심해열수유래초고온성효소의산업화 김현규슈퍼바이오 ( 주 ) 목차 1. 서론 2. 탄수화물및단백질분해효소 2-1. 전분분해효소 2-2. Cyclodextrin glycosyltransferase (CGTases) 2-3. Cellulase 2-4. 단백질분해효소 2-5. DNA polymerase 3. 참고문헌
1. 서론 극한환경 (Extreme environments) 에서생육가능한생물을극한생물 (Extremophiles) 이라고한다. 이러한극한생물은높은온도, 압력, ph, 염농도에서생육하거나혹은반대로낮은온도나 ph, 영양분등의비일반적인환경에서생육할수있다. 또한높은수치의방사선이나독성물질의노출에도견딜수있는생물체등인간의환경에비교하여상대적으로극한환경에생육할수있는생물체를통칭하여극한생물이라한다. 이들극한생물을표현하는용어는 Table 1 과같다. 이러한극한생물의대부분은미생물이며고온성미생물이가장많이연구되어왔다. 미생물은생육온도에따라저온균 (Psychrophile, 15-20 이하 ), 중온균 (mesophile, 20-45 ), 고온균 (thermophile, 45-80 ) 으로분류되며고온균은다시 moderate thermophile, extreme thermophile(65-85 ), hyperthermophile(85-110 ) 으로분류한다. 고온균은약 30 년전처음으로 Thomas D. Brock 에의해 Yellowstone National Park 의 hot spring 의미생물연구로시작하여 1969 년 70 이상에서생육하는 Thermus aquaticus (Block and Freeze, 1969) 를동정하였고, 1973 년에는 85 이상에서생육하는 Sulfolobus acidocaldarius 가보고되었다. 1970 년대후반에는심해의 hydrothermal vents 주변의고온에서생육가능한초고온균에대한연구가시작되어현재까지 60 종류이상의 hyperthermophiles 이분리되었다. Table 1. Terms used to describe extremophiles. Terms Describe Alkaliphile An organism with optimal growth at ph values above 10 Barophile Endolith Extreme Acidophile Extremophile Halophile Hyperthermophile Oligotroph Psychrophile Toxitolerant Xerotolerant An organism that lives optimally at high hydrostatic pressure An organism that lives in rocks An organism with a ph optimum for growth at, or below, ph 3 An organism that is isolated from an extreme environment and often require the extreme condition for growth. Extreme is anthropocentrically derived An organism requiring at least 0.2 M salt for growth An organism having a growth temperature optimum of 80 or higher An organism with optimal growth in nutrient limited conditions An organism having a growth temperature optimum of 15 or lower, and a maximum temperature of 20 An organism able to withstand high levels of damaging agents. For example, living in water saturated with benzene, or in the water-core of a nuclear reactor An organism capable of growth at low water activity. For example, extreme halophlie or endolith 이는대부분독일 Regensburg 대학의 Karl O. Stetter 에의해보고되었으며
DSMZ 등에기탁되어있다. 이중대표적인초고온성미생물의생육온도및생육환경에따라살펴보면 Table 2 와같다. Table 2. Growth temperature and habitats of hyperthermophilic microorganisms 극한미생물 생육온도 서식지 생육환경 Thermotoga maritima 55~90 해양 혐기상태 Aquifex pyrophilus 67~95 해양 호기상태 Pyrobaculum aerophilum 75~104 해양 호기 / 혐기상태 Staphylothemus marinus 65~98 해양 혐기상태 Pyrodictium occultum 82~110 해양 혐기상태 Thermodiscus maritimus 75~98 해양 혐기상태 Pyrococcus furiosus 70~105 해양 혐기상태 Archaeoglobus fulgidus 60~95 해양 혐기상태 Methanopyrus kandleri 84~110 해양 혐기상태 Methanococcus igneus 45~91 해양 혐기상태 Sulfolobus acidocaldarius 60~85 육지 호기상태 Metallosphaera sedula 50~80 육지 호기상태 Acidianus infemus 60~95 육지 호기 / 혐기상태 Stygiolobus azoricus 57~89 육지 혐기상태 Thermoproteus tenax 70~97 육지 혐기상태 Pyrobaculum islandicum 74~103 육지 혐기상태 Themofilum pendens 70~95 육지 혐기상태 Desulfuroccus mobilis 70~95 육지 혐기상태 Methanothermus sociabilis 65~97 육지 혐기상태 Eubacteria 중초고온성미생물로는 1986 년 Stetter 와 Huber 에의해분리된 80 에서생육적온을갖고 90 까지생육가능한 Thermotoga 와 1992 년 Huber 등에의한 Aquifex pyrophilus, 1998 년 Deckert 등에의한 Aquifex aeolicus 가있다. 이두초고온성미생물은 85 의생육적온을갖고 95 까지생육이가능하다. 그러나대부분의초고온균은원시생물 (Archaea) 에속한다. 원시생물은형태적으로는원핵생물 (prokaryote) 과유사하나유전자수준에서비교하면진핵생물 (eukaryote) 에만존재하거나혹은유일하게존재하는유전자를지니고있어진화계통적으로오히려진핵생물에가까운것으로밝혀졌다. 이러한특징으로인해원시생물은생명진화도상에서진핵생물의줄기에서분리되어나옴을알수있다 (Figure 1). 현재까지알려진가장높은생육온도를갖는초고온균은 Pyrolobus fumarii 로최적생육온도가 105, 최대 113 에서도생육이일어나며 90 이하에서는생육이멈추는것으로알려져있다 (Blöchl et al., 1997). 이렇듯초고온균은극한환경과생명의기원에대한이해를넓혀주었을뿐만아니라새로운생물자원으로서의가치를갖고있다. 초고온균에서유래한초고온성효소는내열성, 계면활성제및유기용매등에대한안정성이매우우수하여소량의효소로도장기간사용이가능하고고온에서반응시키므로기질의용해도가낮은
물질의용해도증가, 물질확산속도의증가, 반응속도의증가, 반응시간의단축, 중온성미생물에대한오염방지등의수많은장점을갖고있다. 따라서, 이러한초고온성효소는중온성효소의단점을극복할수있을뿐만아니라, 학문적으로도내열성의원인규명, 구조생물학등의재료로서매우중요 Figure 1. Pylogenetic tree of life. 하며그응용범위가계속적으로넓어지고있다. 이러한중요성을갖는초고온균및초고온성효소로인해전체염기서열의분석이연구의핵이되고있다. 2001년 10월현재미생물 genome 전체염기서열이밝혀진것은 63종이며이들대부분이병원성세균과산업적으로유용한균주들이다. 이중 archaea 는 10종이 genome 서열이완성되었고이중 9종이초고온균이다 (Table 3). 일단 genome 서열이완성되면산업적으로유용한효소의유전자를쉽게 cloning 할수있고이의효소연구가가능해진다. 초고온성효소는이의유전자가대장균을비롯한중온균에서발현되면내열성으로인해 80 이상의고온에서열처리함으로써중온균유래의효소는열에의해변성시키고원하는초고온성효소만을정제하는것이용이하다. 또한유기용매나계면활성제등단백질변성제에대한내성이강하여고온고압뿐아니라유기용매하에서의반응촉매로서의이용가능성도갖고있다. 이러한장점을지닌초고온성효소의산업적으로응용되고있는분야는 Table 4에나타내었다. Table 3. Hyperthermophilic archaea and bacteria complete genomes taxonomy list (Revised Oct 30, 2001) Species NCBI number Genome Size (bp) Deposited Date Aeropyrum pernix NC_000854 1669695 Jul 5, 2001 Archaeoglobus fulgidus NC_000917 2178400 Dec 17, 1997 Halobacterium sp. NRC-1 NC_002607 2014239 Sep 27, 2001 Methanococcus jannaschii NC_000909 1664970 Sep 10, 2001
Pyrococcus abyssi NC_000868 1765118 Jun 21, 2001 Pyrococcus horikoshii NC_000961 1738505 Jul 3, 2001 Sulfolobus solfataricus NC_002754 2992245 Oct 3, 2001 Sulofolobus tokodaii NC_003106 2694765 Sep 15, 2001 Thermoplasma acidophilum NC_002578 1564906 Oct 12, 2001 Thermoplasma volcanium NC_002689 1584804 Mar 22, 2001 Aquifex aeolicus a NC_000918 1551335 Sep 7, 2001 Thermotoga maritima a NC_000853 1860725 sep 10, 2001 a bacteria Table 4. Hyperthermophilic enzyme's uses in biotechnology Hyperthermophilic enzymes Use DNA polymerases DNA amplification by PCR Alkaline phosphatase Diagnostics Proteases and lipases Dairy products Lipases, pullulanases and proteases Detergents Proteases Baking and brewing and amino acid production from keratin Amylases, α -glucosidase, pullulanase Baking and brewing and amino acid and xylose/glucose isomerases production from keratin Alcohol dehydrogenase Chemical synthesis Xylanases Paper bleaching Lenthionin Pharmaceutical 산업적으로유용한초고온성효소중에서몇가지를살펴보면다음과같다. 2. 탄수화물및단백질분해효소 2-1. 전분분해효소 ; amylase, glucoamylase, pullulanase 전분가공은전분의액화 (liquefaction), 당화 (saccharification), 이성화 (isomerization) 에의해최종적으로 starch 를 fructose syrup 으로제조하게된다. 이러한분해공정에최근초고온성효소인 amylase, glucoamylase, pullulanase 등의분리에의해액화공정과당화공정을 90-105 의고온과 ph 4.5-5.0 의약산성조건에서통합공정으로의시도가이루어지고있다. 내열성 α -amylase 는 Pyrococcus woesei (Koch et al., 1991), Pyrococcus furiosus (Laderman et al., 1993a, b) 에서분리되었고이들의최적활성온도는 100 이다. glucoamylase 는 Thermoanaerobacterium thermosaccharolyticum DSM 571 등에서분리되었으며또한 pullulanase 도 Thermococcus (Brown and Kelly, 1993), Staphylothermus, Desulfurococcus (Canganella et al., 1994) 등에서분리되었고이들의최적온도는 calcium ion 이없는상태에서도 90-105 를보였다.
2-2. Cyclodextrin glycosyltransferase (CGTases) CGTase는무작위적으로 polysaccharides내에있는α -1,4-linkages를공격하여 intra- molecular transglycosylation reaction에의해전분을환상으로전환시킨다. Cyclodextrin은환상내부에큰구멍을갖고있기때문에여기에여러가지물질을포입하여복합체를형성할수있으므로식품공업에유용하다. Cyclodextrin 생산공정은첫단계로내열성효소로전분을액화시킨후, 두번째단계로 Baciilus sp. 를이용하여낮은온도에서처리하는공정으로이루어졌다. 그러나, 내열성 CGTase의발견은액화와 cyclization을 105 에서한단계에서처리할수있다. 내열성 CGTase는 Thermoanaerobacter sp. (Norman and Jorgensen, 1992) 와 Thermoanaerobacterium thermosulfurogenes (Wind et al., 1995) 에서발견되었다. 2-3. Cellulase Cellulose 는식물 biomass 의 40% 이상을차지하고있으며, glucose 의 β -1,4-glycosidic bonds 로직선으로연결된 polymer 이다. Cellulose 는 endoglucanase, exoglucanase (cellobihydrolase), β -glucosidase 등의적어도세가지효소들의연속적인작용에의해 glucose 로분해할수있다. Cellulose 에대해활성을보이는내열성 cellulase 가다양한고온균들로부터이들효소의유전자들이클로닝되어효소의특성이밝혀졌다. Thermotoga maritima MSB8 (Bronnenmeier et al., 1995) 에서 95 에서활성을나타내는 cellulase 와 Thermotoga neapolitana 의 cellulase A (29kDa, 95 ) 와 cellulase B (30kDa, 106 ) 가각각정제되었고, 이두유전자들모두 cloning 되었다 (Bok et al., 1998). 고온혐기성세균인 Caldocellum saccharolyticum 의 genome 에서는 cellulase 와 hemicellulase genes 들이 cluster 로함께존재하였다 (Teo et al., 1995). 최근 β -glucans 과 cellulose 의 β -1,4-bonds 분해할수있는내열성 endoglucanase 가 Pyrococcus furiosus 에서분리되어그유전자가대장균에클로닝되어특성이밝혀졌다. 이재조합 endoglucanase 는기질로서 cellopentaose 와 cellohexaose 에특이적인활성을보였다 (Bauer et al., 1999). Cellulose 는높은온도에서알카리를통한전처리과정을통해 ethanol 발효에이용되므로초고온성 cellulase 는이에가장적합한효소로부각된다. 2-4. 단백질분해효소 Protease 들은단백질들의아미노산이나펩타이드들로의전환반응을촉매하는효소로서그들의촉매부위의성질에따라 serine protease, cysteine protease, aspartic protease, metalloprotease 로분류되어진다. 세계적으로산업적으로사용되는단백질분해효소는다른공정에이용되는효소들의양에비하여아주많으며 serine alkaline protease 들은세정을위한가정용세제의첨가물로사용되어지며높은 keratin 분해및 elastin 분해활성을보이는 protease 들은피혁산업에있어 soaking 에사용되어진다. 높은온도나극단적인 ph 조건하에서반응을촉매할수있는 protease 의개발은산업적응용에있어매우유용할것이다. 다양한내열성 protease 들이 Desulfurococcus, Sulfolobus, Staphylothermus, Thermococcus, Pyrobaculum, Pyrococcus 속들에속하는초고온성미생물들에서분리되어져왔다. Extremophile 들로부터의대부분의
protease 들은 serine type 에속하며, 100 이상의고온이나심지어고농도의세제, 변성제의존재하에서도안정한것으로밝혀져왔다. 2-5. DNA polymerase 분자생물학연구에있어내열성효소의중요성을가장부각시킨것은바로 Taq DNA polymerase 로 PCR (Saiki et al., 1988) 의자동화를가능하게한것이다. 내열성 DNA polymerase 로처음특성이밝혀진 Taq DNA polymerase (Chien et al., 1976) 는 Thermus aquaticus 로부터분리되었고이후많은 Thermus 속으로부터 Tfl Pol. (Kaledin et al., 1981), Tth Pol. (Ruttimann et al., 1985), Tca Pol. (Park et al., 1993), Tfi Pol. (Jung et al., 1997), Top Pol. (Kim et al., 1998), Tx1 Pol. (Choi et al., 2001) 등이분리되어 PCR 에적용되고있다. Taq DNA polymerase 를비롯한 Thermus 속의내열성 DNA polymerase 는 5'-3' exonuclease activity 는가지지만, 3'-5' exonuclease activity 는매우약하다. 3'-5' exonuclease activity 의부재로인해이들효소는잘못 insertion 된 nucleotide 를제거하는것이불가능하고, 이결과로 base-insertion fidelity 가낮다. High-fidelity DNA polymerase 들의사용은유전자클로닝, 염기서열의결정이나발현될 PCR 산물들의 error 증가를줄이는데있어필수불가결하다. 3'-5' exonuclease- dependent proofreading activity 를가지는몇몇내열성 DNA polymerase 들이밝혀져왔으며, 이들효소들의 error rate 가결정되어져왔다 (Table 5, 6). T. maritima 로부터의내열성 DNA polymerase (Bost et al., 1994) 와 Aquifex aeolicus 로부터의 Aae Pol. (Chang et al., 2001) 은 3'-5' exonuclease activity 를가지는것으로보고되었다. P. woesei 로부터의 Pwo Pol. (Frey and Suppmann 1995), P. furiosus 로부터의 Pfu Pol. (Lundberg et al., 1991), Pyrococcus strain GB-D 로부터의 Deep Vent Pol. (Perler et al. 1996) 또는 T. litoralis 로부터의 Vent Pol. (Cariello et al., 1991) 과같은 Archaeal proofreading polymerase 들은 Taq polymerase 에비해 10 배정도까지낮은에러율을가진다. 또한 Thermococcus sp. strain 9 N-7 로부터의 9 N-7 DNA polymerase 는 Vent DNA polymerase 에비해 5 배높은 3'-5' exonuclease activity 를가진다 (Southworth et al., 1996). 그러나, 이들 Proofreading polymerase 들은낮은증폭신장율로인해 Taq DNA polymerase 를완전히대체하지못하고있다. 높은 fidelity 를가지는 DNA polymerase 들은그들의강한 exonuclease activity 때문에생긴 DNA 단편들의증폭이나 DNA sequencing 시 ddntp 의도입이잘되지않는다 (Barnes, 1994). 이러한문제들때문에최근의연구는높은신장성과낮은 error 를갖는신규내열성 DNA polymerase 의지속적인개발과더불어돌연변이유도에의한활성의최적화, domain fusion 에의한신장성과정확한증폭활성도입, DNA shuffling (family shuffling) 등의방법에의한내열성 DNA polymerase 의개량연구가활발히진행되고있다. Pyrococcus sp. strain KOD1 으로부터의재조합 KOD1 DNA polymerase 는낮은에러율 (Pfu 의에러율과유사 ) 과높은진행성 ( 뉴클레오타이드중합의지속성 ), 높은신장율을보이는것으로보고되었으며결과적으로 6 kb 까지의목적 DNA 서열들의매우빠르고정확한증폭이가능하다 (Takagi et al., 1997). 또한 Polymerase 와 exonuclease 의미묘한경쟁을최적화하기위해, 9 N-7 DNA polymerase 의
exo-motif 1 은 exonuclease activity 의전체적인제거없이단지활성수준을낮추기위한시도로돌연변이화되었다 (Southworth et al., 1996). 그리고 Taq 또는다른 non-proofreading DNA polymerase 들에 3'-5' exonuclease activity 를가지는 Archaeal proofreading DNA polymerase 의적은양을첨가함에의해긴증폭물 (20-40 kb) 의낮은 error 합성이가능하게하도록향상되어져왔으며 (Barnes, 1994; Cheng et al., 1994) 최근 KOD1 DNA polymerase 에있어서도 exonuclease deficient mutant 와 wild-type KOD1 DNA polymerase 의 mix 를통해 long and accurate PCR 이향상되었다 (Nishioka et al., 2001). 한편 domain fusion ( 또는 domain exchange) 에의해 Taq DNA polymerase 등의증폭효율이높은 polymerase 와 proofreading polymerase 를연결하여 single-type polymerase 로서 PCR 의증폭성과정확성을모두향상시키려는연구가진행되고있다. 몇가지진전된연구로는다음과같다. Tabor and Richardson (1995) 는 active site hybrids 와 mutations 의도입으로 ddntps 의구별을증가시키는결과를얻어 DNA sequencing 에적용하였고 Barnes (1992) 는 Taq DNA polymerase 의 5'-3' exonuclease domain 을제거하여 error 율을저하시켰다. Ignatov 등 (1998) 은 polymerase domain 의아미노산치환으로 long DNA molecules 합성의효율을증가시켰다. 또한 Park 등 (1997) 은 Taq DNA polymerase 에 3'-5' exonuclease domain 의 active site 를도입하여약간의 fidelity 를증가시켰다. 하지만아직까지의미있는결과는도출되지못하였다. Domain fusion 을이용한연구에서는 proofreading domain 으로현재까지는 Pfu, Vent DNA polymerase 를사용하였다. 그러나이는 Taq DNA polymerase (DNA pol I type) 와는다른 Table 5. Error rates of thermostable DNA polymerases Polymerase Error rate Reference Taq (Thermus aquaticus) 1.1 X 10-4 base substitutions/bp Tindall and Kunkel, 1988 2.4 X 10-5 frameshift mutations/bp Tindall and Kunkel, 1988 2.1 X 10-4 errors/bp Keohavang and Thilly, 1989 7.2 X 10-5 errors/bp Ling et al., 1991 8.9 X 10-5 errors/bp Cariello et al., 1991 2.0 X 10-5 errors/bp Lundberg et al., 1991 1.1 X 10-4 errors/bp Barnes, 1992 Klen Taq (Thermus aquaticus, N-terminal deletion mutant) Vent (Thermococcus litoralis) 5.1 X 10-5 errors/bp Barnes, 1992 2.4 X 10-5 errors/bp Cariello et al., 1991 4.5 X 10-5 errors/bp Ling et al., 1991 5.7 X 10-5 errors/bp Matilla et al., 1991
Vent (exo-) (Thermococcus litoralis) 1.9 X 10-4 errors/bp Matilla et al., 1991 Deep Vent (Pyrococcus sp. GB-D) No published literature. (NEB) Deep Vent (exo-) No published literature. Pfu (Pyrococcus furiosus) 1.6 X 10-6 errors/bp Lundberg et al., 1991 Replinase (Thermus flavus) 1.03 X 10-4 errors/bp Matilla et al., 1991 Table 6. Applications of thermostable DNA polymerases High- Reverse Polymerase Origin PCR fidelity transcription Reference PCR DNA pol I-type Taq pol I Thermus aquaticus + - Top pol Thermus thermophilus HB27 Longley et al., 1990 + Kwon et al., 1991 (weak;mn2+) Lawyer et al., 1993 + - NI Kim et al., 1998 Tth pol Thermus thermophilus + - + (Mn2+) Ruttimann et al., 1985 Auer et al., 1995 Tfi pol Thermus filiformis + - + (Mg2+) Jung et al., 1997 Tfl pol Thermus flavus + NI - Kaledin et al., 1981 Tx1 pol Thermus sp. X-1 + - NI Choi et al., 2001 Tca pol Thermus caldophilus GK24 + - + Park et al., 1994 (weak;mn2+) Aae pol Aquifex aeolicus + + NI Chang et al., 2001 Tma pol Thermotoga maritima + + NI Bost et al., 1994 Bst pol Bacillus stearothermophilus+ - - Mead et al., 1994 DNA pol α -type Pwo pol Pyrococcus woesei + + - Frey & Suppmann, 1995 Pfu pol Pyrococcus furiosus + + - Lundberg et al., 1991 Deep Vent pol Pyrococcus sp. GB-D + + - Cline et al., 1996 KOD1 pol Pyrococcus sp. KOD1 + + - Takagi et al., 1997 Vent pol Themococcus litoralis + + - Kong et al., 1993 Perler et al., 1996
9 N-7 pol Thermococcus sp. 9 N-7 + + - Southworth et al., 1996 type 인 DNA pol α -type 으로아미노산서열의상동성이나 3D 구조에있어서차이가매우크다. 이는두개의서로다른 type 의 polymerase 의 proofreading domain 과 polymerase 의각각이 fusion 되었을때구조적으로매우불안정하여지금까지의 fusion polymerase 는내열성이없는것으로나타났다. 따라서최근에는 Taq DNA polymerase 와같은 DNA pol I type 이면서 proofreading 기능에필수적인 3'-5' exonuclease 활성을갖는 eubacterial 초고온성미생물의 DNA polymerase 에관심을갖게되었다. 현재까지알려진이러한 eubacterial 내열성 DNA polymerase 로 Aquifex aeolicus, A. pyrophilus DNA polymerase, Thermotoga neapolitana DNA polymerase (Tne polymerase) (Joyce and Steitz, 1994) 가알려져있다. 독일의 Villbrandt 등 (2000) 은 Tne polymerase 와 E.coli pol I 을이용하여 Taq DNA polymerase 와 domain exchange 에의한 3 가지의 chimeric DNA polymerase : TaqEc1 (exchange of residues 292-423 from Taq polymerase for residues 327-519 of E.coli pol I), TaqTne1 (exchange of residues 292-423 from Taq polymerase for residues 295-485 of Tne polymerase), and TaqTne2 (exchange of residues 292-448 from Taq polymerase for residues 295-510 of Tne polymerase) 제작하였다. 결과에의하면 TaqEc1 은두 polymerase 의중간온도인 50 에서 polymerase activity 와 proofreading 기능을갖고있었다. 반면 TaqTne1 및 TaqTne2 는뚜렷한 3'-5' exonuclease activity 를보이지않았지만 TaqTne2 는 72 에서 polymerase activity 를보였다. 하지만이들 chimeric polymerase 는 PCR 에적용되기위한열안정성이없었다. 한편내열성 polymerase 를이용한 PCR 시 cycling temperature 에도달하기전에 polymerase 에의한신장을막기위해몇몇방법들이개발되어져왔다. DNA 와효소사이에기계적인막으로서 wax 의사용에이어, 중온성온도에서 neutralizing antibody 에의한 Taq polymerase 의억제나고정화효소의열에의한활성화 (Nilsson et al. 1997) 와같은방법들이고안되었으며최근에는 polymerase 를이용한 DNA 증폭기법에대한연구도활발히진행되고있다. 특히진단분야있어서 PCR 기술외의여러가지방법이개발되었다. 이에는 nucleic acid sequence-based amplification (NASBA) (Compton, 1991), self-sustained sequence replication(3sr) (Guatelli et al., 1990) 과 strand displacement amplification (SDA) (Walker et al., 1992), loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000) 등이있다. LAMP method 는 Bacillus stearothermophilus (Bst) DNA polymerase 를사용하여 109 까지증폭시킬수있다. 본연구소에서는가장이상적인내열성 DNA polymerase 로서 single-type 의높은신장성과정확성을함께갖는 DNA polymerase 를개발하고자연구하고있다. DNA pol I-type 과 α -type 은 polymerase activity 에중요한 motif 들은동일하게가지고있지만전체적인아미노산서열의상동성이나 3 차구조에서는매우차이가있다. 따라서 DNA shuffling 과같은 genetic evolution 방법이나
domain fusion 방법을사용하더라도기대하는결과는도출하기어렵다. 따라서본사에서보유하고있는 Taq DNA polymerase 와같은 DNA pol I-type 인 Aquifex aeolicus 또는 A. pyrophilus DNA polymerase 의 3'-5' exonuclease domain 을이용하고또한 Taq DNA polymerase 외의 Top, Tfi, Tca Tx1 등자체보유하고있는 Thermus 속계열의증폭성이우수한 polymerase 과의 domain fusion 과구조적으로안정된 folding 을위하여전체적인혹은 fusion 부위에대한 error prone PCR 또는 DNA shuffling 방법등을이용하여 mutation 을도입하고있다. 현재까지는제작된 fusion polymerase 가안정적으로발현됨을확인하였고내열성을증가시키기위한실험을진행하고있다. 3. 참고문헌 Barnes, W.M. (1992) The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene 112: 29-35 Barnes, W.M. (1994) PCR amplification of up to 35-kb DNA fragments with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA 91: 2216-2220 Bauer, M.W., Driskill, L.E., Callen, W., Snead, M.A., Mathur, E.J., and Kelly, R.M. (1999) An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolizes α -1,4 bonds in mixed-linkage (1 3), (1 4)-β -D-glucans and cellulose. J Bacteriol. 181: 284-290 Bl?chl, E., Rachel, R., Burffraf, S., Hafenbradl, D., Jannasch, H.W., and Stetter, K.O. (1997) Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113. Extremophiles 1:14-21 Block, T.D. and Freeze, H. (1969) Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J. Bacteriol. 98:289-297 Bok, J.D., Dinesh, A., Yernool, D.A., and Eveleigh, D. (1998) Purification, characterization and molecular analysis of thermostable cellulases Cel A and Cel B from Thermotoga neapolitana. Appl Environ Microbiol. 64: 4774-4781 Bronnenmeier, K., Kern, A., Liebl, W., and Staudenbauer, W.L. (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulose materials. Appl Environ Microbiol. 61: 1399-1407 Brown, S.H., Sjholm, C., and Kelly, R.M. (1993) Purification and characterization of a highly thermostable glucose isomerase produced by the
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