh conditions of an industrial process and the limited stability of enzymes has long been recognised. Extremozymes, with their increased stability and activity under extreme conditions, provide a possible solution to this problem. In this review, we have described the biodiversity of extremophiles and the diversity of their metabolic organisation. Also, we have noted that only a small number of species from this large group of organisms have been isolated and characterised in detail. Thus, it is clear that a large number of extremozymes are as yet undiscovered. As more organisms are isolated and more extremozymes are characterised, the possibilities for application of extremozymes in biocatalysis and biotransformations are bound to increase. It is possible that the boundaries defining the limits of life will be extended to even greater extremes. It is also clear that our understanding of the structural basis of enzyme stability and activity under extreme conditions is

6.

7.

8.

Hugenholtz P, Pitulle C, Hershberger KL, Pace NR: Novel division level bacterial diversity in a Yellowstone hot spring. J Bacteriol 1998, 180:366-376. A culture-independent molecular phylogenetic survey that significantly expands our perception of bacterial diversity and calls into question the commonly held belief that Archaea dominate hydrothermal environments. 9. Connaris H, West SM, Hough DW, Danson MJ: Cloning and expression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Extremophiles 1998, 2:61-66.

10. Kim YO, Lee JK, Kim HK, Yu JH, Oh TK: Cloning of a thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiol Lett 1998, 162:185-191. The first reported nucleic acid sequence of a bacterial phytase. 11. Winson MK, Kell DB: If youve got it, flaunt it rapid screening for microbial biocatalysts. Trends Biotechnol 1997, 15:120-122.

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12. Okuta A, Ohnishi K, Harayama S: PCR isolation of catechol 2,3 dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene 1998, 212:221-228. The central and terminal fragment of the catechol 2,3-dioxygenase gene were amplified from environmental DNA, annealed to form full-length hybrids and expressed in E. coli. A library of functional hybrid genes was created without the need to isolate the parent bacteria. 13. Bult CJ, White O, Olsen GJ, Zhou LX, Fleischmann RD, Sutton GG, Blake JA, FitzGerald LM, Clayton RA, Gocayne JD et al.: Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 1996, 273:1058-1073. 14. Smith DR, Doucette Stamm LA, Deloughery C, Lee HM, Dubois J, Aldredge T, Bashirzadeh R, Blakely D, Cook R, Gilbert K et al.: Complete genome sequence of Methanobacterium thermoautotrophicum delta H: functional analysis and comparative genomics. J Bacteriol 1997, 179:7135-7155. 15. Klenk HP, Clayton RA, Tomb JF, White O, Nelson KE, Ketchum KA, Dodson RJ, Gwinn M, Hickey EK, Peterson JD et al.: The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 1997, 390:364-370. 16. Deckert G, Warren PV, Gaasterland T, Young WG, Lenox AL, Graham DE, Overbeek R, Snead MA, Keller M, Aujay M et al.: The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 1998, 392:353-358. 17. Kawarabayasi Y, Sawada M, Horikawa H, Haikawa Y, Hino Y, Yamamoto S, Sekine M, Baba S, Kosugi H, Hosoyama A et al.: Complete sequence and gene organization of the genome of a hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3. DNA Res 1998, 5:55-76.

there were no crystal structures of psychrophilic enzymes and all the structures discussed were generated by homology modelling. 27. Russell RJM, Gerike U, Danson MJ, Hough DW, Taylor GL: Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 1998, 6:351-361. The first report of a crystal structure of a psychrophilic enzyme, together with an analysis of its structure in terms of the proteins cold activity, thermolability and resistance to cold denaturation. 28. Aghajari I, Feller G, Gerday C, Haser R: Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci 1998, 7:564-572. A second psychrophilic enzyme structure, although its analysis in terms of its cold-active properties will be the subject of a forthcoming paper. 29. Danson MJ, Hough DW: The structural basis of protein halophilicity. Comp Biochem Physiol A 1997, 117A:307-312. This paper reviews the structures of the enzymes malate dehydrogenase, dihydrofolate reductase and dihydrolipoamide dehydrogenase from halophilic Archaea and discusses these in the light of models for the structural basis of halophilicity. 30. Dym O, Mevarech M, Sussman JL: Structural features that stabilise halophilic malate dehydrogenase from an Aarchaebacterium. Science 1995, 267:1344-1346. 31. Frolow F, Harel M, Sussman JL, Mevarech M, Shoham M: Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe2S ferredoxin. Nat Struct Biol 1996, 3:452-457. 32. Jolley KA, Russell RJM, Hough DW, Danson MJ: Site-directed mutagenesis and halophilicity of dihydrolipoamde dehydrogenase from the halophilic Archaeon Haloferax volcanii. Eur J Biochem 1997, 248:362-368. 33. Ermler U, Merckel MC, Thauer RK, Shima S: Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri new insights into salt-dependence and thermostability. Structure 1997, 5:635-646. This paper describes the crystal structure of a formyltransferase from a methanogenic Archaeon that grows optimally at 98C and has an internal salt concentration of 1.5 M phosphate. The structure is therefore analysed in terms of the enzymes combined thermophilic and halophilic nature. 34. Huang S-H, Chou W-Y, Lin S-I, Chang G-G: Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein. Proteins 1998, 31:61-73. A double mutant of pigeon liver malic enzyme was designed to introduce an additional ion-pair. The mutant was much more stable to thermal or urea-induced denaturation but was less active than the wild type enzyme. 35. Pappenberger G, Schurig H, Jaenicke R: Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. J Mol Biol 1997, 274:676-683. One of the most lucid accounts of analysing the changes in kinetics and thermodynamics of protein thermostability after site-directed mutagenesis. 36. Shoichet BK, Baase WA, Kuroki R, Matthews BW: A relationship between protein stability and protein function. Proc Natl Acad Sci USA 1995, 92:452-456. Zavodsky P, Kardos J, Svingor A, Petsko G: Adjustment of conformational flexibility is a key event in the thermal adaption of proteins. Proc Nat Acad Sci USA 1998, 95:7406-7411. Isopropylmalate dehydrogenase from Thermus thermophilus has optimum activity at 2225C above the E. coli enzyme and a 17C higher melting temperature. HD exchange showed that the Thermus enzyme is significantly more rigid at room temperature, whereas both enzymes have similar flexibility at their respective optimum temperatures. 38. Giver L, Gershenson A, Freskgard P-O, Arnold FH: Directed evolution of a thermostable esterase. Proc Natl Acad Sci USA 1998, 95:12809-12813. Directed evolution of p-nitrobenzyl esterase from Bacillus subtilis produced a mutant with a 14C increase in melting temperature with no reduction in catalytic activity at lower temperatures. This suggests that these properties are not necessarily inversely correlated, although mutations that increased stability without compromising activity were rare. 39. Van den Burg B, Vriend G, Veltman OR, Venema G, Eijsink VGH: Engineering an enzyme to resist boiling. Proc Natl Acad Sci USA 1998, 95:2056-2060. One of the most impressive examples of enzyme engineering that combines rationally designed mutations with changes based on sequence compar37.

18. FitzGibbon S, Choi AJ, Miller JH, Stetter KO, Simon MI, Swanson R, Kim UJ: A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum. Extremophiles 1997, 1:36-51. 19. Sensen CW, Klenk HP, Singh RK, Allard G, Chan CCY, Liu QY, Penny SL, Young F, Schenk ME, Gaasterland T et al.: Organizational characteristics and information content of an archaeal genome: 156 kb of sequence from Sulfolobus solfataricus P2. Mol Microbiol 1996, 22:175-191. 20. Danson MJ, Hough DW: Structure, function and stability of enzymes from the Archaea. Trends Microbiol 1998, 6:307-314. This is one of the most recent reviews of the structural basis of archaeal enzyme stability, covering thermostability, psychrophily and halophilicity. 21. Yip KSP, Britton KL, Stillman TJ, Lebbink J, de Vos WM, Robb FT, Vetriani C, Maeder D, Rice DW: Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family. Eur J Biochem 1998, 255:336-346. A homology-based modelling study of glutamate dehydrogenases from species spanning a wide range of growth temperatures. The role of ion-pair networks in hyperstability is emphasised. 22. Russell RJM, Ferguson JMC, Hough DW, Danson MJ, Taylor GL: The crystal structure of citrate synthase from the hyperthermophilic Archaeon Pyrococcus furiosus at 1.9 resolution. Biochemistry 1997, 36:9983-9994. 23. Perutz M, Raidt H: Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2. Nature 1975, 255:256-259. 24. Knapp S, de Vos WM, Rice D, Ladenstein R: Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima at 3.0 resolution. J Mol Biol 1997, 267:916-932. A comparison of the crystal structure of a thermophilic enzyme with hyperthermophilic and mesophilic homologues. Hydrophobic interactions appear to be important in thermophilicity, whereas ionic networks are a common hyperthermophilic feature. 25. Aguilar CF, Sanderson I, Moracci M, Ciaramella M, Nucci R, Rossi M, Pearl LH: Crystal structure of the b-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus: resilience as a key factor in thermostability. J Mol Biol 1997, 271:789-802. The paper introduces the novel concept of resilience to thermal denaturation through surface ion-pair and buried solvent networks, and contrasts this with idea of protein thermophilicity being linked to increased rigidity. 26. Gerday C, Aittaleb M, Arpigny JL, Baise E, Chessa JP, Garsoux G, Petrescu I, Feller G: Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1997, 1342:119-131. This is a good review of cold environments, the organisms that grow in them, and the properties of their psychrophilic enzymes. At the time of publication,

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isons with thermostable homologues. A protease is produced that is able to function at 100C but which retains wild type activity at 37C. 40. Akanuma S, Yamagishi A, Tanaka N, Oshima T: Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution. Protein Sci 1998, 7:698-705. An excellent example of in vivo evolutionary engineering to increase the thermostability of an enzyme while at the same time improving the catalytic activity. 41. Cannio R, Contursi P, Rossi M, Bartolucci S: An autonomously replicating transforming vector for Sulfolobus solfataricus. J Bacteriol. 1998, 180:3237-3240. A plasmid shuttle vector able to transform and to be stably maintained both in S. solfataricus and in E. coli was constructed. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. 42. Kuchner O, Arnold FH: Directed evolution of enzyme catalysts. Trends Biotechnol 1997, 15:523-530. Reviews the directed evolution approach to engineering biocatalysts, including techniques for creating and screening combinatorial enzyme libraries. Examples include the evolution of thermostability, resistance to organic solvents, substrate specificity and enantioselectivity. 43. Kano H, Taguchi S, Momose H: Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Appl Microbiol Biotechnol 1997, 47:46-51. Random mutagenesis of subtilisin BPN resulted in the isolation of a mutant with increased activity at low temperature (110C). The thermostability of the mutant was similar to the wild type enzyme. 44. Shafikhani S, Siegel RA, Ferrari E, Schellenberger V: Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. Biotechniques 1997, 23:304-310. 45. Moore JC, Jin H-M, Kuchner O, Arnold FH: Stategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J Mol Biol 1997, 272:336-347. This paper describes an enhancement of the direct evolution approach in order to produce further improvements in the properties of enzyme biocatalysts. Following initial rounds of random mutagenesis, the benefits of random recombination of gene fragments in vitro (DNA shuffling) are compared with additional rounds of random mutagenesis. 46. Crameri A, Raillard SA, Bermudez E, Stemmer WP: DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 1997, 391:288-291. Reviews the DNA shuffling approach, by which libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer WPC: Molecular evolution of an arsenate detoxification pathway DNA shuffling. Nat Biotechnol 1997, 15:436-438. This paper describes the functional evolution of an arsenic resistance operon by DNA shuffling and homologous recombination. It demonstrates how DNA shuffling can improve the function of a pathway by complex and unexpected mutational mechanisms that are likely to be overlooked by rational design. 48. Ito S, Kobayashi T, Ara K, Ozaki K, Kawai S, Hatada Y: Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics and structures. Extremophiles 1998, 2:185-190. 49. Pereira RA, Graham D, Rainey FA, Cowan DA: A novel thermostable nitrile hydratase. Extremophiles 1998, 2:347-357. 50. Dong GQ, Vieille C, Savchenko A, Zeikus JG: Cloning, sequencing, and expression of the gene encoding extracellular alphaamylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 1997, 63:3569-3576. The expressed enzyme (-amylase) has optimal activity at 100C and is much more thermostable than commercially available amylases. 47.

51. Jorgensen S, Vorgias CE, Antranikian G: Cloning, sequencing, characterization and expression of an extracellular alpha-amylase from the hyperthermophilic Archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J Biol Chem 1997, 272:16335-16342. 52. Dong GQ, Vieille C, Zeikus JG: Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 1997, 63:3577-3584. 53. Ikeda M, Clark DS: Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotechnol Bioeng 1998, 57:624-629. A thermophilic esterase was identified by screening a genomic library of P. furiosus. The enzyme has the highest temperature optimum and thermal stability of all esterases reported to date. 54. Fischer L, Bromann R, Kengen SWM, de Vos WM, Wagner F: Catalytical potency of beta-glucosidase from the extremophile Pyrococcus furiosus in glucoconjugate synthesis. Biotechnology 1996, 14:88-91. 55. Maloney SE, Marks TS, Sharp RJ: Detoxification of synthetic pyrethroid insecticides by thermophilic microorganisms. J Chem Technol Biotechnol 1997, 68:357-360. 56. Maloney SE, Marks TS, Sharp RJ: Degradation of 3-chlorobenzoate by thermophilic microorganisms. Lett Appl Microbiol 1997, 24:441-444. 57. Reinscheid UM, Bauer MP, Muller R: Biotransformation of halophenols by a thermophilic Bacillus sp. Biodegradation 1997, 7:445-461.

58. Holst O, Manelius , Krahe M, Mrkl H, Raven N, Sharp R: Thermophiles and fermentation technology. Comp Biochem Physiol 1997, 118A:415-422. This paper describes methods to overcome the problems associated with the growth of thermophiles to high cell density in a fermentor by the use of gas-lift fermentors, dialysis fermentors or cell recycling systems. 59. Hess JM, Tchernajenko V, Vieille C, Zeikus JG, Kelly RM: Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli. Appl Environ Microbiol 1998, 64:2357-2360. Expression of the T. neapolitana xylose isomerase gene in E. coli produced a dimeric recombinant with similar stability and biocatalytic properties to the native, tetrameric enzyme. 60. Gerike U, Danson MJ, Russell NJ, Hough DW: Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R. Eur J Biochem 1997, 248:49-57. One of the first reports of the expression of a psychrophilic enzyme in E.coli, highlighting the need to grow the host at low (27C) temperatures to get maximum yields of active recombinant protein. 61. Feller G, LeBussy O, Gerday C: Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol 1998, 64:1163-1165. 62. Cendrin F, Chroboczek J, Zaccai G, Eisenberg H, Mevarech M: Cloning, sequencing and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui. Biochemistry 1993, 32:4308-4313. 63. Blecher O, Goldman S, Mevarech M: High expression in Escherichia coli of the gene encoding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii. Eur J Biochem 1993, 216:199-203. 64. Taupin CM-J, Hartlein M, Leberman R: Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui; isolation, characterization and sequencing of the gene and its expression in Escherichia coli. Eur J Biochem 1997, 243:141-150.