Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1996-09-09
1999-03-16
Weber, Jon P.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435202, 4352521, 127 38, C12P 706, C12N 928, C12N 112, C13K 106
Patent
active
058829067
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK95/00096 filed 2 Mar., 1995, the text of which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a novel thermostable amylase and its use in the production of sweeteners and ethanol from starch.
BACKGROUND OF THE INVENTION
The production of sweeteners from starch has been largely improved by application of different microbial enzymes to obtain better quality and yields, but the necessity of performing several steps of the starch-hydrolysing process at elevated temperatures means that there is still a need for new starch-hydrolysing enzymes with increased thermal stability.
It is known that Pyrococcus, e.g. Pyrococcus wosei and Pyrococcus furiosus, for reference see Arch. Microbiol. 155, 1991, pp. 572-578, and Appl. Env. Microbiol. 56, 1990, pp. 1985-1991, can produce highly thermostable amylases.
It is the object of this invention to provide an amylase with temperature optimum at 80.degree. C. or above 80.degree. C.
SUMMARY OF THE INVENTION
We have unexpectedly found that a novel thermostable amylase can be obtained from the genus Staphylothermus, a genus not previously reported to produce thermostable amylase; this new enzyme has temperature optimum around 100.degree. C.
Accordingly, the invention provides an amylase preparation, characterized by being produced by cultivation of an amylase producing strain of the genus Staphylothermus.
BRIEF DESCRIPTION OF DRAWINGS
The present invention is further illustrated by reference to the accompanying drawings, in which:
FIG. 1 shows the relative activity (% rel.) of an amylase (.sunburst.) of the invention at various temperatures (determined at pH 5.5 with starch as substrate).
FIG. 2 shows the relative activity (% rel.) of an amylase (.sunburst.) of the invention at various pH, determined at 95.degree. C. with starch as substrate.
DETAILED DISCLOSURE OF THE INVENTION
According to the invention, amylase is derived from an amylase producing strain of the genus Staphylothermus, in particular Staphylothermus marinus.
A strain representative of Staphylothermus marinus has been made publicly available under Accession No. DSM 3639. The number is published in the DSM Catalogue of Strains, 1993.
Amylase of the invention may be produced by anaerobic cultivation of the above mentioned strain on a nutrient medium containing suitable carbon and nitrogen sources, such media being known in the art. Anaerobic conditions may be achieved during the preparation of media by sparging with N.sub.2 and following the anaerobic techniques as described by Balch and Wolfe in Appl. Env. Microbiol. 32, 1976, pp. 781-791.
Alternatively, amylase of the invention can be produced by aerobic cultivation of a transformed host organism containing the appropriate genetic information from the above mentioned strain. Such transformants can be prepared and cultivated by methods known in the art.
The amylase may be recovered by removing the cells from the fermentation medium (e.g. by centrifugation or filtration) and then concentrating the broth (e.g. by ultrafiltration). If desired, the amylase may be further purified by known methods.
The amylase of the invention has immunochemical properties identical or partially identical (i.e. at least partially identical) to those of an amylase derived from the strain Staphylothermus marinus, DSM 3639.
The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectrophoresis according to Axelsen N. H.; Handbook of Immunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications (1983), chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, Chapters 5, 19 and 20.
Monospecific antisera are generated according to the above mentioned method by immunizing rabbits with the purified
REFERENCES:
patent: 4284722 (1981-08-01), Tamuri et al.
patent: 4810647 (1989-03-01), Monceaux et al.
patent: 5059430 (1991-10-01), Bowles
patent: 5370997 (1994-12-01), Antranikian et al.
Canganella et al. "characterization of amylolytic and pullylytic enzymes from thermophilic archaea and from a new Fervidobacterium species", Appl. Microbiol. Biotech. (1994) 42:239-245.
Brown et al., "Characterization of Amylolytic Enzyme Activities Associated With The Hyperthermophilic Archaebacterium Pyrococcus furiosus", Applied And Environmental Microbiology, Jul. 1990, pp. 1985-1991. vol. 56 No. 7.
Koch et al., "Purification And Properties Of A Hyperthermoactive .alpha.-amylase From The Archaeobacterium Pyrococcus woesei", Arch Microbiol. 1991, vol. 155 : pp. 572-578.
Saha et al., "Novel Highly Thermostable Pullulanase From Thermophiles", Tibech-Sep. 1989, vol. 7, pp. 234-238.
Abstract-Dialog--Med. 92--Dialog Accession No. 94079331.
Abstract-Dialog--File:55 Biosis No. 98091261.
Antranikian Garabed
Sj.o slashed.holm Carsten
Gregg, Esq. Valeta
Hanley Susan
Novo Nordisk A S
Weber Jon P.
Zelson Esq. Steve T.
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