hyperthermophilic enzyme

Protein & Peptide Letters, 2006, 13, 645-651 645

0929-8665/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

Industrial Applications of Hyperthermophilic Enzymes: A Review

Trinidad de Miguel Bouzas1, Jorge Barros-Velázquez2 and Tomás González Villa1,*

1Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, E- 15782 Santiago de Com-postela, Spain; and 2Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Veterinary Sciences,University of Santiago de Compostela, E-27002 Lugo, Spain

Abstract: Over the past two decades, research scientists have been involved in the investigation of thermophilic and hy-perthermophilic microorganisms owing to the unique features of their enzymic systems. Such in-depth investigations arenow on their way to mastering the cloning and industrial exploitation of a broad variety of genes encoding enzymes in-volved in starch hydrolysis, amino acid biosynthesis, protein hydrolysis, etc. In this work, we review the state of the artand future perspectives of industrial applications of enzymes from hyperthermophilic and extreme thermophilic microor-ganisms, special attention being paid to the biotechnological methods involved in their industrial exploitation.

Keywords: Thermophilic enzymes, Molecular cloning, Microorganisms, Industrial technology, Food industry, Chemical in-dustry.

INTRODUCTION

In 1967, Thomas D. Brock of the University of Wiscon-sin reported for the first time the existence of microorgan-isms with optimal growth temperatures higher than 75ºC,growing in the hot springs of Yellowstone National Park [1].Since then until now, the work of diverse research groupshas led to the discovery of more than 20 different genera ableto grow above 80ºC. Such organisms are now termed “hy-perthermophiles”.

The enormous difficulty involved in obtaining pure cul-tures from these microorganisms, together with their compli-cated large-scale culture, initially limited the study of theirenzymes. Nevertheless, in the late 1980s the first “hyper-thermophilic enzymes” were purified, which proved to beextremely stable at high temperatures and to exhibit no orvery low activity at temperatures below the growth condi-tions of the organisms from which they were obtained.

The unexpected finding that these enzymes can be clonedand expressed in mesophilic hosts without losing their activeconformation and thermostability solved the initial produc-tion problem. In this sense, the availability of an increasingnumber of thermostable enzymes opened new possibilitiesfor industrial processes. Due to their overall inherent stabil-ity, such enzymes have found commercial applications in thestarch industry, in the synthesis of amino acids, in the petro-leum, chemical, pulp and paper industries, and indeed inmany other fields.

Microorganisms Growing at Extreme Temperatures

The current classification considers thermophiles all or-ganisms growing above 55ºC, moderate thermophiles above65ºC, extreme thermophiles above 75ºC and hyperthermo-

*Address correspondence to this author at the Department of Microbiology,Faculty of Pharmacy, University of Santiago de Compostela, E- 15782Santiago de Compostela, Spain; Tel: +34.981.563100, Ext. 14949; Fax:+34.981.582490; E-mail: [email protected]

philes above 90ºC, although some authors make a singlegroup of hyperthermophiles also including extreme thermo-philes [2, 3]. In this review, we will name extremophiles allmicroorganisms with an optimal growth temperature above80ºC. These microorganisms, represented only by Bacteriaand Archaea, have been isolated from a large variety of natu-ral marine and water-containing terrestrial environments, aswell as from artificial environments [4]. Natural terrestrialbiotopes of hyperthermophiles include hot springs and sol-fataric fields with a wide range of pH (0.5-9.0), low salinity(0.1-0.5%) and deep geothermally heated oil-containingstratifications. Natural marine biotopes are shallow hydro-thermal systems, abyssal hot vents (“black smokers”) andactive seamounts where the salinity levels are high (3%) andthe pH values range from 5.0 to 8.5. Artificial environmentsin which hyperthermophiles have been found includedsmouldering coal refuse piles and hot outflows from geo-thermal and nuclear power plants.

The great interest shown by the scientific community inhyperthermophilic microorganisms is reflected in the in-creasing number of microbial species that have been de-scribed over the last 30 years and the number of genomesequences that have been completed since the beginning ofthe worldwide genome sequencing projects. Thus, currentlymore than 70 species of hyperthermophiles have been de-scribed; 14 genome-sequencing projects have been com-pleted (Aeropyrum pernix K1, Aquifex aeolicus VF5, Ar-chaeoglobus fulgidus VC-16, Methanocaldococcus jan-naschii DSM2661, Methanopyrus kandleri AV19, Pyroba-culum aerophilum IM2, Pyrococcus abyssi GE5, P. furiosusJCM8422, P. horikoshii OT3, Sulfolobus acidocaldariusDSM639, S. solfataricus P2, S. tokodaii 7, JCM10545,Thermococcus kodakaerensis KOD1, Thermotoga maritimaMSB8), and more than 20 others are in progress [4, 5].

One of the main challenges for scientists studying hy-perthermophiles is the difficulty involved in obtaining purecultures of such microorganisms. In situ studies based on thesequencing of 16S ribosomal RNA (rRNA) obtained directly

646 Protein & Peptide Letters, 2006, Vol. 13, No. 7 Bouzas et al.

from hyperthermophilic biotopes have demonstrated the ex-istence of an enormous variety of microorganisms growingat high temperatures, not isolated to date [6, 7]. Remarkably,the design of suitable culture media for hyperthermophiles isnot easy, because these organisms grow in microbial com-munities in which some species need others to satisfy theirnutritional requirements. For example, hyperthermophilicheterotrophs obtain their carbon source from a complexmixture of peptides derived from the decomposition of pri-mary producers [8]. Plating hyperthermophiles to obtain purecultures is also a difficult task. Since most solidifying agentsare unstable at very high temperatures during long incuba-tion periods, thermostable self-gelling agents such aspolysilicate or gellan gum are used for this purpose [4]. Nev-ertheless, this method may not be successful in all casesowing to the inability of some organisms to grow on solidsurfaces [9].

A novel micromanipulation method that allows the isola-tion of a single cell from a mixed culture using “opticaltweezers” has been developed [10]. This method uses theforces exerted by a strongly focused beam of laser light totrap and move objects ranging in size from tens of nanome-tres to tens of micrometres, such as single cells and otherbiological particles.

Recently, an isolation strategy that combines in situ 16SrRNA sequence analysis and specific whole-cell hybridiza-tion, with the isolation of the identified single cell by “opti-cal tweezers” has been proposed. This procedure allows thecollection of pure cultures of unknown organisms harbouringspecific 16S rRNA sequences previously identified by in situsequence analysis [11].

Phylogenetics of Hyperthermophiles

Until the 60s, the available tools for taxonomic study didnot allow to determine the evolutionary relationships amongmicroorganisms. Morphological comparisons and study ofthe fossil evidence were unable to offer profound contribu-tions to the taxonomy of microorganisms. In fact, the onlyaccurate classification available at that time was the differ-entiation of microorganisms as prokaryotes or eukaryotes. In1965, it was found that certain molecules could act as evolu-tionary chronometers, such that the evolutionary distancebetween two organisms is inferred from the differences inthe amino acid or nucleotide sequences of homologous mac-romolecules isolated from both [12]. Several molecules wereproposed as good evolutionary chronometers [13], amongthem ATPase, the RecA protein, and the rRNAs. All of themwere probably essential even for the most primitive cells,and so the changes in the sequence of the genes encoded bythem should allow us to establish the evolutionary relation-ships among different microorganisms. In 1977, Carl Woeseproposed as an optimal prokaryote chronometer the 16SrRNA molecule; the fruit of this was the prokaryote phylo-genetic tree and the discovery of a new group of microor-ganisms: the Archaea [14]. The phylogenetic tree of life,constructed from prokaryote 16S rRNA sequences and eu-karyote 18S rRNA sequences subdivided all living organ-isms into three Domains: Bacteria, Archaea and Eukarya[15].

Hyperthermophiles are found in both the Archaea andBacteria. Only the members of the Eukarya domain have notbeen isolated to date from hyperthermophilic environments.Hyperthermophiles occupy the most basal positions of thephylogenetic tree of life [16]. In the Bacteria, only two gen-era, Thermotoga and Aquifex are hyperthermophiles. Theyare the most deeply rooted in the tree of the domain. Thecultured Archaea can be divided into two lineages: Crenar-chaeota and Euryarchaeota. The kingdom Crenarchaeota,consisting entirely of extreme thermophiles and hyperther-mophiles, encompasses genera with the deepest and shortestbranch lengths in the rRNA-based trees. Some of the hyper-thermophilic genera contained within the Crenarchaeota areSulfolobus, Desulfurococcus, Pyrodictium, Thermofilum,Thermoproteus and Pyrolobus. Members belonging to theEuryarchaeota are more ubiquitous in their ecological distri-bution. One more time, however, the most deeply rootedeuryarchaeotal lineage in the rRNA tree corresponds to hy-perthermophilic genera, among them Methanococcus, Ther-mococcus, Methanopyrus and Pyrococcus. In recent years,some authors have reported the existence of non-culturedArchaea, known only from in situ 16S rRNA analysis andwhich do not match any of the former lineages. Barns et al.[6] suggested the existence of a third lineage, the Korar-chaeota, and Huber et al. [17] proposed the new phylumNanoarchaeota.

There has been much speculation and controversy aboutthe origins of life on Earth. Some estimations set the time oflife’s emergence at about 3.8 billion years ago. The condi-tions of the Earth at that time are uncertain, but it seemsquite clear that temperature and pressure would have beenhigh [18]. Thus, if such speculations are correct life on Earthwould have arisen in habitats very similar to those of presentday hyperthermophiles. Since in the universal rRNA tree thephyla containing hyperthermophilic bacteria and archaea areusually the most deeply rooted and have the shortest branchlengths, several authors have suggested that hyperthermo-philes would be the closest relatives to the most recent com-mon ancestor (MRCA) of all present organisms [18, 19].Nevertheless, the hyperthermophilic origin of life is subjectto considerable debate. Some authors have criticized thishypothesis, arguing that a hot origin of life is not compatiblewith the RNA world hypothesis [20]. Thus, such authorsstate that hyperthermophiles are not primitive but insteadhave adapted to temperatures above the limits imposed onother life forms [21], and that the estimated G+C content ofthe MRCA is not compatible with survival at high tempera-tures [22].

Accordingly, alternative hypotheses concerning the na-ture of the MRCA have been proposed. Most of them placethe root of the life tree in the bacterial branch, the MRCAbeing a hyperthermophile [23], although the origin of lifewould not necessarily have to be hot. Other hypotheses pointto a mesophile bacterial origin of life and MRCA [22], thisimplying that the hyperthermophilic phenotype arose inde-pendently within the Bacteria and Archaea. Finally, the mostcontroversial hypothesis [20] suggests a mesophilic Eu-karyote as the common ancestor of all extant life, only themost recent prokaryotic ancestor being a hyperthermophile.

Industrial Applications of Hyperthermophilic Enzymes: A Review Protein & Peptide Letters, 2006, Vol. 13, No. 7 647

MECHANISMS OF THERMOSTABILITY

Stability of Hyperthermophilic Microorganisms

There are several mechanisms used by hyperthermo-philes to maintain the stability of their DNA at high tem-peratures. Thus, all hyperthermophiles produce a particulartype of DNA topoisomerase, called DNA reverse gyrase,which introduces positive supercoils in the DNA molecule atthe expense of ATP, this conferring a greater stability andrendering it more resistant to thermal denaturation [24, 25].Another mechanism, which is not present in all hyperther-mophiles, involves some histone-like proteins that help topreserve the duplex structure of DNA. Such proteins in-crease the fusion temperature of DNA up to 40ºC abovenormal values [26, 27]. A third mechanism to protect DNAfrom denaturation is the presence in the cytoplasm of somehyperthermophilic methanogenes of large amounts of cyclic2,3-potasium-bi-phospho-glycerate, which avoids chemicaldamage such as DNA depurinization [28].

Since most hyperthermophiles belong to the Archaea, itis necessary to refer the increased stability of archaeal cyto-plasmic membranes with respect to bacterial counterparts,because of their different composition. Thus, whereas thetypical fatty acid bilayer structure of the bacterial cytoplas-mic membrane would become disrupted at extreme tem-peratures, the archaeal monolayer membrane composed ofphytanyl chains connected to glycerol with ether links ismuch more resistant [29].

The most hyperthermophilic organism described andclassified so far is Pyrolobus fumarii, which is able to growat 113ºC [30], although recently an organism called “strain121” was isolated and shown to be able to grow at 121ºC[31].

The upper temperature at which life is possible is stillunknown. However, since ATP is spontaneously hydrolyzedin aqueous solution at temperatures of about 140ºC, one can-not expect to find any living organism above such tempera-ture. If life above 140ºC is found, such finding would con-tribute entirely new principles to Biochemistry and Biology.

Enzyme Thermostability

Thermostability and optimal activity at high temperaturesare inherent properties of the proteins isolated from hyper-thermophilic microorganisms. Because most proteins areinactive at such temperatures, the mechanisms of the thermalstability of hyperthermophilic proteins have attracted theinterest of the scientific community ever since they werefirstly discovered. On one hand, their amino acid sequenceanalysis revealed nothing unusual. On the other hand, thesequences from homologous hyperthermophilic and meso-philic proteins resulted to be highly similar, their three-dimensional structures are superimposable and they exhibitthe same catalytic mechanisms [8]. Nevertheless, the factthat such proteins remain thermostable once expressed inmesophilic hosts suggested that thermotolerance resides inthe gene sequence. In general, it was observed that thermo-philic proteins tend to have hydrophobic cores due to a dif-ferent folding with respect to mesophilic ones, which proba-bly affords them more stability [32, 33]. Therefore, the merefact of replacing one amino acid by another affecting the

folding is enough to render a protein thermorresistant. Al-though no specific rules for thermostability or generaliza-tions can be derived from a systematic analysis of the se-quences of homologous mesophilic and thermophilic pro-teins, some hydrophobic substitutions in the protein coreinvestigated by site-directed mutagenesis (SDM) revealed anenhanced thermostability [34].

As mentioned above, a wide variety of genes from hy-perthermophiles have been successfully cloned and ex-pressed in mesophiles. Thus, most archaeal genes cloned inE. coli have been successfully expressed under the control ofstrong promoters such as pLac, pTac or the T7 RNA polym-erase promoter, owing to the large differences between thearchaeal and bacterial transcription systems [8]. A few genesfrom hyperthermophilic Archaea have also been successfullyexpressed in yeast systems, which harbour transcription sys-tems more closely related to theirs [35]. Of all the hyper-thermophilic enzymes expressed so far in E. coli, less than10% show properties and stabilities different from those ofthe native versions [8]. Such enzymes might require post-translational modifications or molecular chaperones in orderto attain their stable folded state.

Thermophiles (organisms growing between 50 and 80ºC)and hyperthermophiles (above 80ºC) are reported to havespecialized proteins, known as “chaperonins”, that are ther-mostable and resistant to denaturation. Such proteins helpothers to refold to their native form after denaturation and torestore their functions [36]. A large number of features havebeen identified as possible contributors to protein stability athigh temperatures, such as disulfide bridges, aromatic inter-actions or hydrogen bonds [8]. However, there are substan-tial differences between the conclusions reached with differ-ent proteins, and therefore, there is not sufficient evidencefor a universal rule for the structural basis of stability.

Advantages of Working with Thermostable Enzymes

As many common enzymatic industrial reactions are per-formed at high temperatures, thermostable enzymes haveattracted much attention in biotechnology and industry inrecent years. Their increased stability with respect to meso-philic enzymes makes them more suitable for harsh indus-trial processes. In addition, their thermostability is usuallyassociated with a higher resistance to chemical denaturantscommonly used in many industrial reactions.

Performing enzymatic reactions at high temperaturesallows higher reaction rates and process yields because of: (i)a decrease in viscosity, (ii) an increase in the diffusion coef-ficient of substrates, (iii) an increase in the solubility of sub-strates and products, and (iv) a favourable equilibrium dis-placement in endothermal reactions [37]. Moreover, the riskof contamination by common mesophiles decreases withincreases in the reaction temperature. Finally, an importantadvantage from the point of view of enzyme production andpurification is that, once expressed in mesophilic hosts,thermophilic and hyperthermophilic enzymes are easier topurify by heat treatment [8].

STARCH DEGRADATION

Starch is a ubiquitous polysaccharide composed of twohigh-molecular weight components: amylose and amy-


lopectin. Amylose is a linear polymer consisting of units ofα-glucose linked by α-1,4 glycosidic bonds. Amylopectin isa branched polymer, also consisting of units of α-glucose,but containing, in addition to α-1,4 glycosidic bonds, α-1,6linking branch points every 17-26 glucose residues.

Owing to its complex structure, the starch polymer re-quires a combination of enzymes for its complete hydrolysis:α-amylases, glucoamylases or β-amylases, and isoamylasesor pullulanases. α-Amylase is an endo-acting enzyme, whichmeans that it hydrolyses linkages in a random fashion, lead-ing to the formation of linear and branched oligosaccharides.The rest are exo-acting enzymes, which attack the substrateonly from the non-reducing end, producing oligo and mono-saccharides.

These enzymes are very abundant in thermophiles andhyperthermophiles, suggesting an important role in theirmetabolism. Since these microorganisms live in deep-seahydrothermal vents, where only very small amounts of starchare found, it is assumed that they need the starch degradingenzymes to degrade glycogen, which serves as a storagematerial in the cell [38].

The enzymatic hydrolysis of starch is of great interest inthe field of biotechnology. In fact, 30% of the global enzymeconsumption is for this purpose. The most important indus-trial processes in which starch is used as a natural source ofsugars are the production of glucose/ fructose syrup, thesynthesis of non-fermentable carbohydrates, and the synthe-sis of anti-staling agents in baking [39]. In addition, the glu-cose resulting from these processes can be used to produceethanol, amino acids or organic acids [40].

Although thermophiles and hyperthermophiles producelarge amounts of starch-degrading enzymes, production isstill not high enough to satisfy industrial requirements. Thisproblem has been circumvented by cloning and expressingthe genes encoding amylolytic enzymes in mesophilic hosts.In most cases, thermostable enzymes expressed in meso-philic hosts can be purified easily, and the degree of purityobtained is suitable for industrial applications.

The enzymatic starch conversion process involves threesteps: (i) gelatinization: the dissolution of starch granules,forming a viscous suspension; this step is achieved by heat-ing starch with water at high temperatures; the use of a ther-mostable enzyme would save a lot of cooling time, this al-lowing to proceed directly with the hydrolysis after gelatini-zation; (ii) liquefaction: partial hydrolysis of starch, with aloss of viscosity and, (iii) saccharification: the production ofglucose and maltose via further hydrolysis [37].

The first thermostable α-amylases were isolated fromBacillus subtilis, Bacillus amyloliquefaciens and Bacilluslicheniformis. The industrial use of these enzymes presents aproblem that is common for most α-amylases: they requirecalcium for activity. The calcium added to the reaction pre-cipitates as calcium oxalate, blocking process pipes and heatexchangers, and leading some products, for example beer, tobe unacceptable for consumption. Since the concentration ofcalcium oxalate can be reduced by lowering the reaction pH,it is important the search for acid-stable α-amylases. Themost hyperthermophilic α-amylases have been obtainedfrom Pyrococcus woesei, Pyrococcus furiosus, Thermococ-

cus profundus and Thermococcus hydrothermalis [41, 42,43, 44]. The optimal temperature for the activity of theseenzymes is 100ºC, and the optimal pH is lower than thatfound for Bacillus α-amylases. The extracellular α-amylasefrom Pyrococcus furiosus has been cloned and expressed inB. subtilis and E. coli [44, 45], as has also been an intracel-lular α-amylase from the same microorganism [46]. Bothenzymes have been shown to differ widely from each other,the latter one being of interest because it does not require thepresence of calcium for activity.

Unlike α-amylases, glucoamylases seem to be very rarein hyperthermophiles. The only report so far of a hyperther-mophilic glucoamylase is that of a thermostable glucoamy-lase recently isolated from the archaeon Sulfolobus solfatari-cus. The gene coding for this enzyme has been cloned andexpressed in E. coli. The enzyme exhibits an optimal tem-perature of 90ºC and a pH range from 5.5 to 6.0 [40].

α-Glucosidases are generally involved in the last step ofstarch degradation. A bacterial hyperthermophilic α-glucosidase has been isolated from Thermotoga maritima[47]. This enzyme is very unusual in that it requires NAD

+

and Mn+2 for its activity. Hyperthermophilic archaeal α-

glucosidases have been detected in P. furiosus, P. woesei,Sulfolobus shibatae, S. solfataricus, Thermococcus strainAN1 and T. hydrothermalis [38, 48-50]. The most hyper-thermophilic is the intracellular α-glucosidase from P. furio-sus, which exhibits optimal activity in a temperature rangefrom 105

oC to 115

oC and a pH range from 5.0 to 6.0. The

gene encoding α-glucosidase from T. hydrothermalis hasbeen successfully cloned by complementation of a maltase-deficient S. cerevisiae mutant [50].

Pullulan-degrading enzymes belong to the α-amylasefamily. These enzymes are widely distributed in nature andare produced by a very broad variety of species, among themthe hyperthermophilic Archaea. They degrade pullulan, alinear polymer composed of maltotriose units with α-1,6glucoside linkages, produced by the fungus Aureobasidiumpullulans, which shows a variety of potential and medicalapplications. Nevertheless, in natural ecosystems there aredifferent polysaccharides, such as starch or glycogen, thatare also degraded by pullulanases [51]. Pullulanases havebeen classified in two groups: type I pullulanases, that hy-drolyze the α-1,6 -linkages in pullulan as well as in branchedoligosaccharides, producing maltotriose and linear oligosac-charides; type II pullulanases, that attack α-1,6-glycosidelinkages in pullulan and branched polysaccharides and alsoα-1,4-glycosidic linkages in branched and linear polysaccha-rides, leading to the formation of small sugars such as glu-cose, maltose and maltotriose. Thermostable pullulanasesfrom hyperthermophilic archaea and bacteria have been iso-lated from Thermococcus celer, Desulfurococcus mucosus,Staphylothermus marinus, Thermococcus agregans, P. furio-sus, Thermococcus litoralis, T. hydrothermalis, Pyrococcusstrain ES4 and Thermotoga maritima. These enzymes exhibitgreat stability at temperatures above 100

oC, even in the ab-

sence of substrate or calcium ions [52-55]. Type I and IIpullulanases from P. furiosus, P. woesei, Thermotoga mari-tima and T. hydrothermalis have been expressed in E. coli[52, 56-58].


PROTEASES

The production of proteases for commercial purposes isgreater than that of any other enzyme group of biotechno-logical relevance. They are rapidly becoming the most im-portant industrial enzymes, constituting more than 65% ofthe world market [59], and this biotechnological interest hasfurther driven the search for thermostable proteases able towithstand the harsh conditions of industrial processes [37,60]. Proteases are generally classified in two groups: en-dopeptidases –proteases that cleave peptide bonds within theprotein– and exopeptidases, which cleave off amino acidsfrom the ends of the protein. Many proteolytic enzymes fromhyperthermophilic archaea and bacteria have been identified.Most of them are not only catalytically active at high tem-peratures, but they also retain their catalytic activity in thepresence of detergents and other denaturing substances, suchas urea, guanidine-HCl, dithiotreitol or 2-mercaptoethanol.Hyperthermophilic proteases have been identified in T.maritima, T. aggregans, T. celer, T. litoralis and Pyrococcussp. KODI [61, 62]. The thiol protease from Pyrococcus sp.KODI constitutes the most hyperthermophilic proteaseamong them, showing an optimal temperature of 110ºC.Several serine proteases from P. furiosus have been clonedand expressed in E. coli [63-65]. The industrial use of hy-perthermophilic proteases that have not yet been cloned in E.coli is hindered by the fact that they come from anaerobichyperthermophilic microorganisms, whose culture at a scalerequired to meet industrial needs is difficult. Recently, how-ever, proteases from the aerobic hyperthermophile Aeropy-rum pernix K1 have been isolated [66]. This microorganismgrows optimally at 90ºC and its growth pattern and nutri-tional requirements are amenable to large-scale culture tech-niques, making the strain an attractive source of thermosta-ble proteases.

HYPERTHERMOPHILIC ENZYMES FOR THEMOLECULAR BIOLOGY INDUSTRY

Thermophilic DNA polymerases are responsible for theelongation of the primer strand of a growing DNA moleculeduring DNA amplification by PCR. The isolation of TaqDNA polymerase from the thermophilic bacterium Thermusaquaticus, and in particular its cloning and expression in E.coli [67], was truly a revolutionary event in the field of Bio-technology. Since then, many other DNA polymerases havebeen isolated from thermophilic and hyperthermophilic ar-chaea and are now available commercially: P. furiosus Pfupolymerase [68], T. litoralis Vent polymerase [69, 70], P.woesei Pwo polymerase [71], Pyrococcus strain GB-D DeepVent polymerase [72], and a DNA polymerase from Ther-mococcus sp. strain 9N-7 [73]. Although all such enzymeshave error rates that are much lower than Taq polymerase,owing to their low extension rates and unsuitability for theamplification of long DNA fragments none of them has re-placed the use of the T. aquaticus enzyme. Proofreading en-zymes, such as Pfu or Vent and Deep Vent polymerases, arepreferred only if very high fidelity is required. Recently, ahyperthermophilic archaeal DNA polymerase isolated fromPyrococcus sp. strain KOD1 [74] exhibits a low error rateand high process and extension rates. Commercially avail-able thermophilic DNA ligases are the Pfu DNA ligase fromP. furiosus (Stratagene) and the Tcs DNA ligase from Ther-

mus scodoductus (Roche Molecular Biochemicals). In addi-tion, a hyperthermophilic ligase has been identified in Acidi-anus ambivalens [75].

APPLICATIONS TO THE PULP AND PAPER INDUS-TRY

The enzymatic treatment of wood for the production ofpulp is based on hemicellulose hydrolysis with xylanases.These enzymes cleave xylan, the main component of hemi-celluloses and one of the most abundant organic substanceson earth [76]. The treatment of wood to obtain pulp is carriedat very high temperatures and at basic pH to help disrupt thecell wall structure. Thus, an interesting xylanase from theindustrial point of view should be very thermostable andactive at neutral to alkaline pH. Additionally, the enzymesused in these processes should lack cellulolytic activity inorder to avoid hydrolysis of the cellulose fibres. They shouldalso be of low molecular mass in order to diffuse well in thepulp fibres and be easy to obtain at low cost. [52]. Mostcommercially available xylanases do not meet all these re-quirements. Although only few extreme thermophilic micro-organisms are able to secrete such enzymes, some xylanaseshave been reported to operate at very high optimal tempera-tures. These enzymes reduce chlorine consumption in thebleaching process, thus lowering environmental damage dueto the presence of organic halogens [77].

Hyperthermophilic xylanases from Thermotoga, Dyctio-glomus, Sulfolobus, Pyrococcus and Pyrodictium were iso-lated with optimal temperatures above 90ºC [77-83]. Amongthem, the xylanase from Thermotoga sp. strain FjSS3-B1 isthe most hyperthermophilic xylanase so far reported, with anoptimal temperature of 115ºC [81]. Hyperthermophilic xy-lanases from Dyctioglomus sp., Thermotoga sp., and Pyro-coccus furiosus have been cloned and successfully expressedin E. coli [84-87, 79].

LIPASES

Lipases are the most widely used group of biocatalystsfor biotechnology. They catalyse both the hydrolysis of long-chain acylglycerols into glycerol and fatty acids and the re-verse reaction. Thus, these enzymes can be applied industri-ally as hydrolases and as synthetases [88]. Microbial lipaseshave an enormous biotechnological potential owing to theirunique characteristics, including stability in organic solvents,broad substrate specificity, and high enantioselectivity [89].

Among the many applications of lipases as hydrolases istheir use as additives in the detergent industry. In the foodindustry, lipases are also used to modify the structure ofsome triglycerides trying to enhance their physical, nutri-tional or sensory properties. Additionally, these enzymes arealso used to obtain poly-unsaturated fatty acids (PUFAs)from plant and animal lipids, which can be subsequentlyused to produce pharmaceuticals. Another application oflipases as hydrolases is their use in removing the hydropho-bic components of the wood (“pitch”) in the pulp and paperindustry [89].

Lipases and esterases are also being used for the synthe-sis of new biopolymeric materials, such as polyphenols,polysaccharides and polyesters. These compounds are of


special interest from the environmental point of view be-cause they are biodegradable and can be produced from re-newable natural resources. Recently, microbial lipases havebeen used to obtain biodiesel by transesterification of plantfats. Biodiesel is an alternative source of energy that reducessulphur oxide production. It is currently being used in publictransport, although its long-scale production is still hamperedby the high costs of the biocatalyst.

Another main characteristic of microbial lipases is theirenantioselectivity, which makes them suitable for the pro-duction of a number of fine chemicals. This is of great im-portance in the case of certain pharmaceuticals and agro-chemicals, which need to be enantiopure to be functional[88].

Thermostable lipases from hyperthermophilic archaeahave been isolated from Pyrobaculum calidifontis, Pyrococ-cus furiosus and Pyrococcus horikoshii and cloned in E. coli[90, 91, 92]. Although reports on lipases from hyperthermo-philic microorganisms are scant, some such enzymes havebeen isolated from moderately thermophilic microorganismsand exhibit activity at temperatures of 90ºC [93, 94].

CONCLUSIONS

One of the main problems in most enzymatic industrialprocesses is how to achieve an appropriate compromise be-tween harsh working conditions and enzyme stability. En-zymes from hyperthermophilic microorganisms offer a po-tential solution for industrial processes carried out at hightemperatures. Since only a very few species from this groupof microorganisms have been isolated to date, there seems tobe a large number of hyperthermophilic catalysts withunique properties awaiting discovery. The development ofmolecular tools together with biotechnological researchshould lead to the future availability of new hyperthermo-philic enzymes to meet all types of industrial demands.

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Lett. 22, 495-498.

Received: March 16, 2006 Revised: April 06, 2006 Accepted: May 02, 2006

hyperthermophilic enzyme

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