IIIUSO05756339A United States Patent (19) 11) Patent Number: 5,756,339 Mitta et al. 45 Date of Patent: May 26, 1998

54 HYPERTHERMOSTABLE PROTEASE GENE Rik Eggen et al. "Characterization of pyrolysin, a hyper thermoactive serine protease from the archaebacteruim (75) Inventors: Masanori Mitta. Tsuzuki-gun; Pyrococcus furiosus".FEMS Microbiology Letter. vol. 71. Katsuhiko Yamamoto; Mio Morishita. pp. 17-20, 1990. both of Otsu; Kiyozo Asada. Koka-gun; Susumu Tsunasawa, Kusatsu; Helen Connaris et al. "Heterogeneity of proteinases from the Ikunoshin Kato. Uji, all of Japan hyperthermophilic archaeobacterium pyrococcus furiosus". Journal of General Microbiology, vol. 137, pp. 1193-1199. 73) Assignee: Takara Shuzo Co., Ltd., Kyoto, Japan 1991. 21 Appl. No.: 750,532 Michael Klingegerg et al. "Properties of extremely thermo stable proteeases from anaerobic hyperthermophilic bacte 22 PCT Filed: Jun. 5, 1995 ria”, Applied Microbiology and Biotechnology, vol. 34, pp. 86 PCT No.: PCT/P95/01095 715-719, 1991. $371 Date: Dec. 13, 1996 Roland J. Siezen et al. "Homology modelling and protein engineering strategy of subtiliases. the family of subtilisin S 102(e) Date: Dec. 13, 1996 -like serine proteinases". Protein Engineering vol. 4, pp. (87. PCT Pub. No.: WO95/34645 719-737 1991. PCT Pub. Date: Dec. 21, 1995 Robinson et al. (1995) A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous (30) Foreign Application Priority Data to members of the prolyl oligopeptidase family of proteases, Jun. 13, 1994 (JP Japan ...... 6-130236 Gene 152: 103-106, Jan. 11, 1995. Jul. 26, 1994 JP Japan ...... 6-173912 Voorhoorst et al. (1996) Isolation and Characterization of the (51) Int. Cl...... C07H 21/04; C12N 1/20; Hyperthermostable Serine Protease, Pyrolysin. and Its Gene C12N 9/50; C12N 9/52 from the Hyperthermophilic Archaeon Pyrococcus furiosus. 52) U.S. Cl...... 435/220; 435/219; 435/252.3; 435/320.1; 435/325; 536/23.2 J. Biol. Chem. 271 (34): 20426-20431. Aug. 23, 1996. 58) Field of Search ...... 435/219, 220, 435/320.1, 240.1. 252.3, 325:536/23.2: Primary Examiner Robert A. Wax 93.5/22 Assistant Examiner-Einar Stole Attorney, Agent, or Firm-Browdy and Neimark (56) References Cited 57 ABSTRACT U.S. PATENT DOCUMENTS

5.242,817 9/1993 Kelly et al...... 435/220 There is disclosed a hyperthermostable protease gene origi 5,391,489 2/1995 Kelly et al...... 435/220 nating in Pyrococcus furiosus, in particular, a hyperthermo stable protease gene encoding the amino acid sequence FOREIGN PATENT DOCUMENTS represented by the SEQID NO 1 in the Sequence Listing or 6-197770 7/1994 Japan. a part thereof which retains the activity of the hyperther mostable protease. There is also disclosed a process for OTHER PUBLICATIONS producing the protease by culturing a transformant trans Ilse I. Blumentals et al. "Charaterization of Sodium Dodecyl formed with a plasmid into which the above gene has been Sulfate-Resistant Proteolytic Activity in the Hyperthermo inserted. philic Archaebacterium". Applied and Environmental Micro biology, vol. 56, pp. 1992-1998, 1990. 7 Claims, 9 Drawing Sheets U.S. Patent May 26, 1998 Sheet 1 of 9 5,756,339

A/G / XboI Sph I

Not I

Xbo I

Sph I

A/G 2 Xbo.

U.S. Patent May 26, 1998 Sheet 2 of 9 5,756,339

A/G 3 Not

Kpn

Smo I / Pvu D.

Sph I

Pst I PSt I p TPR 1 C T N. Pst Pst I Xbo I *r SphI pTPR 9

PSt Pst pTPR 12 Not Xbo SirXbol Psil Sph I Pvu U.S. Patent May 26, 1998 Sheet 3 of 9 5,756,339

A/G 5 Xbo I

Sph I

Kpn I Smol Pw u

A/G 6 Xbo I

Dro I

Dr O.

Dro

Smo I M P W II Xbo

Sph I Oro I U.S. Patent May 26, 1998 Sheet 4 of 9 5,756,339

Fig. 7

KpnI Smal/Dral

Xba Bam HI Sea4.8 kb EcoRI Sma I/Dra ECOR EcoRIR Sph

Fig. 8 Smal/Dral

U.S. Patent May 26, 1998 Sheet 5 of 9 5,756,339

Fig. 9

Smal/Dral

Xba /EcoRI

Fig. l O

70 175 80 Asp Gly Ser Gly Wal Wa Wa Ala Wai Leu Asp Thr Gly Wa 5'-GAT GGT AGT GGT GTT GTT GTT GCA GTA CTT GAC ACG GGA CTT-3'

PRO-F 5'-GGY WSD RRT GTT RRH GTH GCD GTD MTY GAC ACS GG-3' U.S. Patent May 26, 1998 Sheet 6 of 9 5,756,339

365 370 375 His Giy His Gly Thr His Val Ala Gly Thr Val A la Gly Tyr 5'-CAC GCT CAC GGA ACT CAC GTA GCT GGA ACT GTT GCT GGT TAC-3'

PRO-2F 5'-KST CAC GGA ACT CAC GTD GCB GGM A CD GTT GC-3' PRO-2R 3'-CTG CCT TCA GTG CAH CGW CCK TGH CAA CGM CSA-5'

590 595 Ser Gy. Thr Ser Met Ala Thr Pro E is Wal Ser Giy Wal Wal 5'-TCT GCA ACT TCG ATG GCT ACT CCA CAT GTC ACC GGT GTC GT T-3'

PRO-4R 3'-CCD TGV AGB TAC CGD WGA GCB GTR CAW YSG CCH C-5 U.S. Patent May 26, 1998 Sheet 7 of 9 5,756,339

Kpn

Sph I Pst Bam HI

Sphl Bam H. Barn HI PSt I U.S. Patent May 26, 1998 Sheet 8 of 9 5,756,339

Fig. 15

KpnI

Hpal SacI

Hpal

Sac Sac

Fig. 16

5O

o

100 g > r J U rts g 50 •r rts E () A.

Time (h) U.S. Patent May 26, 1998 Sheet 9 of 9 5,756,339

Fig. 17

1 OO -O

5 O

O

-e- 67K

-ie- 43K

-(-30K Enzyme Enzyme Enzyme Sample Sample Sample PF PF-36 PF-13 BS 13 5,756,339 1 2 HYPERTHERMOSTABLE PROTEASE GENE FIG. 4 illustrates comparison of restriction maps of DNA's derived from Pyrococcus furiosus contained in the FIELD OF THE INVENTION plasmids. The present invention relates to a gene encoding a hyper FIG. 5 illustrates a restriction map of the plasmid thermostable protease which is useful as an enzyme for pTPR15. industrial application and a process for producing the FIG. 6 illustrates a restriction map of the plasmid enzyme by genetic engineering. pTPR15. FIG. 7 illustrates a BACKGROUND OF THE INVENTION restriction map of the plasmid O pTPR13. Proteases are enzymes which cleave peptide bonds in FIG. 8 illustrates a restriction map of the plasmid proteins and various proteases have been found in animals, pUBR13. plants and microorganisms. They are used not only as FIG. 9 illustrates a restriction map of the plasmid reagents for research works and medical supplies, but also in pUBR36. industrial fields such as additives for detergents, food pro cessing and chemical syntheses utilizing their reverse reac 15 FIG. 10 illustrates a design of an oligonucleotide PRO-1F. tions and it can be said that they are very important enzymes FIG. 11 illustrates designs of oligonucleotides PRO-2F from an industrial viewpoint. For proteases to be used in and PRO-2R. industrial fields, since very high physical and chemical FIG. 12 illustrates a design of an oligonucleotide PRO stabilities are required. in particular. enzymes having high 20 4R. thermostability are preferred to use. At present, proteases FIG. 13 illustrates a restriction map of the plasmid p2F predominantly used in industrial fields are those produced 4R. by bacteria of the genus Bacillus because they have rela FIG. 14 illustrates a restriction map of the plasmid pTC1. tively high thermostabilities. FIG. 15 illustrates a restriction map of the plasmid ptC3. However, enzymes having further superior properties are 25 desired and activities have been attempted to obtain FIG, 16 illustrates thermostability of the hyperthermo enzymes from microorganisms which can grow at high stable protease obtained in the present invention. temperatures, for example, thermophiles of the genus Bacil FIG. 17 illustrates the optimum pH of the hyperthermo lus. stable protease obtained in the present invention. On the other hand, a group of microorganisms, named as FIG. 18 illustrates the activity staining pattern after hyperthermophiles, are well adapted themselves to high gelatin-containing SDS-polyacrylamide gel electrophoresis temperature environment and therefore they are expected to of the hyperthermostable protease obtained in the present be supply sources for various thermostable enzymes. It has invention. been known that one of these hyperthermophiles, Pyrococ DISCLOSURE OF THE INVENTION cus furiosus, produces proteases (Appl. Environ. Microbiol. 35 In order to obtain a hyperthermostable protease gene, the 56, 1992-1998 (1990); FEMS Microbiol. Letters, 71.17-20 present inventors attempted to purify a protease from micro (1990); J. Gen. Microbiol. 137. 1193-1199 (1991)). bial cells and a culture supernatant of Pyrococcus furiosus In addition, as for hyperthermophiles of the genera DSM 3638 so as to determine a partial amino acid sequence Thermococcus. Staphylothermus and Thermobacteroides, of the enzyme, independently. However, purification of the the production of proteases have also been known Applied protease was very difficult in either case of using the Microbiology and Biotechnology, 34, 715–719 (1991). microbial cells or the culture supernatant and the present OBJECTS OF THE INVENTION inventors failed to obtain an enzyme sample having suffi Since proteases produced by these hyperthermophiles cient purity for determination of its partial amino acid 45 sequence. have high thermostabilities, they are expected to be appli As a method for cloning a gene for an objective enzyme cable to new applications to which any known enzyme has without any information about a primary structure of the not been utilized. However, the above publications merely enzyme, there is an expression cloning method and, for teach that thermostable protease activities are present in example, a pullulanase gene originating in Pyrococcus cell-free extracts or crude enzyme solutions obtained from culture supernatants and there is no disclosure about prop 50 woesei has been obtained according to this method (WO erties of isolated and purified enzymes and the like. 92/02614). However, in an expression cloning method, a Moreover, since a cultivation of microorganisms at high plasmid vector is generally used and, in such case, it is temperature is required to obtain enzymes from these necessary to use restriction enzymes which can cleave an hyperthermophiles, there is a problem in industrial produc objective gene into relatively small DNA fragments so that 55 the fragments can be inserted into the plasmid vector with tion of the enzymes. out cleavage of any internal portion of the objective gene. In order to solve the above problems, an object of the Then, the method is not always applicable to cloning of all present invention is to isolate a gene encoding a protease of kinds of enzyme genes. Furthermore, it is necessary to test a hyperthermophile. Another object of the present invention for an enzyme activity of a large number of clones and this is to provide a process for producing the protease by genetic operation is complicated. engineering using the gene. The present inventors have attempted to isolate a protease BRIEF DESCRIPTION OF THE DRAWINGS gene by using a cosmid vector which can maintain a larger DNA fragment (35-50 kb) instead of a plasmid vector to FIG. 1 illustrates a restriction map of the plasmid pTPR1. prepare a cosmid library of Pyrococcus furiosus genome and FIG. 2 illustrates a restriction map of the plasmid pTPR9. 65 investigating cosmid clones in the library to find out a clone FIG. 3 illustrates a restriction map of the plasmid expressing a protease activity. By using a cosmid vector, the pTPR12. number of transformants to be screened can be reduced in 5,756,339 3 4 addition to lowering of possibilities of cleavage of an furiosus, in particular, a hyperthermostable protease gene internal portion of the enzyme gene. On the other hand, since which comprises the amino acid sequence represented by the copy number of a cosmid vector in a host is not higher SEQ ID NO 1 in the Sequence Listing or a part thereof than that of a plasmid vector, it may be that an amount of the encoding the active portion of the hyperthermostable enzyme expressed is too small to detect its enzyme activity. protease, especially, the hyperthermostable protease gene In view of high thermostability of the objective enzyme. having the DNA sequence represented by SEQID NO 2 in firstly, the present inventors have cultured respective trans the Sequence Listing. formants in a cosmid library, separately, and have combined In addition, the present invention provides hyperthermo this step with a step for preparing lysates containinging only stable protease genes hybridizable with the above hyper thermostable proteins from the microbial cells thus obtained. 10 thermostable protease genes. For example, there is provided This group of lysates have been named as a cosmid protein a hyperthermostable protease gene containing the nucleotide library. By using the cosmid protein library in detection of sequence represented by SEQ ID NO 7 in the Sequence the enzyme activity, detection sensitivity can be increased Listing. higher than that of a method using transformant colonies. Moreover, the present invention provides a process for In addition, the present inventors have made possible to 5 producing the hyperthermostable protease which comprising detect a trace amount of the enzyme activity by performing culturing a transformant transformed with a recombinant SDS-polyacrylamide gel-electrophoresis with a gel contain plasmid into which the hyperthermostable protease gene of ing gelatin. According to this method, a trace amount of a the present invention has been inserted, and collecting the protease activity contained in a sample can be detected with hyperthermostable protease from the culture. high sensitivity as a band concentrated in the gel. 20 The hyperthermostable protease genes of the present In this manner, the present inventors have screened a invention can be obtained by screening of gene libraries of cosmid protein library originating in Pyrococcus furiosus hyperthermophiles. As the hyperthermophiles, bacteria and have obtained several cosmid clones which express the belonging to the genus Pyrococcus can be used and the desired genes can be obtained by screening a cosmid library protease activity. 25 Furthermore, the present inventors have succeeded in of Pyrococcus furiosus genome. isolation of hyperthermostable protease genes from inserted For example. Pyrococcus furiosus DSM3638 can be used DNA fragments contained in the clones by utilizing various as Pyrococcus furiosus and this strain is available from gene engineering techniques and also have found that prod Deutsch Sammlung von Microorganismen und Zellkulturen ucts expressed from the genes are resistant to surfactants. 30 GmbH. By comparing an amino acid sequence of the hyperther One example of cosmid libraries of Pyrococcus furiosus mostable protease deduced from the nucleotide sequence of can be obtained by partially digesting the genomic DNA of the gene with amino acid sequences of known proteases Pyrococcus furiosus DSM3638 with a restriction enzyme, originating in microorganisms. homology of the amino acid Sau3AI (manufactured by Takara Shuzo, Co. Ltd.), to sequence of the front halfportion of the protease encoded by 35 obtain DNA fragments, ligating the DNA fragments with a the gene with those of a group of alkaline proteases, whose triple helix cosmid vector (manufactured by Stratagene) and representative example is subtilisin. has been shown and, in packaging into lambda phage particles by in vitro packaging particular, very high homology has been found at each method. Then, the library is transduced into a suitable E. region around the four amino acid residues which are known coli, for example. E. coli DH5oMCR (manufactured by to be of importance for a catalytic activity of the enzymes. BRL) to obtain transformants, followed by culturing them. Thus, since the protease produced by Pyrococcus furiosus, collecting the microbial cells, subjecting them to heat treat which is active at such high temperatures that proteases ment (100° C. for 10 minutes), sonicating and subjecting originating in mesophiles are readily inactivated, has been heat treatment (100° C. for 10 minutes) again. The lysates shown to retain a structure similar to those of enzymes from thus obtained can be subjected to screening for the protease mesophiles, it has been suggested that similar proteases 45 activity by performing SDS-polyamide gel-electrophoresis would also be produced by hyperthermophiles other than with a gel containing gelatin. Pyrococcus furiosus. In this manner, a cosmid clone containing a hyperther Then, the present inventors have noted possibilities that. mostable protease gene capable of expressing a protease in the nucleotide sequence of the hyperthermostable pro which is resistant to the above heat treatment can be tease gene obtained, the nucleotide sequences encoding 50 obtained. regions showing high homology with subtilisin and the like Furthermore, a cosmid DNA prepared from the above can be used as probes for investigating hyperthermostable obtained cosmid clone can be digested with suitable restric protease genes and have attempted to detect protease genes tion enzymes to form fragments to prepare recombinant originating in hyperthermophiles by PCR using synthetic plasmids into which the respective fragments thus obtained DNA designed based on the above nucleotide sequences as 55 are inserted. A recombinant plasmid containing the desired primers so as to clone DNA fragments containing the hyperthermostable protease gene can be obtained by trans protease genes. As a result, the present inventors have found forming a suitable microorganism with the above-obtained a protease gene in a hyperthermophile. Thermococcus celer plasmids and testing for the protease activity expressed by DSM2476, and have obtained a DNA fragment containing the resultant transformants. the gene. Furthermore, the present inventors have confirmed That is, a cosmid DNA prepared from one of the above that an amino acid sequence encoded by the DNA fragment obtained cosmid clones can be digested with SphI contains amino acids sequences having high homology with (manufactured by Takara Shuzo. Co. Ltd.). followed by the amino acid sequences of the hyperthermostable protease inserting the resultant DNA fragment into Sph site of a represented by SEQNO 1 of the Sequence Listing. Thus, the plasmid vector. pUC119 (manufactured by Takara Shuzo, present inventors have completed the present invention. 65 Co. Ltd.) to obtain a recombinant plasmid. Then, the That is, the present invention provides an isolated hyper recombinant plasmid is introduced into E. coli JM109 thermostable protease genes originating in Pyrococcus (manufactured by Takara Shuzo. Co., Ltd.) and the protease 5,756,339 S 6 activity of the resultant transformant is tested by the same A lysate of Escherichia coli JM109/pTPR12 shows the method as that used for screening of the cosmid protein protease activity similar to that of the cosmid clone on a library. The transformant having the activity is used for SDS-polyacrylamide gel containing gelatin. FIG. 3 illus preparation of a plasmid. trates a restriction map of the plasmid pTPR12. In FIG. 3, As is seen from Examples hereinafter. one of the recom the thick solid line is the DNA fragment inserted into the binant plasmids has been named as pTPR1 and E. coli plasmid vector puC19. JM109 transformed with the plasmid has been named as FIG. 4 illustrates restriction maps of the DNA fragments Escherichia coli JM109/pTPR1. FIG. 1 illustrates a restric originating in Pyrococcus furiosus which are inserted into tion map of the plasmid pTPR1. In FIG. 1. the thick solid the plasmids pTPR1, ptPR9 and pIPR12, respectively. line represents the DNA fragment inserted into the plasmid 10 According to FIG. 4, a fragment of about 1 kb which does vector pCC119. The recombinant plasmid contains SphI not contain a hyperthermostable protease gene can be fragment of about 7.0 kb. removed from the DNA fragment inserted into the plasmid In addition, a DNA fragment of about 2.5 kb which does pTPR12. That is, the plasmid pIPR12 is digested with Xbal not contain the hyperthermostable protease gene can be and KpnI (manufactured by Takara Shuzo. Co., Ltd.) and removed from the recombinant plasmid. That is, among 15 thus-obtained Xbal-Xbal fragment of about 3.3 kb and three fragments of about 2.5 kb, about 3.3 kb and about 4.3 Xbal-KpnI fragment of about 3.2 kb are isolated, respec kb obtained by digesting the above plasmid pIPR1 with tively. Then, firstly, the Xba-KpnIfragment of about 3.2 kb Xbal (manufactured by Takara Shuzo. Co. Ltd.), only the is inserted into the plasmid vector puC19 at a site between DNA fragment of about 2.5 kb is removed and the remaining Xbal and KpnI to prepare a recombinant plasmid. This fragments are ligated and introduced into E. coli JM109. The plasmid has been named as pTPR14 and FIG. 5 illustrates its protease activity of the resultant transformant is tested by the restriction map. In FIG. 5, the thick solid line represents the same method as that used for screening of the cosmid protein DNA fragment inserted into the plasmid vector puC19. library. The resultant transformant having the protease activ Then, the above Xbal-Xbal fragment of about 3.3 kb is ity is used for preparation of a plasmid. The plasmid has inserted into the plasmid pTPR14 at Xbal site and intro been named as pTPR9 and E. coli JM109 transformed with 25 duced into E. coli JM109. The protease activity of the the plasmid has been named as Escherichia coli JM109/ transformant is tested by using the method used for screen pTPR9. FIG. 2 illustrates a restriction map of the plasmid ing the cosmid protein library. A plasmid is prepared by the pTPR9. In FIG. 2. the thick solid line represents the DNA transformant having the activity. The plasmid has been fragment inserted into the plasmid vector pUC119. named as pTPR15 and E. coli JM109 transformed with the The protease activities expressed by both plasmids pTPR1 30 and pIPR9 show high thermostability. However, since the plasmid has been named as Escherichia coli JM1097 activities are observed at positions different from that for the pTPR15. FIG. 6 illustrates a restriction map of pIPR15. In protease activity expressed by above cosmid clone on a FIG. 6, the thick solid line represents the DNA fragment SDS-polyacrylamide gel containing gelatin, these plasmids inserted into the plasmid vector p C19. are estimated to be defect in a part of the protease gene on 35 Further. in the nucleotide sequences of the DNA fragment the cosmid DNA. A DNA fragment containing the whole originating in Pyrococcus furiosus and inserted into the length of the protease gene can be obtained from the cosmid plasmid pTPR15, the nucleotide sequence of the DNA DNA by, for example, using a part of the inserted DNA fragment of about 4.8 kb between two Drasites are shown fragment of the above plasmid pTPR9 as a probe. That is, as SEQID NO 8 in the Sequence Listing. That is, SEQID the cosmid DNA used for preparation of the plasmid pIPR1 NO 8 of the Sequence Listing is an example of the nucle is digested with Not (manufactured by Takara Shuzo, Co., otide sequence of the hyperthermostable protease gene of Ltd.) and several restriction enzymes which do not cleave the present invention. And, an amino acid sequence of a any internal portion of the DNA fragment inserted into the product of the gene deduced from the nucleotide sequence of plasmid pTPR1. After agarose gel-electrophoresis, the DNA SEQID NO 8 is shown as SEQID NO 9 in the Sequence fragments in the gel are blotted on a nylon membrane. 45 Listing. That is, SEQID NO 9 in the Sequence Listing is an Regarding the membrane thus obtained, hybridization is example of the amino acid sequence of an enzyme protein carried out by using a Pst-Xbal fragment of about 0.7 kb produced by using the hyperthermostable protease gene obtained from the DNA fragment inserted into the plasmid obtained according to the present invention. pTPR9 as a probe to detect a DNA fragment containing the Because it has been found that the hyperthermostable same sequence as that of the Pst-Xbal fragment. 50 protease gene of the present invention is contained in Dra In the cosmid DNA digested with two enzymes. Not and fragment of about 4.8 kb in the DNA fragment inserted into Pvu I (manufactured by Takara Shuzo Co., Ltd.), a DNA the above plasmid ptPR15, a recombinant plasmid contain fragment of about 7.5 kb is hybridized with the Pst-Xbal ing only this Dral fragment can be prepared. fragment. This fragment of about 7.5 kb can be isolated to That is, the above plasmid pTPR15 is digested with DraI insert into a plasmid vector, pUC19 (manufactured by 55 (manufactured by Takara Shuzo Co., Ltd.) to isolate the Takara Shuzo, Co., Ltd.) into which Not linker resultant DNA fragment of about 4.8 kb. Then, it can be (manufactured by Takara Shuzo Co., Ltd.) has been intro inserted into the plasmid vector puC19 at SmaI site to duced at a Hinc site, at a site between Not and Sna. The prepare a recombinant plasmid. The recombinant plasmid plasmid has been named as pTPR12 and E. coli JM109 has been named as pTPR13 and E. coli JM109 transformed transformed by the plasmid has been named and indicated as with the plasmid has been named as Escherichia coli Escherichia coli JM109?pTPR12. This strain has been JM109/pTPR13. deposited with National Institute of Bioscience and Human A lysate of Escherichia coli JM109/pTPR13 shows the Technology (NIBH). Agency of Industrial Science & same protease activity as that of the cosmid clone on a Technology. Ministry of International Trade & Industry SDS-polyacrylamide gel containing gelatin. FIG. 7 illus under the accession number of FERM BP-5103 under 65 trates a restriction map of the plasmid pTPR13. In FIG. 7. Budapest Treaty since May 24, 1994 (the date of the original the thick solid line represents the DNA fragment inserted deposit). into the plasmid vector puC19. 5,756.339 7 8 In addition, the hyperthermostable protease gene of the stable protease genes obtained in the present invention. In present invention can be expressed in Bacillus subtilis. As addition, SEQID NO 1 of the Sequence Listing is an amino the Bacillus subtilis, Bacillus subtilis DB104 can be used acid sequence of the gene product deduced from the nucle and this strain is a known strain described in Gene, Vol. 83, otide sequence of SEQID NO 2. That is, SEQID NO 1 of pp. 215-233 (1989). As a cloning vector, a plasmid puB18 the Sequence Listing is an example of amino acid sequences P43 can be used and this plasmid has been given by Dr. of enzyme proteins produced by using hyperthermostable Sui-Lam Wong of Calgary University. This plasmid contains protease genes obtained by the present invention. a kanamycin resistant gene as a selection marker. As described above. it has been found that the regions The above-described plasmid ptPR13 can be digested commonly present in alkaline serine proteases originating in with KpnI (manufactured by Takara Shuzo Co., Ltd.) and O mesophiles are conserved in the amino acid sequence of the BamHI (manufactured by Takara Shuzo Co., Ltd.) to obtain hyperthermostable protease produced by the hyperthermo a DNA fragment of about 4.8 kb, followed by isolating and phile Pyrococcus furiosus. Therefore, the presence of the ligating the fragment between KpnI site and BamHI site of regions is expected in the same kind of proteases produced the plasmid pUB18-P43 to prepare a recombinant plasmid. by hyperthermophiles other than Pyrococcus furiosus. That The plasmid has been named as pubP13 and Bacillus 15 is, it is possible to obtain genes for hyperthermostable subtilis DB104 transformed with the plasmid has been proteases similar to the above-described hyperthermostable named as Bacillus subtilis DB104/pUBP13. A lysate of protease by preparing suitable synthetic DNA fragments Bacillus subtilis DB104/pUBP13 shows the same protease based on parts of the nucleotide sequence of SEQ ID NO 2 activity as that of the cosmid clone on a SDS of the Sequence Listing which encode amino acid sequences polyacrylamide gel containing gelatin. FIG. 8 illustrates a having high homology with those of subtilisin and the like, restriction map of the plasmid puBP13. In Fig. 8, the thick and using them as probes or primers. solid line represents the DNA fragment inserted into the FIGS. 10, 11 and 12 illustrate the relation among the plasmid vector puB18-P43. amino acid sequences of regions in the amino acid sequence By comparing the amino acid sequence shown by SEQID of the hyperthermostable protease of the present invention NO 9 of the Sequence Listing with amino acid sequences of 25 which have high homology with those of subtilisin and the proteases originating in known microorganisms. it is shown like, the nucleotide sequences of the hyperthermostable that there is homology between the front half portion of the protease gene of the present invention which encode the sequence of the hyperthermostable protease of the present regions, and the nucleotide sequences of oligonucleotides invention and those of a group of alkaline serine proteases PRO-1F PRO-2F, PRO-2R and PRO-4R synthesized based whose representative example is subtilisin Protein 30 on the above sequences, respectively. In addition, SEQ ID Engineering. Vol. 4, pp. 719–737 (1991)), in particular, there NO 3, 4, 5 and 6 of the Sequence Listing illustrate the is high homology between each region around the four nucleotide sequences of the oligonucleotides PRO-1F. PRO amino acid residues which are known to be of importance 2F PRO-2R and PRO-4R. That is, SEQ ID NO 3, 4, 5 and for protease activity. On the other hand, such homology 6 of the Sequence Listing are examples of oligonucleotides cannot be observed between the back half portions of the 35 which can be used for detection of the hyperthermostable amino acid sequences and it is considered that this portion protease genes of the present invention by hybridization. may not be essential to a protease activity. Therefore, a A combination of above oligonucleotides can be used as mutant protease wherein an appropriate peptide chain is primers to carry out PCR using genomic DNA of various removed from its back half portion is expected to show the hyperthermophiles as templates to detect protease genes enzymatic activity. Examples of such mutant protease present in hyperthermophiles. As the hyperthermophiles, include a protease having an amino acid sequence corre bacteria belonging to the genera Pyrococcus. Thermococcus. sponding to SEQ D. NO 9 of the Sequence Listing from Staphylothermus. Thermobacteroides and the like can be which the 904th amino acid, Ser, and the subsequent used. As bacteria belonging to the genus Thermococcus, sequence has been removed. This can be prepared by the Thermococcus celer DSM2476 can be used and the strain is following process. 45. available from Deutsch Sammlung von Microorganismen Firstly, a Kpn-EcoRI fragment of about 2.8 kb wherein und Zellkulturen GmbH. When PCR is carried out by using the EcoRI site is blunted is prepared from the above plasmid genomic DNA of Thermococcus celer DSM2476 as a tem pTPR13 and the fragment is ligated between the KpnI site plate and a combination of the above oligonucleotides and the blunted Xbal site of the plasmid vector pUC119. A PRO-1F and PRO-2R or a combination of PRO-2F and protease gene contained in the recombinant plasmid thus 50 PRO-4R as primers, specific amplification of DNA frag obtained encodes an amino acid sequence corresponding to ments is observed and the presence of a protease gene can the SEQID NO 9 of the Sequence Listing except that the be indicated. In addition, an amino acid sequence encoded nucleotide sequence TCA encoding the 904th amino acid, by the fragment can be estimated by ligating the fragment to Ser. has been replaced with the termination codon TAG and suitable plasmid vector to prepare a recombinant plasmid the subsequent nucleotide sequence has been deleted. The 55 and determining the nucleotide sequence of the inserted plasmid has been named as pTPR36 and E. coli JM109 DNA fragment by dideoxy method. transformed with the plasmid has been named Escherichia A DNA fragment of about 150 bp amplified by using the coli JM109/pTPR36. A lysate of Escherichia coli JM109/ oligonucleotides PRO-1F and PRO-2R and a DNA fragment pTPR36 shows an protease activity on a SDS of about 550 bp amplified by using the oligonucleotides polyacrylamide gel containing gelatin. FIG. 9 illustrates a PRO-2F and PRO-4R are ligated to HincII site of the restriction map of the plasmid ptPR36. In FIG.9. the thick plasmid vector pUC18 to obtain recombinant plasmids. solid line represents the DNA fragment inserted into the respectively. The recombinant plasmids have been named as plasmid vector pUC119. SEQ ID NO 2 in the Sequence plF-2R(2) and p2F4R, respectively, SEQID NO 10 of the Listing is a nucleotide sequence of the open reading frame Sequence Listing illustrates the nucleotide sequence of the contained in the DNA fragment inserted in the plasmid 65 DNA fragment inserted into the plasmid plP-2R(2) and an pTPR36. That is. SEQ D NO 2 of the Sequence Listing is amino acid sequence deduced therefrom. SEQID NO 11 of an example of nucleotide sequences of the hyperthermo the Sequence Listing illustrates the nucleotide sequence of 5,756,339 9 10 the DNA fragment inserted into the plasmid p2F-4R and an and this fragment of about 9 kb can be isolated and inserted amino acid sequence deduced therefrom. In the nucleotide into KpnI site of the plasmid vector pUC119 to obtain a sequence shown by SEQID NO 10 of the Sequence Listing. recombinant plasmid. This plasmid has been named as pTC1 the sequence from the 1st to the 21st nucleotides and the and E. coli JM109 transformed with this plasmid has been sequence from the 113th to the 145th nucleotides and, in the named as Escherichia coli JM109/pTC1. nucleotide sequence shown by SEQ ID NO 11 of the FIG. 14 illustrates a restriction map of the plasmid pIC1. Sequence Listing. the sequence from the 1st to the 32nd In FIG. 14, the thick solid line represents the DNA fragment nucleotides and the sequence from the 532nd to the 564th inserted into the plasmid vector plC119. nucleotides are the nucleotide sequences derived from the Furthermore. a DNA fragment of about 4 kb which does oligonucleotides used as the primers (corresponding to the not contain the hyperthermostable protease gene can be oligonucleotides PRO-1F PRO-2R, PRO-2F and PRO-4R, O removed from the plasmid pIC1. That is, plasmid pCi is respectively). In the amino acid sequences shown by SEQ digested with KpnI and several restriction enzymes which D NO 10 and 11, there are sequences having homology with cleave the region within the fragment inserted into the amino acid sequences of the hyperthermostable protease plasmid pTC1 and, after subjecting to agarose gel originating in Pyrococuss furiosus of the present invention electrophoresis, detection of a DNA fragment containing the as well as alkaline serine protease originating in various 15 protease gene is carried out according to the same manner as microorganisms and it has been shown that the above DNA that for the above phage DNA. When the plasmid pTC1 is fragments amplified by PCR are those amplified utilizing the digested with KpnI and BamHI, a DNA fragment of about protease gene as the template. 5 kb is hybridized with the probe and this fragment of about FIG. 13 illustrates a restriction map of the plasmid p2F 5 kb can be isolated and introduced into Kpn-BamH site of 4R. In FIG. 13, the thick solid line represents the DNA 20 the plasmid vector plJC119 to obtain a recombinant plasmid. fragment inserted into the plasmid vector puC18. This plasmid has been named as pTC1 and E. coli JM109 transformed with this plasmid has been named as Escheri On the other hand, when genomic DNA of Thermo chia coli JM109/pTC3. FIG. 15 illustrates a restriction map bacteroides proteoliticus DSM5265 and Staphylothermus of the plasmid pTC3. In FIG. 15. the thick solid line marinus DSM3639 are used as templates, amplification as 25 represents the DNA fragment inserted into the plasmid observed in case of Thermococcus celer has not been vector pUC119. recognized. The nucleotide sequence of the hyperthermostable pro It has been known that efficiency of gene amplification by tease gene contained in the DNA fragment inserted into the PCR is influenced by annealing efficiency of a 3'-terminal plasmid pTC3 can be determined by using specific primers, portion of a primer and a template DNA. Even when 30 i.e., by using suitable oligonucleotides synthesized based on amplification of DNA fragment is not observed in the above the nucleotide sequences as shown by SEQID NO 10 and PCR, protease genes can be detected by synthesizing oligo 11 of the Sequence Listing as primers. SEQD NO 12, 13. nucleotides having different sequences but encoding the 14, 15, 16 and 17 represent the nucleotide sequences of the same amino acid sequence and using them as primers. In oligonucleotides TCE-2. TCE-4. SEF-3, SER-1, SER-3 and addition, protease genes can also be detected by using these 35 TCE-6R which have been used as the primers for determi oligonucleotides as probes and carrying out Southern nation of the nucleotide sequence of the hyperthermostable hybridization with genomic DNA of various hyperthermo protease gene. In addition, SEQ ID NO 7 of the Sequence philes. Listing represents a part of the nucleotide sequence of the Then, the above-described oligonucleotides or amplified hyperthermostable protease gene thus obtained. That is. DNA fragments obtained by the above PCR can be used as SEQ ID NO 7 is a part of the nucleotide sequence of the probes for screening genomic DNA libraries of hyperther hyperthermostable protease gene of the present invention. mophiles to obtain hyperthermostable protease genes, for Moreover, SEQ ID NO 18 represents an amino acid example, the hyperthermostable protease gene produced by sequence of an example of the enzyme encoded by the Thermococcus celer. hyperthermostable protease gene obtained by the present As an example of genomic DNA libraries of Thermococ 45 invention. In the DNA fragment inserted into the plasmid cus celer, there is a library prepared by partially digesting a pTC3, the sequence derived from lambda GEM-11 vector is genomic DNA of Thermococcus celer DSM2476 with a adjacent to the 5'-end of the nucleotide sequence represented restriction enzyme Sau3AI to obtain a DNA fragment, sented by SEQID NO 7 of the Sequence Listing, indicating ligating the fragment with lambda GEM-11 vector a defect in a part of the 5'-region of the protease gene. In (manufactured by Promega) and packaging it into lambda 50 addition, by comparing the nucleotide sequence with those phage particles according to in vitro packaging method. of SEQIDNO 10 and 11 of the Sequence Listing, it has been Then, the library is transduced into a suitable E. coli, for found that the DNA fragment inserted into the plasmidpTC3 example. E. coli LE392 (manufactured by Promega) to form contains the 41st and the subsequent nucleotides of the plaques on a plate and then plaque hybridization is carried nucleotide sequence represented by SEQID NO 10 and the out by using amplified DNA fragments obtained in the 55 whole nucleotide sequence of SEQID NO 11. above-described PCR. In this manner, phage clones contain Although the hyperthermostable protease gene obtained ing hyperthermostable protease genes can be obtained. from Thermococcus celer is defect in a part thereof, as is Further, the phage DNA prepared from the clone thus obvious to a person skilled in the art, a DNA fragment obtained is digested with suitable restriction enzymes and, containing the whole length of the hyperthermostable pro after subjecting to agarose gel-electrophoresis. DNA frag tease gene can be obtained, for example, (1) by repeating ments in the gel are blotted on a nylon membrane. Regarding screening of a genomic DNA library, (2) by carrying out the membrane thus obtained, hybridization is carried out Southern hybridization with genomic DNA. (3) by obtaining using amplified DNA fragments obtained according to the a DNA fragment of the 5'-upstream region by PCR with a above PCR as probes to detect a ENA fragments containing cassette (manufactured by Takara Shuzo Co., Ltd.) and the protease gene. 65 cassette primers (manufactured by Takara Shuzo Co. Ltd.) When the above phage DNA is digested with KpnI, a (Takara Shuzo's Genetic Engineering Products Guide. DNA fragment of about 9 kb is hybridized with the probe 1994-1995 ed., pp. 250-251), and the like. 5,756,339 11 12 A transformant into which a recombinant plasmid con a 0.1% SDS-10% polyacrylamide gel containing 0.05% of taining with the hyperthermostable protease gene is casein was used. The enzyme samples obtained by the transduced, for example, Escherichia coli JM109?pTPR13 present invention, PF-13, PF-36 and PF-BS13, had casein or Escherichia coli JM109/pTPR36, can be cultured under hydrolyzing activity at 95° C. conventional conditions, for example, by culturing the trans Moreover, casein hydrolyzing activity of the enzyme formant in LB medium trypton (10 g/liter), yeast extract (5 sample obtained by the present invention, PF-BS13. was g/liter), NaCl (5 g/liter); pH 7.2) containing 100 pg/ml of determined by the following method. To 100 ul of 0.1M ampicillin at 37° C. to express the hyperthermostable pro potassium phosphate buffer (pH 7.0) containing 0.2% casein tease in the culture. After completion of culture, the cultured cells are harvested and the cells are sonicated and centri was added 100 ul of a suitably diluted enzyme solution and fuged. The supernatant is subjected to heat treatment at 100° O incubated at 95°C. for 1 hour. The reaction was stopped by C. for 5 minutes to denature and remove contaminated addition of 100 ul of 15% trichloroacetic acid and the proteins. In this way, a crude enzyme sample can be reaction mixture was centrifuged. The amount of acid obtained. The crude enzyme samples thus obtained from soluble short chain polypeptides contained in the superna Escherichia coli JM109?pTPR13 and Escherichia coli tant was determined by measuring absorbance at 280 nm and JM109?pTPR36 have been named as PF-13 and PF-36. 15 the enzyme activity was determined by comparing the Further, a transformant into which a recombinant plasmid absorbance with that of an enzyme free control. The enzyme containing the hyperthermostable protease gene is sample obtained by the present invention, PF-BS13. had transduced, Bacillus subtilis DB104/pUBP13, can be cul casein hydrolyzing activity under the experimental condi tured under conventional conditions, for example, by cul tions of pH 7.0 at 95° C. turing the transformant in LB medium containing 10 g/ml (3) Stability Stability of the enzyme was examined by of kanamycin at 37° C. to express the hyperthermostable detecting remaining enzymatic activity of heat treated protease in the culture. After completion of culture, the enzymes by the above-described method (2) using SDS cultured cells are harvested and the cells are sonicated and polyacrylamide gel containing gelatin. Namely, the enzyme sample was incubated at 95° C. for 3 hours and then a centrifuged. The supernatant is subjected to heat treatment at 25 100° C. for 5 minutes to denature and remove contaminated suitable amount thereof was subjected to detection of the proteins, followed by salting out with ammonium sulfate enzymatic activity to compare its activity with that without and dialysis. In this way, a partially purified enzyme sample treatment at 95°C. Although the position of enzyme activity can be obtained. The roughly purified enzyme sample thus on the gel was somewhat changed due to incubation at 95 C., lowering of the enzyme activity was scarcely observed. obtained from Bacillus subtilis DB104/pUBP13 has been 30 named as PF-BS13. The enzyme samples obtained by the present invention. The enzymatic and physicochemical properties of the PF-13 PF-36 and PF-BS13, were stable to heat treatment at hyperthermostable protease samples produced by the trans 95° C. for 3 hours. formants into which the recombinant plasmids containing In addition. stability of the enzyme samples obtained by the hyperthermostable protease genes derived from Pyro 35 the present invention. PF-13 and PF-36, in the presence of coccus furiosus obtained by the present invention, for surfactants were tested. Namely, Triton X-100. SDS or example, PF-13. PF-36 and PF-BS13 are as follows. benzalkonium chloride was added to the enzyme samples in the final concentration of 0.1%. The mixture was incubated (1) Activity at 95° C. for 3 hours and a suitable amount thereof was The enzymes obtained by the present invention hydrolyze subjected to detection of the enzymatic activity. For each gelatin to form short chain polypeptides. In addition, they surfactant, no substantial change in the enzyme activity was hydrolyze casein to form short chain polypeptides. found in comparison with that in the absence of the surfac (2) Method for detecting enzyme activity The detection of tant. Then, the enzyme samples obtained by the present enzyme activity was carried out by detection of hydrolysis invention, PF-13 and PF 36, were stable to heat treatment at of gelatin with the enzyme on a SDS-polyacrylamide gel. 95° C. for 3 hours in the presence of surfactants. Namely, an enzyme sample to be tested was suitably diluted 45 and to 10 ul of the sample diluted solution was added 2.5ul Moreover, stability of the enzyme sample obtained by the of a sample buffer solution (50 mM Tris-HCl pH 7.6.5% present invention, PF-BS13, was tested by the following SDS. 5% 2-mercaptoethanol, 0.005% Bromophenol Blue, method. Namely, the enzyme sample as such or with addi 50% glycerol). The mixture was subjected to heat treatment tion of SDS in the final concentration of 0.1% was incubated at 100° C. for 5 minutes and then electrophoresis by using 50 at 95° C. for various periods of time and the remaining 0.1% SDS-10% polyacrylamide gel containing 0.05% gela activity was determined by the above-described spectropho tin. After completion of electrophoresis, the gel was soaked tometric method (2) based on increase in the amount of acid in 50 mM potassium phosphate buffer (pH 7.0) and incu soluble polypeptides. FIG. 16 illustrates thermostability of bated at 95° C. for 2 hours to carry out the enzymatic the enzyme sample obtained by the present invention. reaction. Then, the gel was stained with 2.5% Coomassie 55 PF-BS13. The ordinate indicates the remaining activity (%) Brilliant Blue R-250 in 25% ethanol and 10% acetic acid for and the abscissa indicates incubation time (hr). In FIG. 16, 30 minutes and further the gel was transferred in 25% the open circle represents the results obtained without addi ethanol and 7% acetic acid to remove excess dye over 3 to tion of SDS and the closed circle represents the results in the 15 hours. Gelatin hydrolyzed with the protease into peptides presence of 0.1% SDS. As seen from FIG. 16. PF-BS13 was diffused outside of the gel during the enzymatic reaction maintained almost 100% activity after incubation at 95 C. and the corresponding position was not stained with Coo for 4 hours regardless of the presence or absence of 0.1% massie Brilliant Blue, thereby detecting the presence of the SDS. protease activity. The enzyme samples obtained by the (4) Effect of various reagents present invention. PF-13. PF-36 and PF-BS13. had gelatin The enzyme samples were subjected to SDS hydrolyzing activity at 95°C. 65 polyacrylamide gel containing gelatin and then the enzy In addition, the casein hydrolyzing activity was detected matic reaction was carried out in 50 mM potassium phos according to the same manner as described above except that phate buffer (pH 7.0) containing 2 mM EDTA or 2 mM 5,756,339 13 14 phenylmethanesulfonyl fluoride (PMSF) to test for effect of to surfactants. Therefore, they are particularly useful for both reagents on the enzyme activity. No substantial differ treatment of proteins at high temperatures. ence in the enzyme activities of the enzyme samples In addition, a DNA fragment obtained by hybridization obtained by the present invention, PF-13, PF-36 and with the gene isolated by the present invention or a part of PF-BS13, was observed between the buffer containing 2 mM the nucleotide sequence of the isolated gene as a probe can EDTA and 50 mM potassium phosphate buffer alone. On the be transduced into a suitable microorganism and its heat other hand, when the buffer containing 2 mM PMSF was treated lysate can be prepared according to the same manner used, the amount of hydrolyzed gelatin in the gel was as that described with respect to the cosmid protein library. decreased in all the samples, indicating that the activities of Then, a protease activity is tested by an appropriate method. the enzyme samples were inhibited by PMSF. O In this manner, a hyperthermostable protease gene encoding (5) Molecular weight an enzyme whose sequence is not identical with that of the The molecular weight of the enzyme sample obtained by above enzyme but which has a similar activity can be the present invention on a SDS-polyacrylamide gel contain obtained. ing ing gelatin was estimated. The enzyme sample, PF-13. The above hybridization can be carried out under the showed plural active bands within the range of 95 kDa to 51 15 following conditions. Namely, DNA fixed on a membrane is kDa. Although the migration distance was varied according incubated in 6xSSC containing 0.5% of SDS, 0.1% of to the amount of a sample applied. etc., the major bands of bovine serum albumin, 0.1% of polyvinyl pyrrollidone, 0.1% 84 kDa, 79 kDa. 66 kDa, 54 kDa and 51 kDa were appeared. of Ficoll 400 and 0.01% denatured salmon sperm DNA When the enzyme sample was subjected to electrophoresis (1xSSC represents 0.15MNaCl and 0.015M sodium citrate. after heat treatment at 95°C. for 3 hours in the presence of 20 pH 7.0) together with a probe at 50° C. for 12 to 20 hours. SDS in the final concentration of 0.1%, the bands of 63 kDa After completion of incubation, the membrane is washed in and 51 kDa became intensive. For the enzyme sample, such a manner that washing is started with 2XSSC contain PF-BS13, the same results as that of the above with respect ing 0.5% SDS at 37° C., followed by changing SSC con to the enzyme sample PF-13 were obtained. In case of the centrations within the range to 0.1Xand varying tempera enzyme sample PF-36, several minor bands were observed tures up to 50° C. until a signal from the fixed DNA can be in addition to the main bands of 63 kDa and 59 kDa. distinguished from the background signal. (6) Optimum pH Furthermore, the gene isolated by the present invention, a The optimum pH of the enzyme samples PF-13 and PF-36 DNA fragment obtained by in vitrogene amplification using obtained by the present invention was tested. After subject 30 a part of the isolated gene as a primer, or a DNA fragment ing the enzyme samples to electrophoresis on a SDS obtained by hybridization using the fragment obtained by polyacrylamide gel containing gelatin, the gel was soaked in the above amplification as a probe is transduced into a suitable microorganism and, according to the same manner buffers having different pH and the enzyme reaction was as described above, a protease activity is determined. In this carried out to test for the optimum pH. As the buffers, 50 manner, a hyperthermostable protease gene encoding an mM sodium acetate buffer solution at pH 4.0 to 6.0.50 mM 35 potassium phosphate buffer solution at pH 6.0 to 8.0, 50 mM enzyme whose activity is not identical with that of the above sodium borate buffer solution at pH 9.0 to 10.0 were used. enzyme but is similar can be obtained. Both enzyme samples showed gelatin hydrolyzing activity at The following examples further illustrates the present pH 6.0 to 10.0 and their optimum pH was pH 8.0 to 9.0. invention but are not to be construed to limit the scope In addition, the optimum pH of the enzyme sample thereof. In the examples, all the "percents" are by weight. obtained by the present invention, PF-BS13, was determined EXAMPLE by the above-described spectrophotometric method (2) based on increase in the amount of acid soluble polypep Preparation of genomic DNA of Pyrococcus furiosus tides. 0.2% Casein solutions to be used for the determination Pyrococcus furiosus DSM3638 was cultured as follows. were prepared by using 0.1M sodium acetate butter solution 45 A culture medium composed of 1% of trypton, 0.5% of at pH 4.0 to 6.0, 0.1M potassium phosphate buffer solution yeast extract, 1% of soluble starch. 3.5% of Jamarin S-Solid at pH 6.0 to 8.0, 0.1M sodium borate buffer solution at pH (manufactured by Jamarin Laboratory). 0.5% of Jamarin 9.0 to 10.0 and 0.1M sodium phosphate-sodium hydroxide S-Liquid (manufactured by Jamarin Laboratory), 0.003% of buffer solution at pH 11.0 and they were used for the MgSO, 0.001% of NaCl, 0.0001% of FeSO-7H.O. determination. FIG. 17 illustrates the relation between 50 0.0001% of COSO, 0.0001% of CaCl2.H2O. 0.0001% of casein hydrolyzing activity of the enzyme sample obtained ZnSO, 0.1 ppm of CuSO-5H2O, 0.1 ppm of KAI (SO) by the present invention, PF-BS13 and pH. The ordinate 0.1 ppm of HBO, 0.1 ppm of Na2MoCl2.H2O and 0.25 indicates the relative activity (%) and the abscissa indicates ppm of NiCl6HO was placed in a 2 liter-medium bottle pH. In Fig. 17, the open circle, the closed circle, the open and sterilized at 120° C. for 20 minutes. Then, nitrogen gas square and the closed square represent the results obtained 55 was blown into the medium to purge out dissolved oxygen by using the substrate solutions prepared with 0.1M sodium and the above bacterial strain was inoculated into the acetate buffer solution, 0.1M potassium phosphate buffer medium, followed by subjecting to stationary culture at 95 solution, 0.1M sodium borate buffer solution and 0.1M C. for 16 hours. After completion of culture, bacterial cells sodium phosphate-sodeum hydroxide, respectively. As seen were collected by centrifugation. from FIG. 17, the enzyme sample, PF-BS13, showed casein Then, the collected cells were suspended into 4 ml of decomposing activity at the pH range of 5.0 to 11.0 and its 0.05M Tris-HCl (pH 8.0) containing 25% of sucrose and to optimum pH was pH 9.0 to 10.0. this suspension were added 0.8 ml of lysozyme 5 mg/ml. As described hereinabove in detail, according to the 0.25M Tris-HCl (pH 8.0) and 2 ml of 0.2M EDTA. The present invention, the genes encoding the hyperthermostable mixture was incubated at 20° C. for 1 hour. Then, to the proteases and the industrial process for producing the hyper 65 mixture were added 24 ml of SET solution 150 mM NaCl, thermostable proteases using the genes can be provided. The 1 mM EDTA and 20 mM Tris-HCl (pH 8.0) and further 4 enzymes have high thermostability and also show resistance ml of 5% SDS and 400 pil of Proteinase K (10 mg/ml) and 5,756,339 15 16 the mixture was incubated at 37° C. for 1 hour. After The above plasmid ptPR1 was digested with Xbal and completion of the reaction, the reaction mixture was sub subjected to agarose gel-electrophoresis to separate three jected to phenol-chloroform extraction and then ethanol DNA fragments of about 2.5 kb, about 3.3 kb and about 4.3 precipitation to prepare about 3.2 mg of genomic DNA. kb. Among three fragments thus separated, two fragments of Preparation of cosmid protein library 400 ug of Genomic about 3.3 kb and about 4.3 kb were recovered. The DNA DNA of Pyrococcus furiosus DSM3633 was partially fragment of about 4.3 kb was dephosphorylated with alka digested with Sau3AI and subjected to size-fractionation in line phosphatase (manufactured by Takara Shuzo Co. Ltd.) size of 35 to 50 kb by density-gradient ultra-centrifugation. and then was mixed with the DNA fragment of about 3.3 kb Then, 1 ug of a triple helix cosmid vector was digested with to ligate to each other. This was transduced into E. coli Xba, dephosphorylated with alkaline phosphatase 10 JM109. The protease activity of the resultant transformants (manufactured by Takara Shuzo Co., Ltd.) and further were tested by the same method as that used for screening digested with BamHI. The vector was ligated to 140 ug of of the cosmid protein library. A plasmid was prepared from the above fractionated 35 to 50 kb DNA. The genomic DNA the transformant having the protease activity. The plasmid fragments of Pyrococcus furiosus were packaged into was named as pTPR 9 and E. coli JM109 transformed with lambda phage particles by in vitro packaging method using 15 the plasmid was named as Escherichia coli JM109/pTPR9. Gigapack Gold (manufactured by Stratagene) to prepare a FIG. 2 illustrates a restriction map of the plasmid pTPR library. Then, by using a part of the library thus obtained, 9. transduction into E. coli DH5OMCR was carried out and, Detection of DNA fragment containing whole length of among transformants obtained, several transformants were hyperthermostable protease gene selected to prepare cosmid DNA. After confirmation of the 20 The cosmid DNA used in the preparation of the above presence of inserted fractions having suitable size, again plasmid ptPR1 was digested with Not and then further about 500 transformants were selected from the above digested with BamHI. Binl, EcoT22. Nsp(7524)V. Pvul, library and they were independently cultured in 150 ml of Sal I. SmaI and Spe, respectively. Then, digested DNA was LB medium (tripton 10 g/liter, yeast extract 5 g/liter, NaCl subjected to electrophoresis on a 0.8% agarose gel. After 5 g/liter, pH 7.2) containing 100 ug/ml of amplicillin. Each 25 electrophoresis, the gel was soaked in 0.5N NaOH contain culture was centrifuged, the recovered microbial cells were ing 1.5M NaCl to denature the DNA fragments in the gel and suspended in 1 ml of 20 mM Tris-HCl (pH 8.0) and the then the gel was neutralized in 0.5M Tris-HCl (pH 7.5) suspension was subjected heat treatment at 100° C. for 10 containing 3M NaCl. The DNA fragments in the gel was minutes. Then, the suspension was sonicated and again blotted on a Hybond-N nylon membrane (manufactured by subjected heat treatment at 100° C. for 10 minutes. The 30 Amasham) by Southern blotting. After blotting, the mem lysates obtained as supernatants after centrifugation were brane was washed with 6xSSC (1xSSC represents 0.15M used as the cosmid protein library. NaCl, 0.015M sodium citrate, pH 7.0) and air-dried and Selection of cosmid containing hyperthermostable pro DNA was fixed on the membrane by UV irradiation using a tease gene UV transilluminator for 3 minutes. The protease activity was detected by testing for hydroly 35 On the other hand, the plasmid pTPR9 was digested with sis gelatin in a polyacrylamide gel. Pst and Xbal and subjected to electrophoresis on a 1% Namely, 5 pil aliquots of the lysates from the above agarose gel and the separated DNA fragment of about 0.7 kb cosmid protein library were taken out and subjected to was recovered. A 'P-labeled DNA probe was prepared by electrophoresis by using 0.1% SDS-10% polyacrylamide gel using the DNA fragment as a template and using a random containing 0.05% of gelatin. After completion of primer DNA labeling kit Ver2 (manufactured by Takara electrophoresos, the gel was incubated in 50 mM potassium Shuzo Co., Ltd.) and o-'PldCTP (manufactured by phosphate buffer solution (pH 7.0) at 95°C. for 2 hours. The Amasham). gel was stained in 2.5% Coomassie Brilliant Blue-R-250, The above membrane to which the DNA was fixed was 25% ethanol and 10% acetic acid for 30 minutes. Then, the 45 treated in a hybridization buffer solution (6xSCC containing gel was transferred to 25% methanol and 7% acetic acid to 0.5% SDS, 0.1% bovine serum albumin, 0.1% polyvinyl decolorize for 3 to 15 hours. Eight cosmid clones having the pyrrollidone, 0.1% Ficoll 400 and 0.01% denatured salmon protease activity, which shows the bands not stained with sperm) at 68° C. for 2 hours. Then, it was transferred in a Coomassie Brilliant Blue-R-250 due to hydrolysis of gelatin similar hybridization buffer solution containing the P on the gel were selected, labeled DNA probe to allow to hybridize at 68° C. for 14 Preparation of plasmid plPR1 containing hyperthermo hours. After completion of hybridization, the membrane was stable protease gene washed with 2XSSC containing 0.5% of SDS at room Among the 8 cosmid clones having the protease activity. temperature and then 0.1XSSC containing 0.5% of SDS at one cosmid (cosmid No. 304) was selected to prepare 68 C. After rinsing the membrane with 0.1XSSC, it was cosmid DNA and the cosmid DNA was digested with SphI 55 air-dried. AX-ray film was exposed to the membrane at-80° and then ligated to Sphl site of the plasmid vector puC119. C. for 60 hours. The film was developed to prepare an This recombinant plasmids were transduced into E. coli autoradiogram. This autoradiogram showed that a protease JM109 and the protease activity of the resultant transfor gene was present in the DNA fragment of about 7.5 kb mants were tested according to the same method as that used obtained by digestion of the cosmid DNA with Not and for screening of the cosmid protein library. A plasmid was Pyu. prepared from the transformant having the protease activity Preparation of plasmid pTPR12 containing whole length and the resultant recombinant plasmid was named as of hyperthermostable protease gene pTPR1. E. coli JM109 transformed with the plasmid was The cosmid DNA used for the preparation of the above named as Escherichia JM109/pTPR1. plasmid pIPR1 was digested with Not I and Pvul and FIG. 1 illustrates a restriction map of the plasmid plpR1. 65 subjected to electrophoresis using a 0.8% agarose gel to Preparation of plasmid pIPR9 containing recover DNA fragments of about 7 to 8kball together. These hyperthermostable protease gene DNA fragments were mixed with the plasmid vectorp C19 5,756,339 17 18 into which a Not I linker was introduced at HincII site and The above plasmid pIPR15 was digested with DraI and which was digested with Not and SmaI. Then, ligation was subjected to 1% agarose gel-electrophoresis, followed by carried out. The recombinant plasmids were transduced into recovering the separated DNA fragment of about 4.8 kb, E. coli JM109 and the protease activity of the resultant Then, the plasmid vector puC19 was digested with SmaI transformants were tested by the same method as that used and, after dephosphorylation with alkaline phosphatase, it for screening of the cosmid protein library. A plasmid was was mixed with the above DNA fragment of about 4.8 kb to prepared from the transformant having the protease activity. carry out ligation and transduced into E. coli JM109. The The plasmid was named as pTPR12 and E. coli JM109 protease activity of the resultant transformants were tested transformed with the plasmid was designated as Escherichia by the same method as that used for screening of the cosmid coli JM109/pTPR12. protein library. A plasmid was prepared from a transformant FIG. 3 illustrates a restriction map of the plasmid having the activity. The plasmid was named as pTPR13 and pTPR12. E. coli JM109 transformed with the plasmid was named as Preparation of plasmid pIPR 15 containing whole length Escherichia coli JM109/pTPR13. of hyperthermostable protease gene FIG. 7 illustrates a restriction map of the plasmid The above plasmid ptPR 12 was digested with Xbaland 15 pTPR13. subjected to electrophoresis using a 1% agarose gel to recover separated two DNA fragments of about 3.3 kb and Preparation of plasmid puBP13 containing hyperthermo about 7 kb, respectively. Then, the DNA fragments of about stable protease gene for transforming Bacillus subtilis 7 kb thus recovered was digested with Kpn and again The above plasmid ptPR13 was digested with KpnI and subjected to electrophoresis using a 1% agarose gel to BamHI and then subjected to 1% agarose gel separate two fragments of about 3.2 kb and about 3.8 kb. In electrophoresis, followed by recovering the separated DNA these fragments, the DNA fragment of about 3.2 kb was fragment of about 4.8 kb. Then, the plasmid vector publ&- recovered and ligated to the plasmid vector pUC19 digested P43 was digested with KpnI and BamHI and mixed with the with Xbal and Kpn. This was transduced into E. coli above DNA fragment of about 4.8 kb to carry out ligation. JM109. Plasmids held by the resultant transformants were 25 It was transduced into Bacillus subtilis DB104. The protease prepared and the plasmid containing only one molecular of activity of the resultant transformants having kanamycin the above 3.2 kb fragment was selected. This was named as resistance were tested by the same method as that used for pTPR14. screening of the cosmid protein library. A plasmid was FIG. 5 illustrates a restriction map of the plasmid pTPR prepared from a transformant having the activity. The plas 14. 30 mid was named as pluBP13 and Bacillus subtilis DB104 Then, the above plasmid ptPR 14 was digested with Xbal transformed with the plasmid was named as Bacillus subtilis and dephosphorylated using alkaline phosphatase. This was DB1049/pUBP13. mixed with the above fragment of about 3.3 kb to carry out FIG. 8 illustrates a restriction map of the plasmid ligation and was transduced into E. coli JM109. The protease pUBP13. activity of the resultant transformants were tested by using 35 Preparation of plasmid ptPR36 containing hyperthermo the same method as that used for screening of the cosmid stable protease gene defecting in its back halfportion protein library. A plasmid was prepared from the transfor The above plasmid ptPR13 was digested with EcoRI and mant having the protease activity. This plasmid was named the resultant end was blunted with a DNA blunting kit as pTPR15 and E. coli JM109 transformed with the plasmid (manufactured by Takara Shuzo Co., Ltd.). Further, it was was named as Escherichia coli JM109/pTPR15. digested with Kpn and subjected to 1% agarose gel FIG. 6 illustrates a restriction map of the plasmid pIPR electrophoresis, followed by recovering the separated DNA 15. fragment of about 2.8 kb. Next, the plasmid vector puC119 was digested with Xbal and the resultant end was blunted EXAMPLE 2 and further digested with KpnL followed by mixing with the Determination of nucleotide sequence of hyperthermo 45 above DNA fragment of 2.8 kb to carry out ligation and stable protease gene transducing into E. coli JM109. For determination of the nucleotide sequence of the The protease activity of the resultant transformants were hyperthermostable protease gene inserted into the above tested by the same method as that used for screening of the plasmid pIPR 15, deletion mutants wherein the DNA frag cosmid protein library. A plasmid was prepared from a ment portion inserted into the plasmid had been deleted in 50 various lengths were prepared by using Kilo sequence transformant having the activity. The plasmid was named as deletion kit (manufactured by Takara Shuzo Co. Ltd.). pTPR36 and E. coli JM109 transformed with the plasmid Among them, several mutants having suitable lengths of was named as Escherichia coli JM109/pTPR36. deletion were selected and nucleotide sequences of respec FIG. 9 illustrates a restriction map of the plasmid tive inserted DNA fragment portions were determined by 55 pTPR36. SEQID NO 2 of the Sequence Listing shows the dideoxy method using BcaBEST dideoxy sequencing kit nucleotide sequence of the DNA fragment inserted into the (manufactured by Takara Shuzo Co., Ltd.). By putting these plasmid pIPR36. Also, SEQID NO 1 shows an amino acid results together, nucleotide sequences of the inserted DNA sequence of the hyperthermostable protease which can be fragment contained in the plasmid pIPR15 were deter encoded by the nucleotide sequence. mined. Among the nucleotide sequences thus obtained, SEQ ID NO 8 of the Sequence Listing shows the fragment of EXAMPLE 3 4765 bp between two Dral sites. Furthermore, SEQID NO Preparation of oligonucleotide for detection of hyperther 9 shows an amino acid sequence of the hyperthermostable mostable protease gene protease encoded by the open reading frame contained in the By comparing the estimated amino acid sequence of the above nucleotide sequence. 65 hyperthermostable protease of the present invention Preparation of plasmid pIPR13 containing hyperthermo obtained in Example 2 with amino acid sequences of known stable protease gene alkaline serine proteases originating in microorganisms, it 5,756,339 19 20 was found that there were homologous amino acid Listing shows the nucleotide sequence of the DNA fragment sequences commonly present in these enzymes. Among inserted into the plasmid p2F-4R and an amino acid them, three regions were selected and oligonucleotides to be sequence deduced from the nucleotide sequence. In the used as primers in detection of hyperthermostable protease nucleotide sequence shown by SEQNO 10 of the Sequence Listing. the sequence from the first to 21st nucleotides and genes by PCR were designed. that from the 113th to 145th nucleotides and, in the SEQNO FIGS. 10. 11 and 12 illustrate the relation among the 11 of the Sequence Listing. the sequence from the first to the amino acid sequences corresponding to the above three 32nd nucleotides and that from the 532nd to the 564th regions of the hyperthermostable protease of the present nucleotides are the sequences of the primers used in the PCR invention, nucleotide sequences of the hyperthermostable (corresponding to the oligonucleotides PRO-1F PRO-2R, protease of the present invention which encode the above regions. and the nucleotide sequences of oligonucleotides PRO-2F and PRO-4R, respectively). PRO-1F PRO-2F, PRO-2R and PRO-4R synthesized based FIG. 13 illustrates a restriction map of the plasmid p2F on the above nucleotide sequences. Also, SEQ NO. 3. 4.5 4R. and 6 show nucleotide sequences of PRO-1F PRO-2F, Screening of protease gene originating in Thermococcus PRO-2R and PRO-4R, respectively. 15 celer Preparation of genomic DNA of Thermococcus celer The above genomic DNA of Thermococcus celer was partially digested with Sau3AI and was treated with Klenow Microbial cells were collected from 10 ml of a culture fragment (manufactured by Takara Shuzo Co. Ltd.) in the broth of Thermococcus celer DSM2476 obtained from Deut presence of dATP and dGTP to partially repair the DNA sch Sammlung von Microorganismen und Zellkulturen ends. The DNA fragments were mixed with a lambda GmbH by centrifugation and suspended in 100 ul of 50 mM GEM-11 XhoI half site arm vector (manufactured by Tris-HCl (pH 8.0) containing 25% sucrose. To the suspen Promega) to carry out ligation. Then, they were subjected to sion were added 20 ul of 0.5M EDTA and 10 ul of lysozyme in vitro packaging using Gigapack Gold to prepare a lambda (10 mg/ml) and the suspension was incubated at 20° C. for phage library containing genomic DNA of Thermococcus 1 hour. To this were added 800 ul of SET solution (150 mM celer. Apart of the library was transduced into E. coli LE392 NaCl. 1 mM EDTA. 20 mM Tris-HCl, pH 8.0), 50 pil of 10% 25 to form plaques on a plate and the plaques were transferred SDS and 10 pil of Proteinase K (20 mg/ml) and the suspen on a Hybond-N'-membrane. After transfer, the membrane sion was further incubated at 37° C. for 1 hour, Chloroform was treated with 0.5 N NaOH containing 1.5M NaCl and phenol extraction was carried out to stop the reaction. The then 0.5M Tris-HCl (pH 7.5) containing 3M NaCl. Further. reaction mixture was subjected to ethanol precipitation and it was washed with 6XSCC. air-dried and irradiated with UV recovered DNA was dissolved in 50 ul of TE buffer solution 30 light on a UV transilluminator to fix phage DNA on the to obtained a genomic DNA solution. membrane. Detection of hyperthermostable protease by PCR On the other hand, the plasmid p2F-4R was digested with A PCR reaction mixture was prepared from the above PmaCI (manufactured by Takara Shuzo Co. Ltd.) and Stu genomic DNA of Thermococcus celer and the oligonucle (manufactured by Takara Shuzo Co., Ltd.) and subjected to otides PRO-1F and PRO-2R or the oligonucleotides PRO 35 1% agarose gel-electrophoresis to recover the separated 2F and PRO-4R and a PCR reaction (one cycle: 94° C. for DNA fragment of about 0.5 kb. By using this fragment as a 1 minute-55° C. for 1 minute-72° C. for 1 minute, 35 template and using a random primer DNA labeling kit Ver2 cycles) was carried out. When aliquots of the reaction mixture were subjected to agarose gel-electrophoresis, ando-PdCTP, a P-labeled DNA probe was prepared. amplification of three DNA fragments in case of using the The above membrane having DNA fixed thereon was oligonucleotides PRO-1F and PRO-2R and one DNA frag treated in a hybridization buffer solution (6xSSC containing ment in case of using the oligonucleotides PRO-2F and 0.5% SDS, 0.1% bovine serum albumin, 0.1% polyvinyl PRO-4R was observed. These amplified fragments were pyrrolidone, 0.1% Ficoll 400 and 0.01% denatured salmon recovered from the agarose gel and their DNA ends were sperm DNA) at 50° C. for 2 hours. It was transferred to the blunted by a DNA blunting kit, followed by phosphorylating same buffer solution containing the P-labeled DNA prove thereof with T4 polynucleotide kinase (manufactured by 45 and hybridization was carried out at 50° C. for 15 hours. Takara Shuzo Co., Ltd.). Then, the plasmid vector puC18 After completion of hybridization, the membrane was was digested with HincII and subjected to dephosphoryla washed with 2XSSC containing 0.5% SDS at room tempera tion with alkaline phosphatase. It was mixed with the above ture and then 1XSSC containing 0.5% SDS at 50° C. Further, PCR amplified DNA fragments to carry out ligation and then after rinsing the membrane with 1XSCC, it was air-dried and transduced into E. coli JM109. Plasmids were prepared from 50 a X-ray film was exposed thereto at -80° C. for 6 hours to the resultant transformants and plasmids into which suitable prepare an autoradiogram. About 4,000 phage clones were DNA fragments were inserted were selected. Nucleotide screened. As a result, one phage clone containing a protease sequences of the inserted DNA fragments were determined gene was obtained. Based on the signal on the by dideoxy method. Among these plasmids, regarding a autoradiogram, the position of this phage clone was found plasmid p1-2R(2) containing a DNA fragment of about 150 and the plaque corresponding on the plate used for transfer bp which was amplified by using the oligonucleotides PRO 55 to the membrane was isolated into 1 ml of SM buffer 1F and PRO-2R and a plasmid p2F-4R containing a DNA solution 50 mM Tris-HCl, 0.1M NaCl, 8 mM MgSO, fragment of about 550 bp which was amplified by using the 0.01% gelatin (pH 7.5) containing 1% of chloroform. oligonucleotides PRO-2F and PRO-4R, it was found that Detection of phage DNA fragment containing protease amino acid sequences estimated from the thus-obtained gene nucleotide sequences contained sequences having homology The above phage clone was transduced in to E. coli with the amino acid sequences of the hyperthermostable LE392 and the transformant was cultured in NZCYM protease originating in Pyrococcus furiosus of the present medium (manufactured by Bio 101) at 37° C. for 15 hours invention, subtilisin and the like, to obtain a culture broth. A supernatant of the culture broth SEQNO 10 of the Sequence Listing shows the nucleotide was collected and phage DNA was prepared by using sequence of the DNA fragment inserted into the plasmid 65 QIAGEN-lambda kit (manufactured by DIAGEN). The p1F-2R(2) and an amino acid sequence deduced from the resultant phage DNA was digested with BamHI, EcoRI, nucleotide sequence. Also, SEQ NO 11 of the Sequence EcoRV. HincII, KpnI, Nicol, Pst, SacI, Sall. SmaI and Sph 5,756.339 21 22 (all manufactured by Takara Shuzo Co., Ltd.), respectively. oligonucleotides as primers and the plasmid pTC3 as a and subjected to 1% agarose-electro-phoresis. Then, a mem template were summarized to determine the nucleotide brane on which DNA fragments were fixed was prepared by sequence of the hyperthermostable protease gene. the same method as that used for the detection of the DNA SEQID NO 7 of the Sequence Listing shows a part of the fragment containing the whole length of the hyperthermo 5 resultant nucleotide sequence. In addition, SEQID NO 18 of stable protease gene of Example 1. The membrane was the Sequence Listing shows an deduced amino acid treated in a hybridization buffer solution at 50° C. for 4 hours sequence encoded by the nucleotide sequence. and then transferred to the same hybridization buffer solu tion containing the same 'P-labeled DNA probe as that used EXAMPLE 4 in the above screening of the protease gene derived form Thermococcus celer. Then, hybridization was carried out at O Preparation of enzyme sample Escherichia coli JM109/ 50° C. for 18 hours. After completion of hybridization, the pTPR36 which was E. coli JM109 into which the plasmid membrane was washed with 1XSSC containing 0.5% SDS at pTPR36 containing the hyperthermostable protease gene of 50° C. and rinsed with 1xSCC. The membrane was air-dried the present invention obtained in Example 2 was transduced and exposed to a X-ray film at-80° C. for 2 hours to prepare was cultured with shaking in 5 ml of LB medium (trypton 10 an autoradiogram. According to this autoradiogram, it was 5 g/liter, yeast extract 5 g/liter, NaCl 5 g/liter, pH 7.2) con found that, in the phage DNA digested with KpnI, the taining 100 ug/ml of amplicillin at 37° C. for 14 hours. In a protease gene was contain in a DNA fragment of about 9 kb. 1 liter-Erlenmeyer flask, 200 ml of the same medium was Preparation of plasmid pTC1 containing protease gene prepared and 2 ml of the above culture broth was inoculated The above phage DNA containing the protease gene was and cultured with shaking at 37° C. for 10 hours. The culture digested with KpnI and subjected to 1% agarose gel broth was centrifuged. The harvested microbial cells (wet electrophoresis to recover a DNA fragment of about 9 kb weight 1.6 g) were suspended in 2 ml of 20 mM Tris-HCl from the gel. Then, the plasmid vectorpUC119 was digested (pH 8.0), sonicated and centrifuged to obtain a supernatant. with Kpnl and dephosphorylated with alkaline phosphatase. The supernatant was treated at 100° C. for 5 minutes and followed by mixing with the above DNA fragment of about centrifuged again. The resultant Supernatant was used as a 9 kb to carry out ligation. Then, it was transduced into E. coli 25 crude enzyme solution (enzyme sample PF-36). JM109. Plasmids were prepared from the resultant transfor In addition, according to the same manner, Escherichia mants and a plasmid containing only the above DNA frag coli JM109?pTPR13 which was E. coli JM109 into which ment of about 9 kb was selected. This plasmid was named the plasmid pTPR13 containing the hyperthermostable pro as pTC1 and E. coli JM109 transformed with the plasmid tease gene of the present invention was transduced was used was named with Escherichia coli JM109/pTC1. 30 to prepare a crude enzyme solution (enzyme sample PF-13). FIG. 14 illustrates a restriction map of the plasmid ptC1. Moreover. Bacillus subtilis DB104/pUBP13 which was Preparation of plasmid pTC3 containing hyperthermo Bacillus subtilis DB104 into which the plasmid pluBP13 stable protease gene containing the hyperthermostable protease gene of the The above plasmid prC1 was digested with KpnI and present invention was transduced was cultured with shaking further digested with BamHI, Pst and SphI, respectively. 35 in 5 ml of LB medium containing 10 pg/ml of kanamycin at After subjecting to 1% agarose gel-electrophoresis, accord 37° C. for 14 hours. In two 2 liter-Erlenmeyer flasks, ing to the same operation as that for detecting the phage respective 600 ml of the same mediums were prepared. To DNA fragment containing the above protease gene, transfer each flask was inoculated with 2 ml of the above culture of DNA fragments to a membrane and detection of DNA broth and cultured with shaking at 37° C. for 26 hours. The fragments containing the hyperthermostable protease gene culture broth was centrifuged. The resultant microbial cells were carried out. By the signal on the resultant were suspended in 15 ml of 20 mM Tris-HCl (pH 8.0). autoradiogram, it was shown that a DNA fragment of about sonicated and centrifuged to obtain a supernatant. The 5 kb which obtained by digesting the plasmid pTC1 with supernatant was treated at 100° C. for 5 minutes and Kpn and BamHI contained the hyperthermostable protease centrifuged again. To the resultant supernatant was added gene, ammonium sulfate to 50% saturation and then the resultant 45 precipitate was recovered by centrifugation. The recovered Then, the plasmid plC1 was digested with KpnI and precipitate was suspended in 2 ml of 20 mM Tris-HCl (pH BamHI and then subjected to 1% agarose gel 8.0) and the suspension was dialyzed against the same buffer electrophoresis to separate and isolate a DNA fragment of solution. The resultant inner solution was used as a partially about 5kb. The plasmid vector puC119 was digested with purified enzyme sample (enzyme sample PF-BS13). KpnI and BamHI and mixed with the above DNA fragment of about 5 kb to carry out ligation. It was transduced into E. 50 The protease activity of these enzyme samples and the coli JM109. Plasmids were prepared form the resultant cosmid clone lysate used for preparation of plasmids were transformants and a plasmid containing the above DNA tested according to the above method for detection of fragment of about 5 kb. This plasmid was named as pTC3 enzyme activity using SDS-polyacrylamide gel containing and E. coli JM109 transformed with the plasmid was named gelatin. as Escherichia coli JM109/pTC3. 55 FIG. 16 illustrates the thermostability of the hyperther FIG. 15 illustrates a restriction map of the plasmidpTC3. mostable protease obtained by the present invention. And, Determination of nucleotide sequence of hyperthermo FIG. 17 illustrates the optimum pH of the hyperthermostable stable protease gene contained in Plasmid ptC3 protease obtained by the present invention. Further, FIG. 18 For determination of the nucleotide sequence of the illustrates the results of activity staining after SDS hyperthermostable protease gene contained in the above polyacrylamide gel electrophoresis of each sample (enzyme plasmid pTC3. 6 oligonucleotides were synthesized based samples PF-36, PF-13 and PF-BS13 and the lysate). Each on the nucleotide sequences shown by SEQID NO 10 and sample shows activity at 95° C. in the presence of SDS. 11 of the Sequence Listing, respectively. The nucleotide As described hereinabove, according to the present sequences of the synthesized oligonucleotides TCE-2. TCE invention, genes encoding hyperthermostable proteases 4, SEF-3, SER-1. SER-3 and TCE-6R were shown by SEQ 65 which show activity at 95°C. were obtained. These genes ID NO 12, 13. 14, 15, 16 and 17 of the Sequence Listing. make possible to supply a large amount of a hyperthermo The results obtained by dideoxy method using the above stable protease having high purity. 5,756,339 23

SEQUENCE LISTING

1 ) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 18

( 2) INFORMATION FOR SEQID NO:1: ( i SEQUENCE CHARACTERISTICS: A LENGTH:903 amino acids (B) TYPE: amino acid ( Cy STRANDEDNESS: single ( D ). TOPOLOGY: linear { i i y MOLECULETYPE: peptide ( x i ) SEQUENCE DESCRIPTION: SEQID NO:1: Le u L e u Mc t As in y s Ly is G ly L e u her Wa P he I 1 e A la I le Me 5 1 0

Wa 1 W a P o Wa H is P he Wa A 1 a G u Th r P o P o 20 3 0

G 1 u As S. c. r Th I h r As in G in G n Wa Wa 1 Th 3 5 4 5

G 1 u Va. 1 G in A l a A a A 1 a I e Me t Lys Gly G 1 a 50 55 6 0.

A s in Me t W a 1 L C u I c Th G 1 u G y L. y s L. e. to G G 1 u A a 65 7 5 8 O

Thr G G 1 u Gly A a G u I 1 e Le u A sp G 1 u A rig 85 9 O

As Me t e u G 1 u Lys Wall G u 1 O O 1 1 0

As a I l e G u A 1 a G 1 Wa 2 O 25

Let u Se I e Wa G A sp Ly is Thr G 1 u 13 O 1 35 1 4 0

S e r G P o Me Thr Wa l l l e A a L e 1 60 1 4 5 1 5 0

G 1 a P he G u P he Gly A sp G 1 y G 1 y V a A 1 a 65 1 O

Let u As G 1 y Wa A s r. H is Phi e L. eu T 8 0 85

A rig A rig e c G A s p P he As p G u 1 9 2 O 5

Gly As p Th P e P he Wa V a 1 As a G 1 y Th 2 5 2 : 0

1 As in Thr Th it P he G n W a 1 A l a S e G 1 y Le u T b . As in 225 2 30 23 5 2 4

G Th Gly Le u Me t G u Wa 1 Th Wa 1 Wa 2 4 5 25 255

As n Wa 1 Th G 1 y As n I le Thr Ser A 1 a As n G 1 y I 1 e His P be 2 65

G 1 y cu Pro G u A rig P he As p As n Ph e A sp As p G in 27 28 0 2. 85

G 1 u Ph Wa Le u Le u Va. 1 As in Thr G 1 y As a G 1 y 2 9 5 3 O O

A sp A 1 Wa As p Thr A sp As p A s p P he T r A sp G 1 u 3 2 O 3 O 5 3 10

5,756,339 37 38 -continued

T C T C CAATTT TT C C CAC TTT TT CTTT TATA A CAT T C CA AG cc TT TT CTTA G. CTTC TT C GC 43 8 0.

T CAT CTATC A G GA GT C CAT 6 GAG GAT CAA A G G T AA GT TC AA C C T C CA CA T C T C T TACT C 4 4 4 0

C T G G GATT T. C. GA GT ACT TTC T C C T CTA CAG C T C T A A GAA G C CA GAGA GT T AAA G GA CA C C 45 0 0

CAGGA GT T G T CAT T G T CAT C TT TATA TATA CC GT TTT G TC A G GATT AATC TT TAG C T CAT 45 6

AAAT AAT C C AAG GT T TACA A CAT C CAT C C CAATT T C T G G GT CGAT AAC C T C C T T TAG CT 4. 6 20

TT T C C A GAA T CAT TT C T CA GT AA TTT CAA G G T C T CAT C T TT G GT TT CT CT CA CAAA C C 4 6 80

(CAATTT CAA C C T G C C T GATA CCTT CTA ACT C C CTA A G CT GT TATATATC T C CAA AA GA G 47 40

T C G CAT CAC AATTTT C T CT T TAAA 4 65

( 2) INFORMATION FOR SEQID NO:9: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 1398 amino acids (B) TYPE: amino acid ( Cy STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( x i y SEQUENCE DESCRIPTION: SEQID NO:9: Me t As in Lys Lys G y L. eu Thr Wa 1 Le u Ph e I e A a 1 e Me t Le u L. eu 5 1 O 5

Ser Wal Wa 1 Pro Wa His Phe Wa 1 Se T A 1 a G Th P o Pro Wa 20 25 30

Ser G u As n Ser Th Th r S e r e e u Pro As in G G1 in Wa Wa Thr 35 4 0 4 5 Lys G i u Wa 1 Ser G in A a A 1 a eu As in A a I e Me t Ly s Gl y G. in 5 O 55 6 0

As in Me t w a 1 Le u e 1 1 e Ly s Th r Lys G 1 u G 1 y Lys Le u G 1 u G 1 u A a 65 7 O 7 5 8 O Lys Thr G 1 u Le u Gi i u Lys Le u G 1 y Al a G 1 u I l e L. eu As p Glu As n A T g 85 9 O 95

Wa 1 Le u As n Met Le u Le u Wa 1 Lys 1 e Lys Pro G 1 u Ly s Wa l l y s G u 0 0 O 5 1 0. Le u As in Tyr I le S e T S e r le u G 1 u y is A 1 a Trip L. eu As in Arg G u Wa 5 2 O 2 5 Lys Le u Se r P T o Pro l l e V a 1 G 1 u Lys As p V a Lys Th T Lys Gil u 13 O 1 35 1 4 0

Ser Le u G i u Pro Lys Me t Ty r As a Ser Thr Trip v a 1 I 1 e A s in A a L C u 1 4 5 1 5 O 1 55 6 0 G 1 in Ph e I le G 1 in G i u P he Gly Ty r A s p G 1 y Ser G 1 y V a Wa Wa A la 65 7 O 1 75 Wa 1 Le u A s p Thr G y Wa 1 A sp Pro A. s in H is Pro Phi e Le S e r e Th 1 8 O 8 S 9 () Pro A s p G y Arg Arg Lys I le I e G 1 u Trip y s A s p Ph e Thr As p G u 1 95 20 0 2 O 5 G 1 y P he wa 1 A s p Th r S e r h Ser Phi e S e r Ly is Val V a 1 A s in G 1 y T b . 2 1 0 2 5 22 0

Le u e I e As in Th T Thir Phe G Wa A 1 a Ser G y Le u Thir Le u A s in 225 23 O 235 2 4 O Glu Ser Thr Gil y Leu Me t c 1 u Ty r V a l V a Ly is Thr V a Ty r Wa S e 2 4 5 25 0 255 As n w a 1 Thir I le G 1 y As n I e Th r S e r A a As in Gly I e Ty r H is Phe 2 6 O 2 65 2 7 O

5,756,339 47 48 -continued

( x i ) SEQUENCE DESCRIPTION: SEQID NO:12:

G G CAAG GT CA TA G GCT G G T A. 2 O

( 2) INFORMATION FOR SEQID No:13: ( i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 20 base pairs ( B. ) TYPE: nucleic acid C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: cDNA ( x i SEQUENCE DESCRIPTION: SEQID NO:13:

C CA GAA CAAG GATA AG TAC G 20

( 2 NFORMATION FOR SEQID NO:14: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single (d) IOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA ( xi ) SEQUENCE DESCRIPTION: SEQID No:14:

GGCA C C C C GA. T AAA CGACTA 2 O

( 2) INFORMATION FOR SEQID No:15: ( i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear { i i MOLECULETYPE: cDNA ( xi ) SEQUENCE DESCRIPTION: SEQED NO:15:

A C GC CTA T G T A C T G G GA GT 20

( 2) INFORMATION FOR SEQID No:16: ( i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i MOLECULETYPE. cDNA ( x i SEQUENCE DESCRIPTION: SEQID NO:16:

C GT ACT TAT C C T T G T T C T G G 2 O

( 2) INFORMATION FOR SEQID NO:17: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE. cDNA ( x i SEQUENCE DESCRIPTION: SEQID No:17:

T G TAG TA GT C GT T TAT CGGG 2 O 5,756,339

-continued

( 2) INFORMATION FOR SEQID No:18: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 amino acids B. ) TYPE: amino acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear

( i i MOLECULE TYPE: cDNA ( x i SEQUENCE DESCRIPTION: SEQED NO:18: A s p L. eu Lys G 1 y Lys w a I e Gil y Trip Ty r A s p A 1 a Wa i As in G 1 y Arg 1. 5 1 0 1 5 S e r Thr Pro Ty r A s p A s p G 1 in G y H is G 1 y T hr H is V a i Al a G 1 y i. 1 e 2 O 25 3 O W a 1 A 1 a G 1 y Thr G 1 y S e r V a As n Ser G 1 in Tyr I 1 e Gil y V a l A 1 a P ro 3 5 4 0 45 G 1 y A a Lys Le u v a 1 G 1 y V a l Lys v a 1 Le u G 1 y A 1 a. A s p G 1 y Ser G 1 y 50 55 60 Ser Wa 1 Ser Thr I 1 e I 1 e A 1 a G 1 y Wa A s p Trip V a Wa 1 G1 in As n Lys 65 0. 7 5 8 A s p Xa a Tyr G 1 y I 1 e Arg w a I e A s in Le u Ser Le u G 1 y Ser Ser G 1 in 85 9 O 95 S e r S e r A s p G 1 y Th r A s p Ser Le u Ser G 1 m A a V a 1. As in As in A 1 a T r p 1 0 0 1 O 5 1 1 0 A s p A 1 a G 1 y | 1 e V a 1 v a 1 Cy s w a 1 A 1 a. A l a G y As n Ser G 1 y Pro As in 1 15 1 20 25 T b r Ty r Thr Val G 1 y Ser Pro A 1 a. A 1 a. A 1 a Ser Lys Wa l l l e T b r Wal 3 0. 35 4 O G l y A a V a A s p Ser As n A s p As n 1 e Al a Set r Pb e S e r S e r A rig Gly 45 50 155 16 O Pro Th r A 1 a. As p G 1 y Arg Le u Lys Pro G 1 u v a 1 Val A 1 a Pro G 1 y V a 1 1 65 1 7 O 1 75 A s p I e e A 1 a Pro Arg A 1 a Ser G 1 y T h r S er Met G 1 y Thr Pro I le 180 18, 5 90 As in A s p Ty r Xa a A s in Lys G 1 y Ser G 1 y Ser Ser Me t A s p Thr Pro H is 95 20 0 2 OS w a Ser G 1 y V a 1 G1 y G 1 y e u 1 e Le u G 1 in A 1 a H is Pro Ser T r p Thr 2 0 2 15 2 20 Pro A s p Lys w a Lys Thr Pro Ser Ser Arg Pro Pro Thr 225 2 30 235

50 What is claimed is: 5. A hyperthermostable protease gene of claim 4 which 1. An isolated hyperthermostable protease gene originat comprises the nucleotide sequence represented by the SEQ ing in Pyrococcus fiariosus. ID NO 7. 2. A hyperthermostable protease gene of claim 1 which 6. A process for producing a hyperthermostable protease encodes the amino acid sequence of SEQ ID NO: 1 or an 55 which comprises culturing a transformant transformed with enzymatically active fragment thereof. a plasmid into which the hyperthermostable protease gene of 3. A hyperthermostable protease gene of claim 1 which claim 1 has been transduced, and collecting the hyperther comprises the nucleotide sequence represented by the SEQ mostable protease from the culture. ID NO 2 in the Sequence Listing. 7. A process for producing a hyperthermostable protease 4. A hyperthermostable protease gene which is the hybrid which comprises culturing a transformant transformed with izable with the hyperthermostable protease gene of claim 2 a plasmid into which the hyperthermostable protease gene of or DNA selected from the group consisting of the nucleotide claim 4 has been transduced, and collecting the hyperther sequences represented by SEQ ID NO 3, 4, 5 and 6 in the mostable protease from the culture. Sequence Listing which are part of the hyperthermostable protease gene of claim 1.