Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-09-09
1998-02-03
Witz, Jean C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4352521, 435161, 435 99, C12N 928, C12N 120, C12P 706, C12P 1914
Patent
active
057143692
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK95/00095 filed Mar. 2, 1995, which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a novel thermostable amylase and a novel thermostable pullulanase and their 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 and a pullulanase 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 Fervidobacterium, a genus not previously reported to produce thermostable amylase; and a novel thermostable pullulanase which can be obtained from Fervidobacterium pennavorans; these new enzymes have temperature optimum around 80.degree.-90.degree. C.
Accordingly, the invention provides an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Fervidobacterium, and a pullulanase preparation, characterized by being producible by cultivation of a pullulanase producing strain of Fervidobacterium pennavorans.
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.) and a pullulanase (.box-solid.) of the invention at various temperatures (determined at pH 5.5 with starch and pullulan, respectively, as substrate).
FIG. 2 shows the relative activity (% rel.) of an amylase (.sunburst.) and a pullulanase (.box-solid.) of the invention at various pH, determined at 90.degree. C. with starch and pullulan, respectively, as substrate.
DETAILED DISCLOSURE OF THE INVENTION
The Microorganism
According to the invention, amylase is derived from an amylase producing strain of the genus Fervidobacterium, in particular Fervidobacterium pennavorans, and pullulanase is derived from a pullulanase producing strain of Fervidobacterium pennavorans.
A strain representative of Fervidobacterium pennavorans has been deposited according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, on 12 Mar., 1992, at Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, under Accession No. DSM 7003.
Production of Amylase and Pullulanase
Amylase and pullulanase 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 and pullulanase 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 and the pullulanase 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 and the pullulanase may be further purified by known methods.
Immunochemical P
REFERENCES:
patent: 4929557 (1990-05-01), Antranikian et al.
Brown et al., "Characterization of Amylolytic Enzyme Activities Associated With The Hyperthermophilic Archaebacterium Pyrococcus furiosus", Applied And Environmental Microbiology, Jul. 1990, pp. 1985-1991.
Koch et al., "Purification And Properties Of A Hyperthermoactive .alpha.-amylase From The Archaeobacterium Pyrococcus woesei", Arch Microbiol. 1991, 155: pp. 572-578.
Dialog, file 357, Derwent Biotechnology Abs, DBA Accession No. 87-07485.
Antranikian et al., Chem. Ing. Tech., vol. 64, No. 6, pp. 548-550 (1992).
Medline--Accession No. 94079331.
Dialog, file 357, Derwent Biotechnology Abs, DBA Accession No. 94-13129.
Dialog, file 55, Biosis Previews, Dialog Accession No. 98081261.
Hartley et al. "Industrial prospects of thermophiles and thermophilic enzymes", Biochem. Soc. Symp. (1983) vol. 48, pp. 133-146.
Plant et al. "Starch degradation by thermophilic anaerobic bacteria," System Appl. Microbiol. (1987) vol. 9, pp. 158-162.
Madi et al. "Identification of a starch degrading anaerobic thermophile producing thermostable alpha-amylase and pullulanase", Appl. Microbiol. Biotechnol. (1989) vol. 30(4), pp. 422-425.
Antranikian Garabed
Sj.o slashed.holm Carsten
Gregg, Esq. Valeta
Hanley Susan
Novo Nordisk A S
Witz Jean C.
Zelson Esq. Steve T.
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