Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-10-13
2001-02-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000, C510S226000, C510S235000, C510S320000, C510S392000
Reexamination Certificate
active
06187576
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, inter alia, to novel variants (mutants) of parent Termamyl-like &agr;-amylases, notably variants exhibiting increased thermostability at acidic pH and/or at low Ca
2+
concentrations (relative to the parent) which are advantageous with respect to applications of the variants in, industrial starch processing particularly (e.g. starch liquefaction or saccharification).
BACKGROUND OF THE INVENTION
&agr;-Amylases (&agr;-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of &agr;-amylase such as Termamyl-like &agr;-amylases variants are known from e.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
Among more recent disclosures relating to &agr;-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like &agr;-amylase which consists of the 300 N-terminal amino acid residues of the
B. amyloliquefaciens
&agr;-amylase and amino acids 301-483 of the C-terminal end of the
B. licheniformis
&agr;-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl™), and which is thus closely related to the industrially important Bacillus &agr;-amylases (which in the present context are embraced within the meaning of the term “Termamyl-like &agr;-amylases”, and which include, inter alia, the
B. licheniformis, B. amyloliquefaciens
and
B. stearothermophilus
&agr;-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like &agr;-amylase, variants of the parent Termamyl-like &agr;-amylase which exhibit altered properties relative to the parent.
WO 95/35382 (Gist Brocades B.V.) concerns amylolytic enzymes derived from
B. licheniformis
with improved properties allowing reduction of the Ca
2+
concentration under application without a loss of performance of the enzyme. The amylolytic enzyme comprises one or more amino acid changes at positions selected from the group of 104, 128, 187, 188 of the
B. licheniformis
&agr;-amylase sequence.
WO 96/23873 (Novo Nordisk) discloses Termamyl-like &agr;-amylase variants which have increased thermostability obtained by pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.
BRIEF DISCLOSURE OF THE INVENTION
The present invention relates to novel &agr;-amylolytic variants (mutants) of a Termamyl-like &agr;-amylase, in particular variants exhibiting increased thermostability (relative to the parent) which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).
The inventors have surprisingly found out that in case of combining two, three, four, five or six mutations (will be described below), the thermostability of Termamyl-like &agr;-amylases is increased at acidic pH and/or at low Ca
2+
concentration in comparison to single mutations, such as the mutation disclosed in WO 96/23873 (Novo Nordisk), i.e. pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.
The invention further relates to DNA constructs encoding variants of the invention, to composition comprising variants of the invention, to methods for preparing variants of the invention, and to the use of variants and compositions of the invention, alone or in combination with other &agr;-amylolytic enzymes, in various industrial processes, e.g., starch liquefaction.
REFERENCES:
patent: WO 90/11352 (1990-10-01), None
patent: WO 91/00353 (1991-01-01), None
patent: WO 95/10603 (1995-04-01), None
patent: WO 95/26397 (1995-10-01), None
patent: WO 95/35382 (1995-12-01), None
patent: WO 96/23874 (1996-08-01), None
patent: WO 96/23873 (1996-08-01), None
Gray G.L. et al. Structural genes encoding the thermophilic alpha-amylases ofBacillus stearothermophilusandB.licheniformis. J.Bacteriol., May 1986, vol. 166(2):635-643.
Bisgard-Frantzen Henrik
Borchert Torben Vedel
Svendsen Allan
Green, Esq. Reza
Lambiris Esq. Elias J.
Novo Nordisk A S
Prouty Rebecca E.
Rao Manjunath
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