Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-09-19
2000-08-15
Nashed, Nashaat T.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435221, 536 232, C12N 950, C12N 954
Patent
active
061035122
DESCRIPTION:
BRIEF SUMMARY
This is a U.S. national stage application of PCT/NL95/00037 filed Jan. 26, 1995.
The present invention relates to metallo-endopeptidases, also called neutral proteases (NP's), that are produced, processed and secreted by members of the bacterial genus Bacillus. More specifically, it relates to the use of genetic manipulation techniques to alter genes encoding neutral proteases, such that the thermostability of the enzymes encoded by these genes is increased. The altered genes are also a part of the present invention, as are vectors and cells comprising said genes. The invention also relates to the production, processing and secretion of more stable neutral proteases derived from B. stearothermophilus by a B. subtilis strain carrying genes encoding these enzymes. More specifically, this invention relates to the introduction of a series of specific site-directed mutations in the gene encoding the neutral protease of B. stearothermophilus, which increase the thermostability of the product encoded by this gene. It also relates to a general analysis of the structural elements that determine the thermostability of the neutral protease of B. stearothermophilus and to the application of the results of this analysis for the stabilization of other neutral proteases, in particular thermolysin. The altered genes coding for the more stable thermolysin, as well as cells and vectors comprising such genes are also disclosed in the present invention. The invention also relates to the products of such genes, vectors and cells.
A general problem in the industrial use of enzymes is the stability of these catalysts. Enzymes are expensive and should preferably be usable in as low quantities and in as many process cycles as feasible. It is often desirable to conduct industrial processes at elevated temperatures (Nosoh and Sekiguchi, 1990; Kristjansson, 1989; Geisow, 1991). This limits the use of enzymes, since many enzymes do not sufficiently tolerate temperatures outside the physiological range. Thermostable enzymes are often more stable in general (Sonnleitner and Fiechter, 1983; Nosoh and Sekiguchi, 1990): they are more stable at any temperature and they have a higher resistance towards other denaturing factors such as extreme pH values, detergents and high salt concentrations. The availability of thermostable enzymes is therefore clearly desirable for the following reasons: conducted is expanded. is the preparation of the artificial sweetener aspartame (Gerhartz, 1990; Isowa et al., 1979). NP's are also employed in the leather and baking industry, in breweries, and in the production of protein hydrolysates in the leather industry and in breweries (Gerhartz, 1990). At present, mostly thermolysin (e.g. in the preparation of aspartame) and the neutral protease of Bacillus subtilis (e.g. for beer-brewing applications) are used in industrial processes. The costs of these processes could be reduced if more stable NP variants would be available. A recent paper by Kubo et al. (1992) is an illustration of the interest of the aspartame industry in this matter.
Several Bacilli are known to produce extra-cellular metallo-endopeptidases, also called neutral proteases. These enzymes contain 300-319 residues and are active in the neutral pH range. The best known NP is thermolysin, the highly thermostable 316 residue NP from Bacillus thermoproteolyticus. Bacilli exhibit considerable differences in growth temperature and the thermostabilities of their neutral proteases differ accordingly. Several Bacillus neutral proteases have been characterized and genes encoding for these enzymes have been cloned and sequenced from e.g. B. subtilis (Yang et al., 1984), B. stearothermophilus CU-21 (Fujii et al., 1983; Takagi et al., 1985), B. stearothermophilus MK-232 (Kubo & Imanaka, 1988), B. thermoproteolyticus rokko (Marquardt et al., 1990) and B. caldolyticus (Van den Burg et al., 1991). Using genetical techniques (such as `site-directed mutagenesis`, e.g. Stanssens et al., 1989) genes encoding neutral proteases have been mutated to change properties
REFERENCES:
patent: 5340735 (1994-08-01), Christianson et al.
Eijsink et al. "Structural determination of the thermostability of thermolysin-like Bacillus neutral proteases" in Stability and stabilization of enzymes, Proceedings of an International symposium held in Maastricht, The Netherland, Nov. 22-25, 1992, 1993.
van den Burg et al. "A highly thermostable neutral protease from Bacillus caldolyticus . . . " J. Bacteriol. 173, 4107-4115, Jul. 1991.
Hardy et al. "Stabilization of Bacillus stearothermophilius neutral protease by introduction of prolines" FEBS Lett. 317, 89-92, Feb. 1993.
Kubo et al. "Alteration of specific activity and stability of . . . " Appl. Envirn. Microbiol. 58, 3779-3783, Nov. 1992.
Eijsink Vincentius
Venema Gerhardus
Michaelson Peter L.
Nashed Nashaat T.
Rijksuniversiteit te Groningen
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