Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-05-20
2001-07-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C536S023200
Reexamination Certificate
active
06261822
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a hyperthermostable protease useful as an industrial enzyme, a gene encoding the same and a method for preparation of the enzyme by the genetic engineering.
BACKGROUND ART
The proteases are the enzymes which cleave peptide bonds in the proteins, and a number of the 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 synthesis utilizing the 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 thermostabilities 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 thermostability.
However, enzymes having further superior properties are desired and activities have been attempted to obtain enzymes from microorganisms which grow at high temperature, 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 environments and therefor they are expected to be a source supplying various thermostable enzymes. It has been known that one of these hyperthermophiles,
Pyrococcus furiosus
, produces proteases [Appl. Environ. Microbiol., volume 56, page 1992-1998 (1990), FEMS Microbiol. Letters, volume 71, page 17-20 (1990), J. Gen. Microbiol., volume 137, page 1193-1199 (1991)].
A hyperthermophile belonging to the genus Pyrococcus, Pyrococcus sp. Strain KOD1 is reported to produce a thiol protease (cysteine protease) [Appl. Environ. Microbiol., volume 60, page 4559-4566 (1994)]. Bacteria belonging to the genus Thermococcus, Staphylothermus and Thermobacteroides, which are also hyperthermophiles, are known to produce a protease [Appl. Microbiol. Biotechnol., volume 34, page 715-719 (1991)].
OBJECTS OF THE INVENTION
As the proteases produced by these hyperthermophiles have high thermostabilities, they are expected to be applicable to new applications to which any known enzymes has not been utilized. However, the above publication merely teach that thermostable protease activities present in cell-free extract or crude enzyme solution obtained from culture supernatant, and there is no disclosure about properties of isolated and purified enzymes and the like. Only a protease produced by strain KOD1 is obtained as the purified form. However, since a cysteine protease has the defect that it easily loses the activity by oxidation, it is disadvantageous in the industrial use. In addition, 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 provide a protease of the hyperthermophiles which is advantageous in the industrial use, to isolate a gene encoding a protease of the hyperthermophiles, and to provide a method for preparation of a hyperthermostable protease using the gene by the genetic engineering.
DISCLOSURE OF THE INVENTION
In order to obtain a hyperthermostable protease gene, the present inventors originally tried to purify a protease from microbial cells and a culture supernatant of
Pyrococcus furiosus
DSM3638 so as to determine a partial amino acid sequence of the enzyme. However, purification of the protease was very difficult in either cases of using the microbial cells or the culture supernatant, and the present inventors failed to obtain such an enzyme sample having sufficient purity for determination of its partial amino acid sequence.
As a method for cloning a gene for an objective enzyme without any information about a primary structure of the enzyme protein, there is an expression cloning method. For example, a pullulanase gene originating in
Pyrococcus woesei
(WO92/02614) has been obtained according to this method. However, in an expression cloning method, a plasmid vector is generally used and, in such case, it is necessary to use restriction enzymes which can cleave an objective gene into relatively small DNA fragments so that the fragments can be inserted into the plasmid vector without cleavage of any internal portion of the objective gene. Therefore, the expression cloning method is not always applicable to cloning of all kind of enzyme genes. Furthermore, it is necessary to test for an enzyme activity of a large number of clones and this operation is complicated.
The present inventors have attempted to isolate a protease gene by using a cosmid vector which can maintain a larger DNA fragment (30-50 kb) instead of a plasmid vector to prepare a cosmid library of
Pyrococcus furiosus
genome and investigating cosmid clone in the library to find out a clone expressing a protease activity. By using the cosmid vector, the number of transformants to be screened can be reduced in addition to lowering of possibilities of cleavage of a internal portion of the enzyme gene. On the other hand, since the copy number of a cosmid vector in a host cell is not higher than that of a plasmid vector, it may be that an amount of the enzyme expressed is too small to detect it.
In view of high thermostability of the objective enzyme, firstly, the present inventors have cultured respective transformants in a cosmid library, separately, and have combined this step with a step for preparing lysates containing only thermostable proteins from the microbial cells thus obtained, and used these lysates for detecting the enzyme activity. Further, the use of the gelatin-containing SDS-polyacrylamide gel electrophoresis for detecting the protease activity allowed the detection of a trace amount of the enzyme activity.
Thus, the present inventors obtained several cosmid clones expressing the protease activity from the cosmid library of
Pyrococcus furiosus
and successfully isolated a gene encoding a protease from the inserted DNA fragment contained in the clones. In addition, the present inventors confirmed that a protease encoded by the gene has the extremely high thermostability.
By comparing an amino acid sequence of the hyperthermostable protease deduced from the nucleotide sequence of the gene with amino acid sequences of known proteases originating in microorganisms, homology of the amino acid sequence of the front half portion of the hyperthermostable protease with those of a group of alkaline serine proteases, a representative of which is subtilisin, has been shown. In particular, the extremely high homology has been found at each region around the four amino acid residues which are known to be important for the catalytic activity of the enzyme. Thus, since the protease produced by
Pyrococcus furiosus
, which is active at such a high temperature that proteases originating in mesophiles are readily inactivated, has been shown to retain a structure similar to those of enzymes from mesophiles, it has been suggested that similar proteases would also be produced by hyperthermophiles other than
Pyrococcus furiosus.
Then, the present inventors have noted possibilities that, in the nucleotide sequence of the hyperthermostable protease gene obtained, the nucleotide sequence encoding regions showing high homology with subtilisin and the like can be used as a probe for detecting hyperthermostable protease gene, and have attempted to detect protease genes originating in hyperthermophiles by PCR using synthetic DNAs designed based on the nucleotide sequences as primers so as to clone the gene. As a result, it was found that a fragment of gene having the homology with the above gene existed in a hyperthermophile,
Thermococcus celer
DSM2476. The cloning of the full length of the gene was difficult and this was thought to be due to tha
Asada Kiyozo
Kato Ikunoshin
Mitta Masanori
Morishita Mio
Takakura Hikaru
Browdy & Neimark
Prouty Rebecca E.
Rao Manjunath N.
Takara Shuzo Co. Ltd.
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