APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1994, p. 4559-4566 Vol. 60, No. 120099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Purification and Characterization of a Thermostable Thiol Proteasefrom a Newly Isolated Hyperthermophilic Pyrococcus sp.MASAAKI MORIKAWA,' YOSHIFUMI IZAWA,' NAEEM RASHID,' TOSHIHIRO HOAKI,2

AND TADAYUKI IMANAKAl*Department of Biotechnology, Faculty of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565,1 and

Manine Biotechnology Institute, Shimizu Laboratory, Shimizu-shi, Shizuoka 424,2 JapanReceived 15 June 1994/Accepted 7 October 1994

A hyperthermophilic archaeon strain, KODI, was isolated from a solfatara at a wharf on Kodakara Island,Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100°C, and the optimaltemperature was 950C. The anaerobic strain was an So-dependent heterotroph. Cells were irregular cocci andwere highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC contentof the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcusabyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively.Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellularproteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determinedto be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtrationchromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibitedits maximum activity at approximately pH 7.0 and at a temperature of 110°C, with azocasein as a substrate.The enzyme activity was completely retained after heat treatment at 90°C for 2 h, and the half-life of enzymaticactivity at 100°C was 60 min. The proteolytic activity was significantly inhibited byp-chloromercuribenzoic acidor E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in thepresence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiolprotease.

Many thermostable enzymes have been isolated, and theirmechanisms of thermotolerance have been investigated withthermophilic bacteria such as Bacillus stearothernophilus (1,16, 25), Thennus aquaticus (9), and others (30, 32). Thermo-stability is usually increased because of additional hydrogenbonds, ionic bonds, hydrophobic bonds, and sometimes disul-fide or coordinate bonds of chelating metal ions. There are alsoseveral reports on enhancing the thermostability of nativeenzymes by altering the amino acid residues through proteinengineering (21, 26, 28) in the light of three-dimensionalstructure. It is widely accepted that protein thermostability isbasically governed by the amino acid sequences.

Recently, hyperthermophiles which can grow at tempera-tures higher than 90°C have been isolated, and the enzymesderived from them have been shown to be extremely thermo-stable. These microorganisms are unusually archaea (38).Although hyperthermophilic archaea are not easy to grow on alarge scale, several enzymes from Sulfolobus sp. (8, 13),Thermococcus sp. (24, 31), Desulfurococcus sp. (12), andPyrococcus sp. (7, 36, 37) have been intensively investigated.Their growth rates are relatively high, and the generation timesrange from 30 to 60 min. The enzymes from these thermo-philes seem not only to be thermostable but also to haveenhanced activities at elevated temperatures. Therefore, theyare good samples for investigation of the stabilizing mecha-nisms of thermophile proteins. In this paper, we report on thecharacteristics of a newly isolated hyperthermophilic archaeon,Pyrococcus sp. strain KOD1, and the successful purification ofa thermostable thiol protease.

* Corresponding author.

MATERIALS AND METHODS

Isolation of the hyperthermophile. Sediments and watersamples from a solfatara (102°C, pH 5.8) at a wharf onKodakara Island (Kagoshima, Japan) were inoculated imme-diately into anaerobic gas-flushed 25-ml test tubes containingmedium. A mixture of anaerobic gases, C02-H2-N2 (5:5:90[vol/vol/vol]), was used. Tubes were closed tightly and incu-bated at 90°C for 2 days. Single-colony isolation was performedby the roll-tube method. Diluted cell culture (10-6 or 10-')was mixed with 0.8% gellan gum solution (>90°C) and thensolidified on the inside wall of a screw-cap test tube (30 mm indiameter). Tubes were flushed with the anaerobic gas mixtureand closed. After another 2 days of incubation at 90°C, severalsmall colonies appeared on the medium.

Culture conditions. Samples and new isolates were culti-vated in a half-strength (0.5X) 2216 marine broth (3). Thebottle contained (per liter) 18.7 g of 2216 marine broth (DifcoLaboratories, Detroit, Mich.), 3.48 g of PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid); Dojindo Laboratories, Kyoto,Japan] (buffer), 0.725 g of CaCl2- 2H20, 0.4 ml of 0.2%resazurin, 475 ml of artificial seawater, and 500 ml of distilledwater, and the pH was adjusted to 7.0 with NaOH. Artificialseawater consisted of (per liter) 28.16 g of NaCl, 0.7 g of KCl,5.5 g of MgCl2 - 6H20, and 6.9 g of MgSO4 * 7H20. The broth,in screw-cap culture bottles, was autoclaved and stood in ananaerobic chamber (filled with anaerobic gas mixture) imme-diately after autoclaving. Na2S - 9H20 (final concentration, 400,uM) and 10 g of sulfur (sterilized separately at 105°C for 20min) were added prior to inoculation. The bottles were tightlyclosed and placed in an incubator, usually at 90°C, overnight.Other buffers, such as MES [2-(N-morpholino)ethanesulfonicacid; pH 5 to 6.5], HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7 to 8.5), or CHES [2-(cyclohexylami-

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no)ethanesulfonic acid, pH 9] (Dojindo Laboratories, Kum-amoto, Japan), were used instead of PIPES to prepare mediaat various pHs as appropriate. Cell numbers were countedmicroscopically with a hemacytometer.

Continuous culture. Cells were grown in a five-neck round-bottom flask (working volume, 1 liter) maintained at 94°C witha heating mantle (Daiken Electric Co., Tokyo, Japan) (6). Theflask was continuously sparged with nitrogen gas. The exhaustgas was connected to a condenser to avoid water loss, and H2Swas trapped in an adjacent gas-washing bottle (containing 3 NNaOH). Cells had been pregrown in a batch mode. When thegrowth reached the late-log phase (ca. 108 cells per ml), freshmedium was added and then the excess culture was sucked outthrough a Teflon tubing pump (Masterflex; Cole Parmer,Chicago, Ill.) at a dilution rate of 0.15 h-1. A reservoir tank forculture was put in an ice bath. Approximately 100 liters ofculture was collected for protease purification.H2S measurement. The amount of H2S was measured by the

spectrophotometric method of Cline (10).Analysis of lipid composition. Lipid fractions were extracted

from dried cells (0.1 g) by the standard method of Bligh andDyer (4). Extracts were separated on a silica gel column (15mm diameter by 300 mm, Wakogel C-200; Wako Pure Chem-icals Co., Osaka, Japan). Simple lipid fractions were elutedwith chloroform first, and then the complex (polar) lipidfractions were eluted with methanol. Methyl-esters of thelatter fractions were analyzed on a Silica Gel 60 thin-layerplate (Merck Co., Berlin, Germany). The plate was developedwith n-hexane-diethylether-acetate (70:30:1 [vol/vol/vol]).Spots were detected by spraying with 30% H2SO4 and charringat 105°C. A polar lipid fraction prepared from a knownarchaeon, Sulfolobus solfataricus ATCC 35091, was used as acontrol.

Analysis of the S-layer fraction. The cell surface (S-layer)fraction was prepared according to the method of Stetter et al.(35). Cells were suspended in 20 mM phosphate buffer (pH7.0), disrupted by sonication, and then treated with deoxyribo-nuclease I (1 mg/liter) and ribonuclease A (1 mg/liter). After 1h of incubation at 37°C, samples were centrifuged at 156,500 xg for 1 h. The resulting pellet was suspended in the same bufferand analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). After the electrophoresis, thegel was fixed overnight with the mixture of ethanol, acetate,and water (40:5:55 [vol/vol/vol]). Glycoprotein bands weredetected as follows: the gel was soaked in 0.7% periodatesolution in 5% acetic acid for 2 h, kept in 0.2% sodiumbisulfide solution in 5% acetic acid for 2 h, and then stainedwith Schiff reagent for 12 h.

Cloning and sequencing of the 16S rRNA gene. The 16SrRNA gene of strain KOD1 was amplified by PCR withforward and reverse primers which correspond to nucleotidepositions 1 to 20 (5'-GGAGGCCACTGCTATGGGGG-3')and 1351 to 1332 (5'-TGACGGGCGGTGTGTGCAAG-3')of Pyrococcus abyssi 16S rRNA (GenBank accession number,19921), respectively. The PCR product (1.3 kb) was labeledand used as a probe for the cloning of full-length 16S rRNA.Southern hybridization was performed for the KOD1 chromo-somal DNA digested with various restriction enzymes. The7-kb EcoRI fragment was cloned in pBR322, and both strandsof the 16S rRNA gene were determined by the dideoxy-chaintermination method with fluorescent primer (A.L.F DNAsequencer; Pharmacia, Uppsala, Sweden). DNA sequence datawere analyzed with DNASIS software (Hitachi Software, To-kyo, Japan).Assay of proteolytic activity. Proteolytic activity was mea-

sured by the hydrolysis of azocasein (5). The reaction mixture

consisted of 5 mg of azocasein per ml in 600 Rl of buffer (50mM Tris-HCl buffer [pH 7.5], 5 mM CaCl2) and 120 RI ofenzyme solution. The reaction was completed at 80°C for 20min unless otherwise stated. A heating block was used attemperatures higher than 60°C. The reaction was stopped byaddition of 480 RI of 15% trichloroacetic acid, and the samplewas placed on ice. The sample was centrifuged at 18,500 x gfor 10 min to remove precipitate. After addition of 30 ,u of 10N NaOH to the supernatant, the A440 was measured with aUV-160 spectrophotometer (Shimadzu Co., Kyoto, Japan). Ina reference tube, enzyme solution was added after trichloro-acetic acid. One unit of proteolytic activity was determined asa change of 0.1 A440 unit at 80°C for 20 min.The amount of protein in the sample was estimated with

bicinchoninic acid assay reagent (Pierce, Rockford, Ill.).Activity staining of protease. After the conventional SDS-

PAGE, the gel was washed in 2.5% Triton X-100-50 mMsodium phosphate buffer (pH 7.0) for 1 h to remove SDS. Thegel was put on filter paper which had been soaked in a 50 mMsodium phosphate buffer (pH 7.0). Gel (12% polyacrylamide, 1mm in thickness) containing 0.5% gelatin was placed on theoriginal plate. Then, a filter paper and a pack of absorbentpapers were placed onto the top gel. After being wrapped witha hybridization bag, this gel sandwich was pressed with a5-mm-thick glass plate. Protease was transferred onto thegelatin gel, and the proteolytic reaction was performed in a75°C incubator for 16 h. Gel was stained with 0.1% amidoblack in 100 ml of 30% methanol, 10% acetic acid, and 60%water. Protease bands were visualized as clear zones resultingfrom the hydrolysis of gelatin (5).

Purification of protease from strain KODL. Prior to thepurification of protease, we measured the proteolytic activitiesof culture supernatant and cell extracts under various temper-atures. Culture supernatant was prepared by centrifugation(10,000 x g for 30 min) of the broth and concentration to 1liter (ca. 100-fold) by the ultrafiltration system (UF-lOPS;Tosoh Co., Tokyo, Japan). Cell extracts were prepared bysonication of the cells and subsequent centrifugation (27,000 xg for 30 min) to remove the cell debris. Proteolytic activitieswere measured as mentioned above. Purification started with 1liter of concentrated culture supernatant. This crude enzymesample was brought to 60% ammonium sulfate saturation andkept at 4°C overnight. The precipitate was collected by cen-trifugation at 27,000 x g for 30 min, dissolved in 25 mMTris-HCl (pH 7.5) containing 5 mM CaCl2, and dialyzedovernight against the same buffer. The dialysate was chromato-graphed with a TSKgel Toyopearl HW-55F 2-cm-diameter by80-cm column (Tosoh) at a flow rate of 1 ml/min. The elutionpattern was monitored by A280, and proteolytic activity wasdetermined. Fractions containing major proteolytic activitywere pooled and applied to the second gel filtration column(fast protein liquid chromatography [FPLC] with Superose 12[Pharmacia]) at a flow rate of 0.5 ml/min. Final purification wasperformed by passage through a hydroxyapatite column in anisocratic mode (particle size, 2 ,um; 7.5 mm diameter by 100mm; Tonen Co., Tokyo, Japan) which had been equilibratedwith 5 mM sodium phosphate (pH 6.8). Impurities of larger-molecular-size proteins in the sample were eluted by a lineargradient of sodium phosphate concentrations up to 200 mM.

Effect of pH on proteolytic activity. Purified enzyme andsubstrate were dissolved either in 50 mM sodium acetate buffer(pH 4.0 to 6.0), sodium phosphate buffer (pH 6.5 to 8.0), orglycine-sodium hydroxide buffer (pH 8.5 to 11.0). Other reac-tion conditions were the same as described earlier.

Effect of inhibitors on proteolytic activity. Enzyme sampleswere incubated at 37°C for 15 min with each inhibitor: DFP

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THERMOSTABLE THIOL PROTEASE 4561

a 10

60 70 80 90100Temperature(°c)

b

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pH NaCI conc. (gfi)FIG. 1. Growth profile of strain KODI. (a) Effects of temperature

on cell growth; (b) growth curve of strain KOD1 at 95°C; (c) effects ofpH on cell growth; (d) effects of salinity on cell growth. Cells were

cultivated in a half-strength 2216 marine broth (a and b) withmodification of pH or NaCl concentration (c and d). Cells were grown

for 20 h (a, c, and d).

(diisopropyl fluorophosphate), PMSF (phenylmethylsulfonylfluoride), pCMB (p-chloromercuribenzoic acid), E-64 (an ef-fective thiol protease inhibitor produced by Aspergillus japoni-cus TPR-64 [18]), or EDTA. Residual activity was then deter-mined.

Thermostability of the protease. The purified protease sam-ple in a buffer (50 mM Tris-HCl [pH 7.5], 8 mM cysteine, 5mM CaCl2) was incubated at 90 or 100°C for an adequateperiod and then put on an ice bath. Residual activity was

measured at 80°C.

RESULTSIsolation and characterization of a hyperthermophile. A

hyperthermophilic archaeon, strain KOD1, was isolated asdescribed in Materials and Methods. The growth temperatureof the strain ranged from 65 to 100°C (Fig. la). The optimalgrowth temperature was 95°C. The growth curve at 95°C isshown in Fig. lb. The doubling time at the exponential-growthphase was estimated to be 40 min. Strain KOD1 could not growat or above 105°C. The effects of pH and salinity on growth areshown in Fig. lc and d, respectively. The optimum pH andNaCl concentration for the growth were found to be 7.0 and 30g/liter, respectively. Sulfur reduction to H2S occurred in theliquid medium during growth of strain KOD1. Carbon source

TABLE 1. Requirement for carbon sources

Cell growth under indicated conditionbCarbon source(s)' CO2, H2, CO2, H2, N2, N N2 and

and N2 and So 2 So

None - - - -Yeast extract + + + + - + +Tryptone + + + + - +++Peptone - + + + - + + +Yeast extract + peptone + + + + + - + + +Casamino Acids - + + -

Glucose - - NT NTSucrose - - NT NTStarch - - _ _

a Each carbon source was added to the culture medium (Materials andMethods), from which 2216 marine broth was omitted. The concentration of thecarbon sources yeast extract, tryptone, peptone, and Casamino Acids was 0.1%;that of glucose, sucrose, and starch was 0.5%.

b Cultivation was done at 95°C for 14 h. Symbols for degrees of cell growth areas follows (cells/ml): + + +, >2 x 107; +, > 1 X 107; +, <1 X 107; -, no cells.NT, not tested.

requirements were also examined. Strain KOD1 could notgrow autotrophically using CO2 as the sole carbon source withor without S (Table 1). S generally enhanced the growth andwas essential if peptone or Casamino Acids were used as thecarbon source. These data indicate that strain KOD1 belongsto the group of sulfur-dependent hyperthermophiles. Thethermophile was found to be sensitive to 100 ,ug of rifampinper ml and insensitive to chloramphenicol, kanamycin, andpenicillin (100 ,ug/ml) at 85°C.

Analyses of lipids and S-layer of the strain KODI. Polarlipid fractions prepared from cells of KOD1 and of Sulfolobussolfataricus ATCC 35091 (as a standard archaeon) showedsimilar separation patterns on thin-layer silica gel chromatog-raphy (Fig. 2a). Strain KOD1 contains tetraether-type lipids

a0000

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FIG. 2. (a) Analysis of membrane lipids of strain KODl: separa-tion pattern of polar lipid fraction on silica gel thin-layer plate. Lane 1,Sulfolobus solfataricus ATCC 35091; lane 2, strain KOD1. Largearrows at Rfs of 0.3 and 0.7 indicate the positions of ether-type lipids.(b) Analysis of S-layer by SDS-PAGE. Lane 1, total proteins stainedwith Coomassie brilliant blue R-250; lane 2, carbohydrate-containingsubstances stained with periodate-Schiff reagent. Arrowheads on theright indicate the positions of glycoproteins.

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4562 MORIKAWA ET AL.

(Rf, 0.3), diether-type lipids (Rf, 0.7), and some other esters orfatty acids (Rf, >0.8).The S-layer fraction was analyzed by SDS-PAGE. Some

protein bands that were positive for Coomassie brilliant blueR-250 staining could also be stained with periodate-Schiff dye(Fig. 2b). Therefore, these proteins seem most likely to beglycoproteins. These data indicated that strain KOD1 can beclassed in the category of extremely thermophilic S°-metabo-lizing archaea (34).

Microscopic observation of strain KOD1. The morphologyof strain KOD1 was examined by electron microscopy (Fig. 3).Cells were irregular cocci (ca. 1 pLm in diameter), sometimes inpairs, and were highly motile with several polar flagella (Fig.3c).GC content of DNA. Chromosomal DNA was prepared from

strain KOD1 by the Sarkosyl method and purified by CsClequilibrium density gradient ultracentrifugation (22). PurifiedDNA was dissolved and diluted to an A260 of 0.1 (at 25°C) with0.15 M NaCl-0.015 M sodium citrate (pH 7.0). The DNAsolution was slowly heated up to 100°C, and the A260 wasmonitored at different temperatures (27). The melting pointwas determined to be 84.95°C, and the GC content of the DNAwas estimated to be 38 mol%.

Sequencing of the 16S rRNA gene. The nucleotide sequenceof 16S rRNA was determined and was compared with those ofother known archaea (Fig. 4). The 16S rRNA sequence ofstrain KOD1 was 95 and 97% homologous with those ofPyrococcus abyssi (15) and Thermococcus celer, respectively.The 16S rRNA sequence homology between T. celer and P.abyssi is 96%. These high scores affiliated the strain KOD1 withthe order Thermococcales, family Thermococcaceae.

Proteolytic activities in cell extract and culture supernatant.The culture supernatant (extracellular fraction) and the cellextract (intracellular fraction) were prepared as describedpreviously. The proteolytic activity of the culture supernatantwas higher at 100°C than it was at 80 or 90°C, whereas theactivity of the cell extract at 100°C was lower than it was at 80or 90°C (data not shown). This suggests that proteases in theculture supernatant were more thermostable than those in thecell extract. Proteolytic activity of the extracellular fractionshowed a maximum value at 105°C and a typical shoulderpattern at 80°C (figure not shown). The temperature profile ofproteolytic activity implied that there were at least two pro-teases, whose optimum temperatures were lower and higherthan 100°C, respectively.

Purification and characterization of major thermostableprotease. Extracellular major protease was purified by ammo-nium sulfate fractionation, TSKgel HW-55F gel filtrationchromatography, FPLC with Superose 12, and hydroxyapatitechromatography. The protein in each fraction was analyzed bySDS-PAGE (Fig. Sa). After the hydroxyapatite chromatogra-phy, the single 44-kDa protease band was confirmed to be oneof the three major proteolytic enzymes of the culture superna-tant (Fig. Sb). This protease is a monomeric enzyme, and themolecular size was estimated by gel filtration chromatographyto be 45 kDa. There was a minor protein band (42 kDa) in Fig.Sa, lane 4. The 44-kDa protease and the 42-kDa protein couldbe separated on the Prosieve gel system (Takara Shuzo Co.,Kyoto, Japan) and further purified by reversed-phase high-performance liquid chromatography. N-terminal amino acid

FIG. 3. Electron microscopic observation of strain KODL. (a andb) Thin sections of strain KOD1; (c) negatively stained (with 1%uranyl acetate) cell of strain KODI.

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FIG. 5. PAGE of enzyme samples at each purification step. (a)Analysis by SDS-PAGE. The gel was stained with Coomassie brilliantblue R-250. Lane 1, after precipitation with ammonium sulfate; lane 2,after gel filtration chromatography on Toyopearl HW-55F; lane 3,after gel filtration chromatography on Superose 12; lane 4, afterhydroxyapatite chromatography. (b) SDS-PAGE and activity staining.Lane 1, Coomassie brilliant blue staining of the 44-kDa protease afterhydroxyapatite chromatography; lane 2, activity staining of the samesample after blotting to a gelatin gel; lane 3, activity staining of a crudeculture supernatant sample (only after precipitation with ammoniumsulfate).

TABLE 2. Effects of inhibitors on the proteolytic activityof the 44-kDa protease

Inhibitor Inhibitor class Concn (mM) % Inhibition

pCMB Cysteine 0.1 821 80

E-64 Cysteine 0.1 251 82

PMSF Serine 5 0DFP Serine 10 38EDTA Metal 10 0

mixture. The optimum temperature for the activity is 110°C, asshown in Fig. 6b. The enzyme was found to be very stable at90°C, and it has a half-life of 60 min even at 100°C (Fig. 6c).The specific activity was determined as 2,160 U/mg of proteinat 80°C.

Inhibition test of the protease. Both pCMB and E-64, whichare specific inhibitors for thiol protease, strongly inhibited theproteolytic activity of this enzyme even at 1 mM (Table 2). Theenzyme was partially inhibited by 10 mM DFP (an inhibitor ofserine protease). EDTA (a metalloprotease inhibitor) andPMSF (a serine protease inhibitor) were found to be ineffec-tive inhibitors for this protease. Proteolytic activity was en-hanced three times when 8 mM cysteine was added to thereaction mixture. These experimental results indicate that theenzyme is a typical thiol protease.

sequences of both the 44-kDa and the 42-kDa proteins wereanalyzed by the automatic protein sequencer ABI 476A (Ap-plied Biosystems, Foster, Calif.). Six amino acid sequencesfrom both N-terminal regions were exactly the same (Val-Glu-Ile-Pro or Gly-Asn-Ile). This indicates that the 42-kDa proteinis a product cleaved from the 44-kDa protease. The 44-kDaprotease did not seem to be a glycoprotein, because it couldnot be stained by periodate-Schiff reagent (data not shown).The effects of pH and temperature on the purified 44-kDa

protease were examined. The enzyme was found to be a typicalneutral protease, whose optimum pH was 7 (Fig. 6a), and itretained high proteolytic activity even at 120°C (Fig. 6b). Aslight instability of azocasein was found at higher temperatures(over 100°C) for 20 min. Therefore, the A440 value of thereference sample was subtracted from that of each reaction

DISCUSSIONThere are two genera, Pyrococcus and Thernococcus, in the

family Thermococcaceae (34). Electron microscopic observa-tion and the optimum growth temperature (95°C), pH (7.0),and GC content (38 mol%) of strain KOD1 strongly suggestedthat it belonged to Pyrococcus rather than Thennococcus,because those values for Thernococcus are 85 to 88°C, 5.8 to6.0, and 56%, respectively (29, 34). Unfortunately, 16S rRNAsequence data for the typical Pyrococcus species Pyrococcusfuriosus and Pyrococcus woesei have not been reported yet. Thescores for 16S rRNA sequence homology between strainKOD1 on the one hand and P. abyssi and T. celer on the otherwere 95 and 97%, respectively. Since the difference in 16SrRNA sequences between P. abyssi and T. celer was so small(96% homologous), it was difficult to determine the genus ofthe strain by using only the sequence data of the 16S rRNA

u' I . . ...

20 40 60 80100120Temperature (OC) 0 30 60 90 120

Time (min)FIG. 6. Characteristics of the 44-kDa protease. (a) Effects of pH on proteolytic activity; (b) effects of temperature on proteolytic activity; (c)

thermostability of the 44-kDa protease. The sample was incubated at 90 (0) or 100°C (A), and then the remaining activity was assayed at 80°C.

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THERMOSTABLE THIOL PROTEASE 4565

TABLE 3. Comparison of TT protease and other thermostable proteases from archaea

Mol mass Optimum Optimum Inhibition by: Thermo-Protease (kDa) pH temp Active site stability Origin Reference(°C) EDTA PMSF pCMB Pepstatin

iT protease 44 7.0 110 - - + NT0 Cysteine 100°C, 60 min Pyrococcus sp. This workThermopsin 45 2.0 90 - - - + Aspartate 90°C, 90 min Sulfolobus acidocaldarius 17Archaelysin 52 7.0 98 - + - NT Serine 1050C, 8-9 min Desulfurococcus sp. 12Pyrolysin 140, 130, 115, 6.5-10.5 115 - + - NT Serine 950C, 9 h Pyrococcus funiosus 11, 14

100, 65S66 66 7.0 105 - + NT NT Serine 98°C, 33 h Pyrococcus furiosus 5

a NT, not tested.

gene in this case. We believe that the GC content and growthprofile (optimum temperature, pH, and NaCl concentration)were as important as the homology data on 16S rRNAsequences for identification of strain KOD1. Strain KOD1 wasvery similar to P. furiosus with respect to the GC content(38%), its ability to ferment yeast extract and tryptone, and thebroad range of ionic strength tolerable for growth. However,several properties of strain KOD1 were not in conformity withthose of P. fuiriosus. The optimum growth temperature (95°C)was lower, and the optimum pH (7.5) was slightly higher thanthose of P. furiosus DSM 3638 (100 and 7.0°C, respectively).Production of extracellular 44-kDa protease was not reportedfor the strain DSM 3638 (11). Although the growth profile ofP. abyssi was quite similar to that of KOD1, its GC content wasobviously higher (45%) than that of KOD1.Most of the So-dependent hyperthermophilic heterotrophs

require complex proteinaceous substrates as a sole carbonsource. This is caused by their requirement for several essentialamino acids (20) and suggests that protease production isessential for their growth. The production of thermostableproteases has been previously reported for many other ar-chaea, as shown in Table 3. The 44-kDa thermostable thiolprotease (TT protease), purified and characterized in thiswork, is one of the most thermostable proteases found thus far.Among the purified forms of proteases, the optimum temper-ature of 1T protease is one of the highest (110°C) determined,and it is the first thiol protease isolated from archaea. Intra-cellular protease of P. furiosus DSM 3638 was found to relateimmunologically to the eukaryotic proteosome (33). Thisindicates an evolutionary relationship between Pyrococcus andthe eucarya. Thiol proteases such as ficin, papain, and cathep-sin C are widely found in the lysosomes of eukaryotic cells andthe fruits of various plants. Cysteine was found to enhance theproteolytic activity of thiol protease (2), and a similar charac-teristic was found with our Ti protease. Thiol proteases arefound to be strongly inhibited by pCMB and E-64 thiolprotease inhibitors. They are often found partly inhibited byPMSF or DFP because of a similar configuration of active sitesfor serine proteases (23). In fact, DFP was found to partiallyinhibit lT protease in our study. Cloning and sequencing ofthe extracellular Ti protease gene are now in progress.

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