Hyperthermostable protease gene

Details Hyperthermostable protease gene Hyperthermostable protease gene

1996-12-13

1998-05-26

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435219, 4352523, 4353201, 435325, 536 232, C07H 2104, C12N 120, C12N 950, C12N 952

Patent

active

057563390

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to a gene encoding a hyperthermostable protease which is useful as an enzyme for industrial application and a process for producing the enzyme by genetic engineering.


BACKGROUND OF THE INVENTION

Proteases are enzymes which cleave peptide bonds in proteins and various proteases have been found in animals, plants and microorganisms. They are used not only as reagents for research works and medical supplies, but also in industrial fields such as additives for detergents, food processing and chemical syntheses utilizing their reverse reactions and it can be said that they are very important enzymes from an industrial viewpoint. For proteases to be used in industrial fields, since very high physical and chemical stabilities are required, in particular, enzymes having high thermostability are preferred to use. At present, proteases predominantly used in industrial fields are those produced by bacteria of the genus Bacillus because they have relatively high thermostabilities.
However, enzymes having further superior properties are desired and activities have been attempted to obtain enzymes from microorganisms which can grow at high temperatures, for example, thermophiles of the genus Bacillus.
On the other hand, a group of microorganisms, named as hyperthermophiles, are well adapted themselves to high temperature environment and therefore they are expected to be supply sources for various thermostable enzymes. It has been known that one of these hyperthermophiles, Pyrococcus (1990); FEMS Microbiol. Letters, 71, 17-20 (1990); J. Gen. Microbiol., 137, 1193-1199 (1991)!.
In addition, as for hyperthermophiles of the genera Thermococcus, Staphylothermus and Thermobacteroides, the production of proteases have (1991)!.


OBJECTS OF THE INVENTION

Since proteases produced by these hyperthermophiles have high thermostabilities, they are expected to be applicable to new applications to which any known enzyme has not been utilized. However, the above publications merely teach that thermostable protease activities are present in cell-free extracts or crude enzyme solutions obtained from culture supernatants and there is no disclosure about properties of isolated and purified enzymes and the like. Moreover, since a cultivation of microorganisms at high temperature is required to obtain enzymes from these hyperthermophiles, there is a problem in industrial production of the enzymes.
In order to solve the above problems, an object of the present invention is to isolate a gene encoding a protease of a hyperthermophile. Another object of the present invention is to provide a process for producing the protease by genetic engineering using the gene.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a restriction map of the plasmid pTPR1.
FIG. 2 illustrates a restriction map of the plasmid pTPR9.
FIG. 3 illustrates a restriction map of the plasmid pTPR12.
FIG. 4 illustrates comparison of restriction maps of DNA's derived from Pyrococcus furiosus contained in the plasmids.
FIG. 5 illustrates a restriction map of the plasmid pTPR15.
FIG. 6 illustrates a restriction map of the plasmid pTPR15.
FIG. 7 illustrates a restriction map of the plasmid pTPR13.
FIG. 8 illustrates a restriction map of the plasmid pUBR13.
FIG. 9 illustrates a restriction map of the plasmid pUBR36.
FIG. 10 illustrates a design of an oligonucleotide PRO-1F.
FIG. 11 illustrates designs of oligonucleotides PRO-2F and PRO-2R.
FIG. 12 illustrates a design of an oligonucleotide PRO-4R.
FIG. 13 illustrates a restriction map of the plasmid p2F-4R.
FIG. 14 illustrates a restriction map of the plasmid pTC1.
FIG. 15 illustrates a restriction map of the plasmid pTC3.
FIG. 16 illustrates thermostability of the hyperthermostable protease obtained in the present invention.
FIG. 17 illustrates the optimum pH of the hyperthermostable protease obtained in the present invention.
FIG. 18 illustrates the activity staining pattern after gelatin-containing SDS-polyacrylamide gel electrophoresis of the hyperthermostable prote

REFERENCES:
patent: 5242817 (1993-09-01), Kelly et al.
patent: 5391489 (1995-02-01), Kelly et al.
Ilse I. Blumentals et al, "Charaterization of Sodium Dodecyl Sulfate-Resistant Proteolytic Activity in the Hyperthermophilic Archaebacterium",Applied and Environmental Microbiology, vol. 56, pp. 1992-1998, 1990.
Rik Eggen et al, "Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacteruim Pyrococcus furiosus",FEMS Microbiology Letter, vol. 71, pp. 17-20, 1990.
Helen Connaris et al, "Heterogeneity of proteinases from the hyperthermophilic archaeobacterium pyrococcus furiosus", Journal of General Microbiology, vol. 137, pp. 1193-1199, 1991.
Michael Klingegerg et al, "Properties of extremely thermostable proteeases from anaerobic hyperthermophilic bacteria", Applied Microbiology and Biotechnology, vol. 34, pp. 715-719, 1991.
Roland J. Siezen et al, "Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases", Protein Engineering, vol. 4, pp. 719-737 1991.
Robinson et al. (1995) A gene from the hyperthermophile Pyrococcus furiosus whose deduced product is homologous to members of the prolyl oligopeptidase family of proteases, Gene 152: 103-106, Jan. 11, 1995.
Voorhoorst et al. (1996) Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus, J. Biol. Chem. 271 (34): 20426-20431, Aug. 23, 1996.

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