Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1993-08-03
1996-01-23
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435200, 435691, 4352523, 43525231, 43525233, 4353201, 536 232, 935 14, 935 29, 935 73, 935 74, C12N 944, C12N 1556, C12N 1570, C12N 1574
Patent
active
054864690
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to thermostable enzymes. More specifically, the invention relates to novel thermostable pullulanases obtainable from Thermotogales, and to processes for the preparation of these enzymes.
The invention also relates to the use of the pullulanases in starch converting processes, and to saccharification processes.
BACKGROUND ART
Thermostable pullulanases are known and have been isolated from e.g. Bacillus acidopullulyticus, and their use in industrial saccharification processes has been described, vide EP patent publication No. 63,909. To comply with the demands for more thermostable enzymes, the search has continued, and it is the purpose of this invention to provide novel pullulanases with improved thermostability.
STATEMENT OF THE INVENTION
We have isolated a microorganism producing a novel pullulanase of surprising thermostability.
Accordingly, the invention provides a pullulanase having temperature optimum in the range 80-90.degree. C., pH optimum in the range of 5-7, at least 60% residual activity, preferably at least 80% residual activity, after 24 hours of incubation at pH 6.0, and having immunochemical properties identical or partially identical to those of the pullulanases derived from the strain Fervidobacterium sp. Ven 5, DSM 6204.
In another aspect, the invention provides a process for the preparation of a pullulanase, which process comprises cultivation of a pullulanase producing strain of Thermotogales under anaerobic conditions in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
In yet another aspect, the invention provides a process for the preparation of a recombinant pullulanase, the process comprising isolating a DNA fragment encoding the pullulanase, combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector, introducing the plasmid vector into an appropriate host either as an autonomously replicating plasmid or integrated into the chromosome, cultivating the host organism under aerobic conditions in a suitable nutrient medium, leading to expression of the pullulanase and recovering the pullulanase from the culture medium.
In a further aspect, the invention relates to the use of these pullulanases for the saccharification of liquefied starch, and the invention provides a process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting the saccharification of starch hydrolysates in the presence of a pullulanase and one or more enzymes selected from the group consisting of glucoamylase, .alpha.-glucosidase, .beta.-amylase, or other saccharifying enzymes.
BRIEF DESCRIPTION OF DRAWINGS
The present invention is further illustrated by reference to the accompanying drawings, in which:
FIG. 1A shows the relative activity (%) of the pullulanase of the invention at different temperatures; 1.2 U/ml were used in incubation for the determination of temperature optimum;
FIG. 1B shows the relative activity (%) of the pullulanase of the invention at different pH values; 1.8 U/ml were used throughout for pH optimum determination;
FIG. 2 shows the analysis of sugars released by the action of the pullulanase of the invention during degradation of pullulan (FIG. 2A), branched oligosaccharides (FIG. 2B), and amylose (FIG. 2C), respectively. Left side without pullulanase, right side after 8 hours of incubation at 80.degree. C. 0.25 U/ml was used throughout incubation;
FIG. 3 shows thermal stability of the pullulanase at 70.degree. C. ( ), 80.degree. C. ( ), and 90.degree. C. (.quadrature.) at pH 6.0 in the absence of starch and metal ions (3A, 0-24 hours; 3B, 0-1 hour). Initial activity of the pullulanase solution used for the investigation of thermal stability was 6.5 U/ml;
FIG. 4 shows thermal stability of the pullulanase at pH 45 and 80.degree. C. in the presence of 5 mM CaCl.sub.2 ( ), 1% starch ( ), 5 mM CaCl.sub.2 and 1% starch ( ), and without metal ions or starch (.quadrature.) (4A, 0-48 h
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Antranikian Garabed
Jorgensen Per L.
Lambiris Elias J.
Moore William W.
Novo Nordisk A S
Wax Robert A.
Zelson Steve T.
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