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

645

Industrial Applications of Hyperthermophilic Enzymes A !e"ie#

Trinidad de Miguel Bouzas , Jorge Barros-Velzquez and Toms Gonzlez Villa *
1

1,

Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, E 1!"#$ Santiago de Com $ postela, Spain% and Department of &nalytical Chemistry, 'utrition and Food Science, Faculty of (eterinary Sciences, University of Santiago de Compostela, E $"))$ *ugo, Spain
A$stract Over the ast t!o de"ades, resear"h s"ientists have #een involved in the investigation o$ thermo hili" and h%erthermo hili" mi"roorganisms o!ing to the unique $eatures o$ their enz%mi" s%stems& 'u"h in-de th investigations are no! on their !a% to mastering the "loning and industrial e( loitation o$ a #road variet% o$ genes en"oding enz%mes involved in star"h h%drol%sis, amino a"id #ios%nthesis, rotein h%drol%sis, et"& )n this !or*, !e revie! the state o$ the art and $uture ers e"tives o$ industrial a li"ations o$ enz%mes $rom h% erthermo hili" and e(treme thermo hili" mi"roorganisms, s e"ial attention #eing aid to the #iote"hnologi"al methods involved in their industrial e( loitation&

%ey#ords Thermo hili" enz%mes, Mole"ular "loning, Mi"roorganisms, )ndustrial te"hnolog%, +ood industr%, ,hemi"al in- dustr%& I&'!()*+'I(& )n 1-./, Thomas 0& Bro"* o$ the 1niversit% o$ 2is"onsin re orted $or the $irst time the e(isten"e o$ mi"roorganisms !ith o timal gro!th tem eratures higher than /34,, gro!ing in the hot s rings o$ 5ello!stone 6ational 7ar* 819& 'in"e then until no!, the !or* o$ diverse resear"h grou s has led to the dis"over% o$ more than 2: di$$erent genera a#le to gro! a#ove ;:4,& 'u"h organisms are no! termed <h%erthermo hiles=& The enormous di$$i"ult% involved in o#taining ure "ultures $rom these mi"roorganisms, together !ith their "om li"ated large-s"ale "ulture, initiall% limited the stud% o$ their enz%mes& 6evertheless, in the late 1-;:s the $irst <h% erthermo hili" enz%mes= !ere uri$ied, !hi"h roved to #e e(tremel% sta#le at high tem eratures and to e(hi#it no or ver% lo! a"tivit% at tem eratures #elo! the gro!th "onditions o$ the organisms $rom !hi"h the% !ere o#tained& The une( e"ted $inding that these enz%mes "an #e "loned and e( ressed in meso hili" hosts !ithout losing their a"tive "on$ormation and thermosta#ilit% solved the initial rodu"tion ro#lem& )n this sense, the availa#ilit% o$ an in"reasing num#er o$ thermosta#le enz%mes o ened ne! ossi#ilities $or industrial ro"esses& 0ue to their overall inherent sta#ilit%, su"h enz%mes have $ound "ommer"ial a li"ations in the star"h industr%, in the s%nthesis o$ amino a"ids, in the etroleum, "hemi"al, ul and a er industries, and indeed in man% other $ields& ,icroor-anisms .ro#in- at E/treme 'emperatures The "urrent "lassi$i"ation "onsiders thermo hiles all or- ganisms gro!ing a#ove 334,, moderate thermo hiles a#ove .34,, e(treme thermo hiles a#ove /34, and h% erthermo*>ddress "orres onden"e to this author at the 0e artment o$ Mi"ro#iolog%, +a"ult% o$ 7harma"%, 1niversit% o$ 'antiago de ,om ostela, ?- 13/;2 'antiago de ,om ostela, ' ain@ TelA BCD&-;1&3.C1::, ?(t& 1D-D-@ +a(A BCD&-;1&3;2D-:@ ?-mailA m villaEus"&es 0020-1665206 3504005400

hiles a#ove -:4,, although some authors ma*e a single grou o$ h% erthermo hiles also in"luding e(treme thermohiles 82, C9& )n this revie!, !e !ill name e(tremo hiles all mi"roorganisms !ith an o timal gro!th tem erature a#ove ;:4,& These mi"roorganisms, re resented onl% #% Ba"teria and >r"haea, have #een isolated $rom a large variet% o$ natural marine and !ater-"ontaining terrestrial environments, as !ell as $rom arti$i"ial environments 8D9& 6atural terrestrial #ioto es o$ h% erthermo hiles in"lude hot s rings and sol$atari" $ields !ith a !ide range o$ F G:&3--&:H, lo! salinit% G:&1-:&3IH and dee geothermall% heated oil-"ontaining strati$i"ations& 6atural marine #ioto es are shallo! h%drothermal s%stems, a#%ssal hot vents G<#la"* smo*ers=H and a"tive seamounts !here the salinit% levels are high GCIH and the F values range $rom 3&: to ;&3& >rti$i"ial environments in !hi"h h% erthermo hiles have #een $ound in"luded smouldering "oal re$use iles and hot out$lo!s $rom geothermal and nu"lear o!er lants& The great interest sho!n #% the s"ienti$i" "ommunit% in h% erthermo hili" mi"roorganisms is re$le"ted in the in"reasing num#er o$ mi"ro#ial s e"ies that have #een des"ri#ed over the last C: %ears and the num#er o$ genome sequen"es that have #een "om leted sin"e the #eginning o$ the !orld!ide genome sequen"ing roJe"ts& Thus, "urrentl% more than /: s e"ies o$ h% erthermo hiles have #een des"ri#ed@ 1D genome-sequen"ing roJe"ts have #een "omleted G&eropyrum perni+ K1, &,uife+ aeolicus V+3, &r chaeoglobus fulgidus V,-1., Methanocaldococcus -an naschii 0'M2..1, Methanopyrus .andleri >V1-, Pyroba culum aerophilum )M2, Pyrococcus abyssi G?3, P/ furiosus J,M;D22, P/ hori.oshii OTC, Sulfolobus acidocaldarius 0'M.C-, S/ solfataricus 72, S/ to.odaii /, J,M1:3D3, 0hermococcus .oda.aerensis KO01, 0hermotoga maritima M'B;H, and more than 2: others are in rogress 8D, 39& One o$ the main "hallenges $or s"ientists stud%ing h%erthermo hiles is the di$$i"ult% involved in o#taining ure "ultures o$ su"h mi"roorganisms& 1n situ studies #ased on the sequen"ing o$ 1.' ri#osomal L6> GrL6>H o#tained dire"tl%

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$rom h% erthermo hili" #ioto es have demonstrated the e(isten"e o$ an enormous variet% o$ mi"roorganisms gro!ing at high tem eratures, not isolated to date 8., /9& Lemar*a#l%, the design o$ suita#le "ulture media $or h% erthermo hiles is not eas%, #e"ause these organisms gro! in mi"ro#ial "ommunities in !hi"h some s e"ies need others to satis$% their nutritional requirements& +or e(am le, h% erthermo hili" heterotro hs o#tain their "ar#on sour"e $rom a "om le( mi(ture o$ e tides derived $rom the de"om osition o$ rimar% rodu"ers 8;9& 7lating h% erthermo hiles to o#tain ure "ultures is also a di$$i"ult tas*& 'in"e most solidi$%ing agents are unsta#le at ver% high tem eratures during long in"u#ation eriods, thermosta#le sel$-gelling agents su"h as ol%sili"ate or gellan gum are used $or this ur ose 8D9& 6evertheless, this method ma% not #e su""ess$ul in all "ases o!ing to the ina#ilit% o$ some organisms to gro! on solid sur$a"es 8-9& > novel mi"romani ulation method that allo!s the isolation o$ a single "ell $rom a mi(ed "ulture using <o ti"al t!eezers= has #een develo ed 81:9& This method uses the $or"es e(erted #% a strongl% $o"used #eam o$ laser light to tra and move o#Je"ts ranging in size $rom tens o$ nanome- tres to tens o$ mi"rometres, su"h as single "ells and other #iologi"al arti"les& Le"entl%, an isolation strateg% that "om#ines in situ 1.' rL6> sequen"e anal%sis and s e"i$i" !hole-"ell h%#ridization, !ith the isolation o$ the identi$ied single "ell #% <o ti"al t!eezers= has #een ro osed& This ro"edure allo!s the "olle"tion o$ ure "ultures o$ un*no!n organisms har#ouring s e"i$i" 1.' rL6> sequen"es reviousl% identi$ied #% in situ sequen"e anal%sis 8119& 9hylo-enetics of Hyperthermophiles 1ntil the .:s, the availa#le tools $or ta(onomi" stud% did not allo! to determine the evolutionar% relationshi s among mi"roorganisms& Mor hologi"al "om arisons and stud% o$ the $ossil eviden"e !ere una#le to o$$er ro$ound "ontri#utions to the ta(onom% o$ mi"roorganisms& )n $a"t, the onl% a""urate "lassi$i"ation availa#le at that time !as the di$$erentiation o$ mi"roorganisms as ro*ar%otes or eu*ar%otes& )n 1-.3, it !as $ound that "ertain mole"ules "ould a"t as evolutionar% "hronometers, su"h that the evolutionar% distan"e #et!een t!o organisms is in$erred $rom the di$$eren"es in the amino a"id or nu"leotide sequen"es o$ homologous ma"romole"ules isolated $rom #oth 8129& 'everal mole"ules !ere ro osed as good evolutionar% "hronometers 81C9, among them >T7ase, the Le"> rotein, and the rL6>s& >ll o$ them !ere ro#a#l% essential even $or the most rimitive "ells, and so the "hanges in the sequen"e o$ the genes en"oded #% them should allo! us to esta#lish the evolutionar% relationshi s among di$$erent mi"roorganisms& )n 1-//, ,arl 2oese ro osed as an o timal ro*ar%ote "hronometer the 1.' rL6> mole"ule@ the $ruit o$ this !as the ro*ar%ote h%logeneti" tree and the dis"over% o$ a ne! grou o$ mi"roor- ganismsA the >r"haea 81D9& The h%logeneti" tree o$ li$e, "onstru"ted $rom ro*ar%ote 1.' rL6> sequen"es and eu- *ar%ote 1;' rL6> sequen"es su#divided all living organ- isms into three 0omainsA Ba"teria, >r"haea and ?u*ar%a 8139&

F% erthermo hiles are $ound in #oth the >r"haea and Ba"teria& Onl% the mem#ers o$ the ?u*ar%a domain have not #een isolated to date $rom h% erthermo hili" environments& F% erthermo hiles o""u % the most #asal ositions o$ the h%logeneti" tree o$ li$e 81.9& )n the Ba"teria, onl% t!o genera, 0hermotoga and &,uife+ are h% erthermo hiles& The% are the most dee l% rooted in the tree o$ the domain& The "ultured >r"haea "an #e divided into t!o lineagesA ,renar- "haeota and ?ur%ar"haeota& The *ingdom ,renar"haeota, "onsisting entirel% o$ e(treme thermo hiles and h% erther- mo hiles, en"om asses genera !ith the dee est and shortest #ran"h lengths in the rL6>-#ased trees& 'ome o$ the h% er- thermo hili" genera "ontained !ithin the ,renar"haeota are Sulfolobus, Desulfurococcus, Pyrodictium, 0hermofilum, 0hermoproteus and Pyrolobus& Mem#ers #elonging to the ?ur%ar"haeota are more u#iquitous in their e"ologi"al distri- #ution& One more time, ho!ever, the most dee l% rooted eur%ar"haeotal lineage in the rL6> tree "orres onds to h%- erthermo hili" genera, among them Methanococcus, 0her mococcus, Methanopyrus and Pyrococcus& )n re"ent %ears, some authors have re orted the e(isten"e o$ non-"ultured >r"haea, *no!n onl% $rom in situ 1.' rL6> anal%sis and !hi"h do not mat"h an% o$ the $ormer lineages& Barns et al/ 8.9 suggested the e(isten"e o$ a third lineage, the Korar"haeota, and Fu#er et al& 81/9 ro osed the ne! h%lum 6anoar"haeota& There has #een mu"h s e"ulation and "ontrovers% a#out the origins o$ li$e on ?arth& 'ome estimations set the time o$ li$eMs emergen"e at a#out C&; #illion %ears ago& The "onditions o$ the ?arth at that time are un"ertain, #ut it seems quite "lear that tem erature and ressure !ould have #een high 81;9& Thus, i$ su"h s e"ulations are "orre"t li$e on ?arth !ould have arisen in ha#itats ver% similar to those o$ resent da% h% erthermo hiles& 'in"e in the universal rL6> tree the h%la "ontaining h% erthermo hili" #a"teria and ar"haea are usuall% the most dee l% rooted and have the shortest #ran"h lengths, several authors have suggested that h% erthermo- hiles !ould #e the "losest relatives to the most re"ent "om- mon an"estor GML,>H o$ all resent organisms 81;, 1-9& 6evertheless, the h% erthermo hili" origin o$ li$e is su#Je"t to "onsidera#le de#ate& 'ome authors have "riti"ized this h% othesis, arguing that a hot origin o$ li$e is not "om ati#le !ith the L6> !orld h% othesis 82:9& Thus, su"h authors state that h% erthermo hiles are not rimitive #ut instead have ada ted to tem eratures a#ove the limits im osed on other li$e $orms 8219, and that the estimated GB, "ontent o$ the ML,> is not "om ati#le !ith survival at high tem eratures 8229& >""ordingl%, alternative h% otheses "on"erning the nature o$ the ML,> have #een ro osed& Most o$ them la"e the root o$ the li$e tree in the #a"terial #ran"h, the ML,> #eing a h% erthermo hile 82C9, although the origin o$ li$e !ould not ne"essaril% have to #e hot& Other h% otheses oint to a meso hile #a"terial origin o$ li$e and ML,> 8229, this im l%ing that the h% erthermo hili" henot% e arose inde- endentl% !ithin the Ba"teria and >r"haea& +inall%, the most "ontroversial h% othesis 82:9 suggests a meso hili" ?u- *ar%ote as the "ommon an"estor o$ all e(tant li$e, onl% the most re"ent ro*ar%oti" an"estor #eing a h% erthermo hile&

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,E+HA&I8,8 (< 'HE!,(8'A7I:I'= 8ta$ility of Hyperthermophilic ,icroor-anisms There are several me"hanisms used #% h% erthermohiles to maintain the sta#ilit% o$ their 06> at high temeratures& Thus, all h% erthermo hiles rodu"e a arti"ular t% e o$ 06> to oisomerase, "alled 06> reverse g%rase, !hi"h introdu"es ositive su er"oils in the 06> mole"ule at the e( ense o$ >T7, this "on$erring a greater sta#ilit% and rendering it more resistant to thermal denaturation 82D, 239& >nother me"hanism, !hi"h is not resent in all h% erthermo hiles, involves some histone-li*e roteins that hel to reserve the du le( stru"ture o$ 06>& 'u"h roteins in"rease the $usion tem erature o$ 06> u to D:4, a#ove normal values 82., 2/9& > third me"hanism to rote"t 06> $rom denaturation is the resen"e in the "%to lasm o$ some h% erthermo hili" methanogenes o$ large amounts o$ "%"li" 2,C- otasium-#i- hos ho-gl%"erate, !hi"h avoids "hemi"al damage su"h as 06> de urinization 82;9& 'in"e most h% erthermo hiles #elong to the >r"haea, it is ne"essar% to re$er the in"reased sta#ilit% o$ ar"haeal "%to- lasmi" mem#ranes !ith res e"t to #a"terial "ounter arts, #e"ause o$ their di$$erent "om osition& Thus, !hereas the t% i"al $att% a"id #ila%er stru"ture o$ the #a"terial "%to las- mi" mem#rane !ould #e"ome disru ted at e(treme tem- eratures, the ar"haeal monola%er mem#rane "om osed o$ h%tan%l "hains "onne"ted to gl%"erol !ith ether lin*s is mu"h more resistant 82-9& The most h% erthermo hili" organism des"ri#ed and "lassi$ied so $ar is Pyrolobus fumarii , !hi"h is a#le to gro! at 11C4, 8C:9, although re"entl% an organism "alled <strain 121= !as isolated and sho!n to #e a#le to gro! at 1214, 8C19& The u er tem erature at !hi"h li$e is ossi#le is still un*no!n& Fo!ever, sin"e >T7 is s ontaneousl% h%drol%zed in aqueous solution at tem eratures o$ a#out 1D:4,, one "an- not e( e"t to $ind an% living organism a#ove su"h tem era- ture& )$ li$e a#ove 1D:4, is $ound, su"h $inding !ould "on- tri#ute entirel% ne! rin"i les to Bio"hemistr% and Biolog%& Enzyme 'hermosta$ility Thermosta#ilit% and o timal a"tivit% at high tem eratures are inherent ro erties o$ the roteins isolated $rom h% erthermo hili" mi"roorganisms& Be"ause most roteins are ina"tive at su"h tem eratures, the me"hanisms o$ the thermal sta#ilit% o$ h% erthermo hili" roteins have attra"ted the interest o$ the s"ienti$i" "ommunit% ever sin"e the% !ere $irstl% dis"overed& On one hand, their amino a"id sequen"e anal%sis revealed nothing unusual& On the other hand, the sequen"es $rom homologous h% erthermo hili" and meso- hili" roteins resulted to #e highl% similar, their three- dimensional stru"tures are su erim osa#le and the% e(hi#it the same "atal%ti" me"hanisms 8;9& 6evertheless, the $a"t that su"h roteins remain thermosta#le on"e e( ressed in meso hili" hosts suggested that thermotoleran"e resides in the gene sequen"e& )n general, it !as o#served that thermo- hili" roteins tend to have h%dro ho#i" "ores due to a di$- $erent $olding !ith res e"t to meso hili" ones, !hi"h ro#a- #l% a$$ords them more sta#ilit% 8C2, CC9& There$ore, the mere $a"t o$ re la"ing one amino a"id #% another a$$e"ting the

$olding is enough to render a rotein thermorresistant& >lthough no s e"i$i" rules $or thermosta#ilit% or generalizations "an #e derived $rom a s%stemati" anal%sis o$ the sequen"es o$ homologous meso hili" and thermo hili" roteins, some h%dro ho#i" su#stitutions in the rotein "ore investigated #% site-dire"ted mutagenesis G'0MH revealed an enhan"ed thermosta#ilit% 8CD9& >s mentioned a#ove, a !ide variet% o$ genes $rom h%erthermo hiles have #een su""ess$ull% "loned and e(ressed in meso hiles& Thus, most ar"haeal genes "loned in E/ coli have #een su""ess$ull% e( ressed under the "ontrol o$ strong romoters su"h as Na", Ta" or the T/ L6> ol%merase romoter, o!ing to the large di$$eren"es #et!een the ar"haeal and #a"terial trans"ri tion s%stems 8;9& > $e! genes $rom h% erthermo hili" >r"haea have also #een su""ess$ull% e( ressed in %east s%stems, !hi"h har#our trans"ri tion s%stems more "losel% related to theirs 8C39& O$ all the h% erthermo hili" enz%mes e( ressed so $ar in E/ coli, less than 1:I sho! ro erties and sta#ilities di$$erent $rom those o$ the native versions 8;9& 'u"h enz%mes might require ost- translational modi$i"ations or mole"ular "ha erones in order to attain their sta#le $olded state& Thermo hiles Gorganisms gro!ing #et!een 3: and ;:4,H and h% erthermo hiles Ga#ove ;:4,H are re orted to have s e"ialized roteins, *no!n as <"ha eronins=, that are thermosta#le and resistant to denaturation& 'u"h roteins hel others to re$old to their native $orm a$ter denaturation and to restore their $un"tions 8C.9& > large num#er o$ $eatures have #een identi$ied as ossi#le "ontri#utors to rotein sta#ilit% at high tem eratures, su"h as disul$ide #ridges, aromati" intera"tions or h%drogen #onds 8;9& Fo!ever, there are su#stantial di$$eren"es #et!een the "on"lusions rea"hed !ith di$$erent roteins, and there$ore, there is not su$$i"ient eviden"e $or a universal rule $or the stru"tural #asis o$ sta#ilit%& Ad"anta-es of >or?in- #ith 'hermosta$le Enzymes >s man% "ommon enz%mati" industrial rea"tions are er$ormed at high tem eratures, thermosta#le enz%mes have attra"ted mu"h attention in #iote"hnolog% and industr% in re"ent %ears& Their in"reased sta#ilit% !ith res e"t to mesohili" enz%mes ma*es them more suita#le $or harsh industrial ro"esses& )n addition, their thermosta#ilit% is usuall% asso"iated !ith a higher resistan"e to "hemi"al denaturants "ommonl% used in man% industrial rea"tions& 7er$orming enz%mati" rea"tions at high tem eratures allo!s higher rea"tion rates and ro"ess %ields #e"ause o$A GiH a de"rease in vis"osit%, GiiH an in"rease in the di$$usion "oe$$i"ient o$ su#strates, GiiiH an in"rease in the solu#ilit% o$ su#strates and rodu"ts, and GivH a $avoura#le equili#rium disla"ement in endothermal rea"tions 8C/9& Moreover, the ris* o$ "ontamination #% "ommon meso hiles de"reases !ith in"reases in the rea"tion tem erature& +inall%, an im ortant advantage $rom the oint o$ vie! o$ enz%me rodu"tion and uri$i"ation is that, on"e e( ressed in meso hili" hosts, thermo hili" and h% erthermo hili" enz%mes are easier to uri$% #% heat treatment 8;9& 8'A!+H )E.!A)A'I(& 'tar"h is a u#iquitous t!o high-mole"ular !eight am%ol%sa""haride "om osed o$ "om onentsA am%lose and

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lo e"tin& >m%lose is a linear ol%mer "onsisting o$ units o$ -glu"ose lin*ed #% -1,D gl%"osidi" #onds& >m%lo e"tin is a #ran"hed ol%mer, also "onsisting o$ units o$ -glu"ose, #ut "ontaining, in addition to -1,D gl%"osidi" #onds, -1,. lin*ing #ran"h oints ever% 1/-2. glu"ose residues& O!ing to its "om le( stru"ture, the star"h ol%mer requires a "om#ination o$ enz%mes $or its "om lete h%drol%sisA -am%lases, glu"oam%lases or -am%lases, and isoam%lases or ullulanases& ->m%lase is an endo-a"ting enz%me, !hi"h means that it h%drol%ses lin*ages in a random $ashion, leading to the $ormation o$ linear and #ran"hed oligosa""harides& The rest are e(o-a"ting enz%mes, !hi"h atta"* the su#strate onl% $rom the non-redu"ing end, rodu"ing oligo and monosa""harides& These enz%mes are ver% a#undant in thermo hiles and h% erthermo hiles, suggesting an im ortant role in their meta#olism& 'in"e these mi"roorganisms live in dee -sea h%drothermal vents, !here onl% ver% small amounts o$ star"h are $ound, it is assumed that the% need the star"h degrading enz%mes to degrade gl%"ogen, !hi"h serves as a storage material in the "ell 8C;9& The enz%mati" h%drol%sis o$ star"h is o$ great interest in the $ield o$ #iote"hnolog%& )n $a"t, C:I o$ the glo#al enz%me "onsum tion is $or this ur ose& The most im ortant industrial ro"esses in !hi"h star"h is used as a natural sour"e o$ sugars are the rodu"tion o$ glu"oseO $ru"tose s%ru , the s%nthesis o$ non-$ermenta#le "ar#oh%drates, and the s%nthesis o$ anti-staling agents in #a*ing 8C-9& )n addition, the glu"ose resulting $rom these ro"esses "an #e used to rodu"e ethanol, amino a"ids or organi" a"ids 8D:9& >lthough thermo hiles and h% erthermo hiles rodu"e large amounts o$ star"h-degrading enz%mes, rodu"tion is still not high enough to satis$% industrial requirements& This ro#lem has #een "ir"umvented #% "loning and e( ressing the genes en"oding am%lol%ti" enz%mes in meso hili" hosts& )n most "ases, thermosta#le enz%mes e( ressed in mesohili" hosts "an #e uri$ied easil%, and the degree o$ urit% o#tained is suita#le $or industrial a li"ations& The enz%mati" star"h "onversion ro"ess involves three ste sA GiH gelatinizationA the dissolution o$ star"h granules, $orming a vis"ous sus ension@ this ste is a"hieved #% heating star"h !ith !ater at high tem eratures@ the use o$ a thermosta#le enz%me !ould save a lot o$ "ooling time, this al- lo!ing to ro"eed dire"tl% !ith the h%drol%sis a$ter gelatini- zation@ GiiH lique$a"tionA artial h%drol%sis o$ star"h, !ith a loss o$ vis"osit% and, GiiiH sa""hari$i"ationA the rodu"tion o$ glu"ose and maltose via $urther h%drol%sis 8C/9& The $irst thermosta#le -am%lases !ere isolated $rom 2acillus subtilis, 2acillus amyloli,uefaciens and 2acillus licheniformis& The industrial use o$ these enz%mes resents a ro#lem that is "ommon $or most -am%lasesA the% require "al"ium $or a"tivit%& The "al"ium added to the rea"tion re"i itates as "al"ium o(alate, #lo"*ing ro"ess i es and heat e("hangers, and leading some rodu"ts, $or e(am le #eer, to #e una""e ta#le $or "onsum tion& 'in"e the "on"entration o$ "al"ium o(alate "an #e redu"ed #% lo!ering the rea"tion F, it is im ortant the sear"h $or a"id-sta#le -am%lases& The most h% erthermo hili" -am%lases have #een o#tained $rom Pyrococcus 3oesei, Pyrococcus furiosus, 0hermococ

cus profundus and 0hermococcus hydrothermalis 8D1, D2, DC, DD9& The o timal tem erature $or the a"tivit% o$ these enz%mes is 1::4,, and the o timal F is lo!er than that $ound $or 2acillus -am%lases& The e(tra"ellular -am%lase $rom Pyrococcus furiosus has #een "loned and e( ressed in 2/ subtilis and E/ coli 8DD, D39, as has also #een an intra"ellular -am%lase $rom the same mi"roorganism 8D.9& Both enz%mes have #een sho!n to di$$er !idel% $rom ea"h other, the latter one #eing o$ interest #e"ause it does not require the resen"e o$ "al"ium $or a"tivit%& 1nli*e -am%lases, glu"oam%lases seem to #e ver% rare in h% erthermo hiles& The onl% re ort so $ar o$ a h% erthermo hili" glu"oam%lase is that o$ a thermosta#le glu"oam%lase re"entl% isolated $rom the ar"haeon Sulfolobus solfatari cus& The gene "oding $or this enz%me has #een "loned and e( ressed in E/ coli& The enz%me e(hi#its an o timal tem- erature o$ -:4, and a F range $rom 3&3 to .&: 8D:9& -Glu"osidases are generall% involved in the last ste o$ star"h degradation& > #a"terial h% erthermo hili" glu"osidase has #een isolated $rom 0hermotoga maritimaB 8D/9& This enz%me is ver% unusual in that it requires 6>0 and Mn $or its a"tivit%& F% erthermo hili" ar"haeal glu"osidases have #een dete"ted in P/ furiosus, P/ 3oesei, Sulfolobus shibatae, S/ solfataricus, 0hermococcus strain >61 and 0/ hydrothermalis 8C;, D;-3:9& The most h% erthermo hili" is the intra"ellular -glu"osidase $rom P/ furio sus, !hi"oh e(hi#itso o timal a"tivit% in a tem erature range $rom 1:3 , to 113 , and a F range $rom 3&: to .&:& The gene en"oding -glu"osidase $rom 0/ hydrothermalis has #een su""ess$ull% "loned #% "om lementation o$ a maltase- de$i"ient S/ cerevisiae mutant 83:9& 7ullulan-degrading enz%mes #elong to the -am%lase $amil%& These enz%mes are !idel% distri#uted in nature and are rodu"ed #% a ver% #road variet% o$ s e"ies, among them the h% erthermo hili" >r"haea& The% degrade ullulan, a linear ol%mer "om osed o$ maltotriose units !ith -1,. glu"oside lin*ages, rodu"ed #% the $ungus &ureobasidium pullulans, !hi"h sho!s a variet% o$ otential and medi"al a li"ations& 6evertheless, in natural e"os%stems there are di$$erent ol%sa""harides, su"h as star"h or gl%"ogen, that are also degraded #% ullulanases 8319& 7ullulanases have #een "lassi$ied in t!o grou sA t% e ) ullulanases, that h%- drol%ze the -1,. -lin*ages in ullulan as !ell as in #ran"hed oligosa""harides, rodu"ing maltotriose and linear oligosa"- "harides@ t% e )) ullulanases, that atta"* 1,.-gl%"oside lin*ages in ullulan and #ran"hed ol%sa""harides and also -1,D-gl%"osidi" lin*ages in #ran"hed and linear ol%sa""harides, leading to the $ormation o$ small sugars su"h as glu"ose, maltose and maltotriose& Thermosta#le ullulanases $rom h% erthermo hili" ar"haea and #a"teria have #een isolated $rom 0hermococcus celer, Desulfurococcus mucosus, Staphylothermus marinus, 0hermococcus agregans, P/ furio sus, 0hermococcus litoralis, 0/ hydrothermalis, Pyrococcus strain ?'D and 0hermotoga maritima& Theose enz%mes e(hi#it great sta#ilit% at tem eratures a#ove 1:: ,, even in the a#- sen"e o$ su#strate or "al"ium ions 832-339& T% e ) and )) ullulanases $rom P/ furiosus, P/ 3oesei, 0hermotoga mari tima and 0/ hydrothermalis have #een e( ressed in E/ coli 832, 3.-3;9&
B2

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9!('EA8E8 The rodu"tion o$ roteases $or "ommer"ial ur oses is greater than that o$ an% other enz%me grou o$ #iote"hno- logi"al relevan"e& The% are ra idl% #e"oming the most im- ortant industrial enz%mes, "onstituting more than .3I o$ the !orld mar*et 83-9, and this #iote"hnologi"al interest has $urther driven the sear"h $or thermosta#le roteases a#le to !ithstand the harsh "onditions o$ industrial ro"esses 8C/, .:9& 7roteases are generall% "lassi$ied in t!o grou sA endo e tidases P roteases that "leave e tide #onds !ithin the roteinP and e(o e tidases, !hi"h "leave o$$ amino a"ids $rom the ends o$ the rotein& Man% roteol%ti" enz%mes $rom h% erthermo hili" ar"haea and #a"teria have #een identi$ied& Most o$ them are not onl% "atal%ti"all% a"tive at high tem- eratures, #ut the% also retain their "atal%ti" a"tivit% in the resen"e o$ detergents and other denaturing su#stan"es, su"h as urea, guanidine-F,l, dithiotreitol or 2mer"a toethanol& F% erthermo hili" roteases have #een identi$ied in 0/ maritima, 0/ aggregans, 0/ celer, 0/ litoralis and Pyrococcus s & KO0) 8.1, .29& The thiol rotease $rom Pyrococcus s & KO0) "onstitutes the most h% erthermo hili" rotease among them, sho!ing an o timal tem erature o$ 11:4,& 'everal serine roteases $rom P/ furiosus have #een "loned and e( ressed in E/ coli 8.C-.39& The industrial use o$ h%- erthermo hili" roteases that have not %et #een "loned in E/ coli is hindered #% the $a"t that the% "ome $rom anaero#i" h% erthermo hili" mi"roorganisms, !hose "ulture at a s"ale required to meet industrial needs is di$$i"ult& Le"entl%, ho!- ever, roteases $rom the aero#i" h% erthermo hile &eropy rum perni+ K1 have #een isolated 8..9& This mi"roorganism gro!s o timall% at -:4, and its gro!th attern and nutri- tional requirements are amena#le to large-s"ale "ulture te"h- niques, ma*ing the strain an attra"tive sour"e o$ thermosta- #le roteases& Hyperthermophilic ,olecular 7iolo-y Industry Enzymes <or 'he

mus scodoductus GLo"he Mole"ular Bio"hemi"alsH& )n addition, a h% erthermo hili" ligase has #een identi$ied in &cidi anus ambivalens 8/39& A99:I+A'I(&8 '( 'HE 9*:9 A&) 9A9E! I&)*8'!= The enz%mati" treatment o$ !ood $or the rodu"tion o$ ul is #ased on hemi"ellulose h%drol%sis !ith (%lanases& These enz%mes "leave (%lan, the main "om onent o$ hemi- "elluloses and one o$ the most a#undant organi" su#stan"es on earth 8/.9& The treatment o$ !ood to o#tain ul is "arried at ver% high tem eratures and at #asi" F to hel disru t the "ell !all stru"ture& Thus, an interesting (%lanase $rom the industrial oint o$ vie! should #e ver% thermosta#le and a"tive at neutral to al*aline F& >dditionall%, the enz%mes used in these ro"esses should la"* "ellulol%ti" a"tivit% in order to avoid h%drol%sis o$ the "ellulose $i#res& The% should also #e o$ lo! mole"ular mass in order to di$$use !ell in the ul $i#res and #e eas% to o#tain at lo! "ost& 8329& Most "ommer"iall% availa#le (%lanases do not meet all these requirements& >lthough onl% $e! e(treme thermo hili" mi"roorganisms are a#le to se"rete su"h enz%mes, some (%lanases have #een re orted to o erate at ver% high o timal tem eratures& These enz%mes redu"e "hlorine "onsum tion in the #lea"hing ro"ess, thus lo!ering environmental damage due to the resen"e o$ organi" halogens 8//9& F% erthermo hili" (%lanases $rom 0hermotoga, Dyctio glomus, Sulfolobus, Pyrococcus and Pyrodictium !ere isolated !ith o timal tem eratures a#ove -:4, 8//-;C9& >mong them, the (%lanase $rom 0hermotoga s & strain +J''C-B1 is the most h% erthermo hili" (%lanase so $ar re orted, !ith an o timal tem erature o$ 1134, 8;19& F% erthermo hili" (%lanases $rom Dyctioglomus s &, 0hermotoga s &, and Pyro coccus furiosus have #een "loned and su""ess$ull% e( ressed in E/ coli 8;D-;/, /-9& :I9A8E8 Ni ases are the most !idel% used grou o$ #io"atal%sts $or #iote"hnolog%& The% "atal%se #oth the h%drol%sis o$ long"hain a"%lgl%"erols into gl%"erol and $att% a"ids and the reverse rea"tion& Thus, these enz%mes "an #e a lied industriall% as h%drolases and as s%nthetases 8;;9& Mi"ro#ial li ases have an enormous #iote"hnologi"al otential o!ing to their unique "hara"teristi"s, in"luding sta#ilit% in organi" solvents, #road su#strate s e"i$i"it%, and high enantiosele"tivit% 8;-9& >mong the man% a li"ations o$ li ases as h%drolases is their use as additives in the detergent industr%& )n the $ood industr%, li ases are also used to modi$% the stru"ture o$ some trigl%"erides tr%ing to enhan"e their h%si"al, nutritional or sensor% ro erties& >dditionall%, these enz%mes are also used to o#tain ol%-unsaturated $att% a"ids G71+>sH $rom lant and animal li ids, !hi"h "an #e su#sequentl% used to rodu"e harma"euti"als& >nother a li"ation o$ li ases as h%drolases is their use in removing the h%dro ho#i" "om onents o$ the !ood G< it"h=H in the ul and a er industr% 8;-9& Ni ases and esterases are also #eing used $or the s%nthesis o$ ne! #io ol%meri" materials, su"h as ol% henols, ol%sa""harides and ol%esters& These "om ounds are o$

Thermo hili" 06> ol%merases are res onsi#le $or the elongation o$ the rimer strand o$ a gro!ing 06> mole"ule during 06> am li$i"ation #% 7,L& The isolation o$ Taq 06> ol%merase $rom the thermo hili" #a"terium 0hermus a,uaticus, and in arti"ular its "loning and e( ression in E/ coli 8./9, !as trul% a revolutionar% event in the $ield o$ Bio- te"hnolog%& 'in"e then, man% other 06> ol%merases have #een isolated $rom thermo hili" and h% erthermo hili" ar- "haea and are no! availa#le "ommer"iall%A P/ furiosus 7$u ol%merase 8.;9, 0/ litoralis Vent ol%merase 8.-, /:9, P/ 3oesei 7!o ol%merase 8/19, Pyrococcus strain GB-0 0ee Vent ol%merase 8/29, and a 06> ol%merase $rom 0her mococcus s & strain -6-/ 8/C9& >lthough all su"h enz%mes have error rates that are mu"h lo!er than Taq ol%merase, o!ing to their lo! e(tension rates and unsuita#ilit% $or the am li$i"ation o$ long 06> $ragments none o$ them has re- la"ed the use o$ the 0/ a,uaticus enz%me& 7roo$reading en- z%mes, su"h as 7$u or Vent and 0ee Vent ol%merases, are re$erred onl% i$ ver% high $idelit% is required& Le"entl%, a h% erthermo hili" ar"haeal 06> ol%merase isolated $rom Pyrococcus s & strain KO01 8/D9 e(hi#its a lo! error rate and high ro"ess and e(tension rates& ,ommer"iall% avail- a#le thermo hili" 06> ligases are the 7$u 06> ligase $rom P/ furiosus G'tratageneH and the T"s 06> ligase $rom 0her

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Protein & Peptide Letters, 2006, Vol. 13, No. 7 81.9 81/9 81;9 81-9 82:9 8219 8229 22:82C9 82D9 8239 82.9 82/9 82;9 82-9 8C:9 8C19 8CC9

Bouzas et al. 0eNong, ?&+& G 2001H in Methods in En6ymology G>dams, M&2&2& and Kell%, L&M&, ?ds&H& & C-11& >"ademi" 7ress, 'an 0iego, ,ali$ornia, 1'>& Fu#er, F&, Fohn, M&J&, La"hel, L&, +u"hs, T&, 2immer, V&,& and 'tetter, K&O& G2002H 'ature 51", .C-./& 7a"e, 6& G1001H Cell 9!, 3C1-3CC& 'tetter, K&O& G1004H in Early life on Earth GBengston, '& ?d&H& & 1DC-131& ,olum#ia 1niversit% 7ress, 6e! 5or*& +orterre, ,&L& G1005H &cad Sci 111 81#, D13-D22& Miller, '&N and Naz"ano, >& G1005H ;/ Mol/ Evol/ 51, .;--.-2& Galtier, 6&, Tourasse, 6& and Gou%, M& G1000H Science $#8, 221& Bro!n, J&L& and 0oolittle, 2&+& G1005H Proc/ 'atl/ &cad/ Sci/ US& 7$, 2DD1-2DD3& +orterre, 7&, Bergerat, >& and NR ez-Gar"Sa, 7& G1006H FEMS Mi crobiol/ 4ev/ 1#, 2C/-2D;& No ez, G& G1000H ;/ Mol/ Evol/ 59, DC--D32& Todorova, L& and >tanosov, B& G2004H 1nt/ ;/ 2iol/ Macromol/ 85, 1C3-1D/& Kimura, M&, Kimura, J&, 0avie, 7&, Leinhardt, L& and 0iJ*, J& G1014H FE2S *ett/ 1"9, 1/.-1/;& Grogan, 0&2& G2004H Curr/ 1ssues/ Mol/ 2iol/ 9, 1C/-1DD& ,omita, 7&B&, Gagosian, L&B&, 7ang, F& and ,ostello, ,&?& G1014H ;/ 2iol/ Chem/ $!7, 132CD-132D1& Blo"h, ?&, La"hel, L&, Burgra$$, '&, Fa$en#radl, 0&, Jannas"h, F&2& and 'tetter, K&O& G100;H E+tremophiles 1, 1D-21& Kashe$i, K& and Novle%, 0&L& G200@H Science 1! GC:1H, 3.C3&Fane%, 7&J&, 'tees, M& and Konis*%, J& G1000H ;/ 2iol/ Chem/ $"5, ;D1-;D;& Kirino, F&, >o*i, M&, >oshima, M&, Fa%ashi, 5&, Oh#a, M&, 5amagishi, T&, 2a*agi, T& and Oshima, T& G1004H Eur/ ;/ 2iochem/ $$), 2/3-2;1& Fough, 0&2& and 0anson, M&J& G1000H Curr/ <pin/ Chem/ 2iol/ 8, C--D.& 0M>uria, '&, Morana, >&, +e##raio, +&, ,arlo, V&, 0e Losa, M& and 6u""i, L& G1006H Protein E+pr Purif/ ", 2---C:;& ?verl%, ,& and >l#erto, J& G2000H Front/ 2iosci/ !, /;:-/;.& Fa*i, G&0& and La*shit, '&K& G200@H 2iores/ 0echnol/ #7, 1/-CD& Bertoldo, ,& and >ntrani*ian, G& G 2002H Curr/ <pin/ Chem/ 2iol/ 131-1.:& Bauer, M&2&, 0ris*ill, N&?& and Kell%, L&M& G1001H Curr/ <pin/ 2iotechnol/ 7, 1D1-1D3& Kim, M&'&, 7ar*, J&T&, Kim, 5&2&, Nee, F&'&, 6%a!ira, L&, 'hin, F&'&, 7ar*, ,&'&, 5oo, '&F&, Kim, 5&L&, Moon, T&2& and 7ar*, K&F& G2004H &ppl/ Environ/ Microbiol/ "), C-CC-C-D:& Ko"h, L&, ' reinat, K&, Nem*e, K& and >ntrani*ian, G& G1001H &rch/ Microbiol/ 1!!, 3/2-3/;& Nee, J&T&, Kanai, F&, Ko#a%ashi, T&, >*i#a, T& and Kudo, T& G1006H ;/ Ferment/ 2ioeng/ #$, DC2-DC;& ,hung, 5&,&, Ko#a%ashi, T&, Kanai, F&, >*i#a, T& and Kudo, T& G1005H &ppl/ Environ/ Microbiol/ 91, 13:2-13:.& 0ong, G&, Vieille, ,&, 'av"hen*o, >& and Qei*us, J& G& G100;H &ppl/ Environ/ Microbiol/ 98, 1.CC3-1.CD2& Jorgensen, '&, Vorgias, ,&?& and >ntrani*ian, G& G100;H ;/ 2iol/ Chem/ $"$, 1.CC3-1.CD2& Naderman, K&>&, 0avis, B&L&, Krutzs"h, F&,&, Ne!is, M&'&, Gri*o, 5&V&, 7rivalov, 7&N& and >n$insen, ,&B& G100@H ;/ 2iol/ Chem/ $9#, 2DC-D-2DD:1& Laas"h, ,&, 'treit, 2&, '"hanzer, J&, Bi#el, M&, Gosslar, 1& and Nie#l, 2& G2000H E+tremophiles 5, 1;--2::& Lol$smeier, M&, Faseltine, ,&, Bini, ?&, ,lar*, >& and Blum, 7& G1001H ;/ 2acteriol/ 1#), 12;/-12-3& 7iller, K&, 0aniel, L&M& and 7eta"h, F&F& G1006H 2iochim/ 2iophys/ &cta 1$7$, 1-/-2:3& Gali"het, >& and Belar#i, >& G1000H FE2S *ett/ 5!#, 1;;-1-2& 0oman-7%t*a, M& and Bardo!s*i, J& G 2004H Crit/ 4ev/ Microbiol/ 8), 1:/-121& 6iehaus, +&, Bertoldo, ,&, KTler, M& and >ntrani*ian, G& G1000H &ppl/ Microbiol/ 2iotechnol/ !1, /11-/2-& Bro!n, '&F& and Kell%, L&M& G100@H &ppl/ Environ/ Microbiol/ !7, 2.1D-2.21& Gantelet, F& and 0u"hiron, +& G1001H &ppl/ Microbiol/ 2iotechnol/ 57, //:-///& '"huliger, J&2&, Bro!n, '&F&, Baross, J&>& and Kell%, L&M& G100@H Mol/ Mar/ 2iol/ 2iotechnol/ $, /.-;/&

s e"ial interest $rom the environmental oint o$ vie! #e"ause the% are #iodegrada#le and "an #e rodu"ed $rom rene!a#le natural resour"es& Le"entl%, mi"ro#ial li ases have #een used to o#tain #iodiesel #% transesteri$i"ation o$ lant $ats& Biodiesel is an alternative sour"e o$ energ% that redu"es sul hur o(ide rodu"tion& )t is "urrentl% #eing used in u#li" trans ort, although its long-s"ale rodu"tion is still ham ered #% the high "osts o$ the #io"atal%st& >nother main "hara"teristi" o$ mi"ro#ial li ases is their enantiosele"tivit%, !hi"h ma*es them suita#le $or the rodu"tion o$ a num#er o$ $ine "hemi"als& This is o$ great imortan"e in the "ase o$ "ertain harma"euti"als and agro"hemi"als, !hi"h need to #e enantio ure to #e $un"tional 8;;9& Thermosta#le li ases $rom h% erthermo hili" ar"haea have #een isolated $rom Pyrobaculum calidifontis, Pyrococ cus furiosus and Pyrococcus hori.oshii and "loned in E/ coli 8-:, -1, -29& >lthough re orts on li ases $rom h% erthermohili" mi"roorganisms are s"ant, some su"h enz%mes have #een isolated $rom moderatel% thermo hili" mi"roorganisms and e(hi#it a"tivit% at tem eratures o$ -:4, 8-C, -D9& +(&+:*8I(&8 One o$ the main ro#lems in most enz%mati" industrial ro"esses is ho! to a"hieve an a ro riate "om romise #et!een harsh !or*ing "onditions and enz%me sta#ilit%& ?nz%mes $rom h% erthermo hili" mi"roorganisms o$$er a otential solution $or industrial ro"esses "arried out at high tem eratures& 'in"e onl% a ver% $e! s e"ies $rom this grou o$ mi"roorganisms have #een isolated to date, there seems to #e a large num#er o$ h% erthermo hili" "atal%sts !ith unique ro erties a!aiting dis"over%& The develo ment o$ mole"ular tools together !ith #iote"hnologi"al resear"h should lead to the $uture availa#ilit% o$ ne! h% erthermohili" enz%mes to meet all t% es o$ industrial demands& !E<E!E&+E8
819 829 8C9 8D9 839 8.9 8/9 8;9 8-9 81:9 88), 8119 8129 81C9 81D9 8139 'atl/ Bro"*, T&0& G106;H Science 1!#, 1:12-1:1)mana*a, T& and >tomi, F& G2002H Chem/ 4ec/ $, 1D--1.C 'tetter, K&O& G1000H FE2S *ett/ 5!$, 22-23& Fu#er, L& and 'tetter, K&O& G2001H in Methods in En6ymology G>dams, M&2&2& and Kell%, L&M&, ?ds&H& Vol& CC:, & 11-2D& >"ademi" 7ress, 'an 0iego, ,ali$ornia, 1'>& Bernal, >&, ?ar, 1& and K%r ides, 6& G2001H 'ucleic &cids 4es/ $7, 12.-12/& G!!!&genomesonline&orgH Barns, '&M&, 0el!i"he, ,&+&, 7almer, J&0& and 7a"e, 6& L& G1006H 7ro"& 'atl/ &cad/ Sci/ US& 78, -1;;--1-C& Fedri"*, 0&B&, 7ledger, L&0&, 2hite, 0&,& and Baross, J&>& G1002H FEMS Microbiol/ Ecol/ 1)1, 1-1:& Vieille, ,& and Qei*us, G&J& G2001H Microbiol/ Mol/ 2iol/ 4ev/ 9!, 1-DC& 'tetter, K&O& G1000H in E+tremophiles: Microbial *ife in E+treme Environments GFori*oshi, K& and Grant, 2&0& ?ds&H& &1-2D& 2ile%, 6e! 5or*& >sh*in, >&, 0ziedzi", J&M& and 5amane, T& G101;H 'ature /.--//1& Fu#er, L&, Burggra$, '&, Ma%er, T&, Barns, '&M&, Lossnagel, 7& and 'tetter, K&O& G1005H 'ature 8"9, 3/-3;& Qu"*er*andl, ?& and 7auling N& G1065H ;/ 0heor/ 2iol/ #, C3/-C..& 2oese, ,&L& G101;H Microbiol/ 4ev/ !1, 221-2/1& 2oese, ,&L& and +o(, G&?& G10;;H Proc/ 'atl/ &cad/ Sci/ US& "5, 3:;;-3:-:& 2oese, ,&L&, Kandler, O& and 2heelis, M&N& G1000H Proc/ &cad/ Sci/ US& #", D3/.-D3/-&

8CD9 8C39 8C.9 8C/9 8C;9 9, 8C-9 8D:9 8D19 8D29 8DC9 8DD9 8D39 8D.9 8D/9 8D;9 8D-9 83:9 8319 8329 83C9 83D9 8339

Industrial Applications of Hypert er!op ilic "nzy!es# A $e%ie& 83.9 83/9 83;9 83-9 8.:9 8.19 8.29 8.C9 ;/ 8.D9 8.39 8..9 8./9 1$", 8.;9 8.-9 8/:9 8/19 8/29 8/C9 8/D9 0ong, G&, Vieille, ,& and Qei*us, J&G& G100;H &ppl/ Environ/ Microbiol/ 98, C3//-C3;D& LUdiger, >&, Jorgensen, 7&N& and >ntrani*ian, G& G1005H &ppl/ Environ/ Microbiol/ 91, 3./-3/3& ?rra-7uJada, M&, 0e#iere, 7&, 0u"hiron, +& and OM0onohue, M&J& G1000H ;/ 2acteriol/ 1#1, C2;D-C2;/& Lao, M&, Tan*asale, >&, Ghatge, M& and 0es hande, V& G1001H Microbiol/ Mol/ 2iol/ 4ev/ 9$, 3-/-.CD& ?i"hler, J& G2001H 2iotechnol/ &dv/ 17, 2.1-2/;& Kling#erg, M&, Fash!a, +& and >ntrani*ian, G& G1001H &ppl/ Mi crobiol/ 2iotechnol/ 85, /13-/1-& +uJi!ara, '&, O*u%ama, '& and )mana*a, T& G1006H =ene 1"7 , 1.31/:& Tsunasa!a, '&, 6a*ura, '&, Taniga!a, T& and Kato, )& G1001H 2iochem& GTo*%oH 1$5, //;-/;C& Far!ood, V&J&, 0enson, J&0&, Lo#inson-Bidle, K&>& and '"hreier, F&J& G100;H ;/ 2acteriol/ 1"7, C.1C-C.1;& Falio, '&B&, Blumentals, )&)&, 'hort, '&>&, Merrill, B&M& and Kell%, L&M& G1006H ;/ 2acteriol/ 1"#, 2.:3-2.12& 'a*o, 5&, ,roo"*er, 7&,& and )shida, 5& G1006H FE2S *ett 51!, C2-CCD& ,hien, >&, ?dgar, 0&B& and Trela, J&M& G10;6H ;/ 2acteriol/ 133:-3/& Nund#erg, K&'&, 'hoema*er, 0&0&, >dams, M&2&, 'hort, J&M&, 'orge, J&>& and Mathur, ?&J& G1001H =ene 1)#, 1-.& ,ariello, 6&+&, '!en#erg, J&>& and '*o e*, T&L& G1001H 'ucleic &cids 4es/ 17, D1-C-D1-;& Mattila, 7&, Kor ela, J&, Ten*anen, T& and 7it*anen, K& G1001H 'u cleic &cids 4es/ 9, D-./-D-/C& +re%, B& and 'u mann, B& G1005H 2iochemica $, CD-C3& 7erler, +&B&, Kumar, '& and Kong, F& G1006H &dv/ Protein/ Chem/ 5#, C//-DC3& 'outh!orth, M&2&, Kong, F&, Ku"era, L&B&, 2are, J&, Jannash, F&2& and 7erler, +&B& G1006H Proc/ 'atl/ &cad/ Sci/ US& 78, 21221-& Ta*agi, M&, 6ishio*a, M&, Ka*ihara, F&, Kita#a%ashi, M&, )noue, F&, Ka!a*ami, B&, O*a, M& and )mana*a, T& G100;H &ppl/ Environ/ Microbiol/ 98, D3:D-D31:& 8/39 8/.9 8//9 8/;9 8/-9 8;:9 8;19 $"", 8;29 8;C9 8;D9

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Kletzin, >& G1002H 'ucleic &cids 4es/ $), 3C;--3C-.& ,hen, ,&, >dol hson, L&, 0ean, +&, ?ri*sson, K&, >dams, M& and 2est heling, J& G100;H En6yme Microb/ 0echnol/ $), C--D3& Vi*ari, N&, Kantelinen, >&, 'undquist, J& and Nin*o, M& G 1004H FEMS Microbiol/ 4ev/ 18, CC3-C3:& MarJaana, L&, )ndra, M&, Birgitte, >& and Niisa, V& G1004H &ppl/ Microbiol/ 2iotechnol/ 51, 1C:-1C-& Bauer, M&, 0ris*il, N&, ,allen, 2&, 'nead, M&, Mathur, ?& and Kell%, L& G1000H ;/ 2acteriol/ 1#1, 2;D-2-:& 6u""i, L&, Mora""i, M&, Va""aro, ,&, Ves a, 6& and Lossi, M& G100@H 2iotechnol/ &ppl/ 2iochem/ 1", 2C--23:& 'im son, F&, Fau$ler, 1& and 0aniel, K& G1001H 2iochem/ ;/ 1//-1;3& >ndrade, ,&M&, >guiar, 2&B& and >ntrani*ian, G& G2001H &ppl/ 2iochem/ 2iotechnol/ 71 78, .33-..-& ,annio, L&, 0i 7ritzio, 6&, Lossi, M& and Morana, >& G2004H E+tre mophiles #, 11/-12D& Morris, 0&, Gi##s, M&, ,hin, ,&, Koh, F&, 2ong, K&, >llison, L&, 6elson, 7& and Bergquist, 7& G1001H &ppl/ Environ/ Microbiol/ 95, 1/3--1/.3& 'aul, 0&, 2illiams, N&, Leeves, L&, Gi##s, M& and Bergquist, 7& G1005H &ppl/ Environ/ Microbiol/ 91, D11:-D11C& Jiang, Q&, Qhu, 5&, Ni, N&, 5u, V&, Kusa#e, )&, Kitao*a, M& and Fa%ashi, K& G2004H ;/ 2iotechnol/ 115, 123-1CD& Vue, 5& and 'hao, 2& G2004H 2iotechnol/ *ett/ 17, 1311-1313& Jaeger, K&?& and ?ggert T& G2002H Curr/ <pin/ 2iotechnol/ 18, C-:C-/& Jaeger, K&?& and Leetz, M&T& G1001H 0rends 2iotechnol/ 19, C-.D:C& Fotta, 5&, ?za*i, '&, >tomi, F& and )mana*a, T& G2002H &ppl/ Envi ron/ Microbiol/ 9#, C-23-C-C1& )*eda, M& and ,lar*, 0& G1001H 2iotechnol/ 2ioeng/ !", .2D-.2-& >ndo, '&, )shida, F&, Kosugi, 5& and )shi*a!a, K& G2002H Environ/ Microbiol/ 9#, DC:-DCC& Ni, F& and Qhang, V& G2005H Protein E+pr/ Purif/ 5$, 13C-13-& Lathi, 7&, 'a na, B&, 'e(ena, L& and Gu ta, L& G2000H 2iotechnol/ *ett/ $$, D-3-D-;&

8;39 8;.9 8;/9 8;;9 8;-9 8-:9 8-19 8-29 &ppl/ 8-C9 8-D9

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