Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-11-16
2001-03-06
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S204000, C435S210000, C510S226000, C510S236000, C510S530000, C510S320000, C510S392000
Reexamination Certificate
active
06197565
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 stability at acidic pH at low calcium concentrations and/or high temperatures (relative to the parent) which are advantageous with respect to applications of the variants in, in particular, industrial starch processing (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.
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 stability at acidic pH at high temperatures (relative to the parent) which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).
In the context of the invention the term “acidic pH” means a pH below 7.0, especially below the pH range in which industrial starch liquefaction processes are normally performed, which is between pH 5.5 and 6.2.
In the context of the present invention the term “low Calcium concentration” means concentrations below the normal level used in industrial starch liquefaction. Normal concentrations vary depending of the concentration of free Ca
2+
in the corn. Normally a dosage corresponding to 1 mM (40 ppm) is added which together with the level in corn gives between 40 and 60 ppm free Ca
2+
.
In the context of the invention the term “high temperature” means temperatures between 95 and 160° C., especially the temperature range in which industrial starch liquefaction processes are normally performed, which is between 95 and 105° C.
The invention further relates to DNA constructs encoding variants of the invention, to methods for preparing variants of the invention, and to the use of variants of the invention, alone or in combination with other &agr;-amylolytic enzymes, in various industrial processes, in particular starch liquefaction.
Nomenclature
In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, &agr;-amylase variants of the invention are described by use of the following nomenclature: Original amino acid(s):position(s):substituted amino acid(s)
According to this nomenclature, for instance a change from alanine to asparagine at position
30
is shown as:
Ala30Asn or A30N
a deletion of alanine in the same position is shown as:
Ala30* or A30*
and insertion of an additional amino acid residue, such as lysine, is shown as:
Ala30AlaLys or A30AK
A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or &Dgr;(A30-N33).
Where a specific &agr;-amylase contains a “deletion” in comparison with other &agr;-amylases and an insertion is made in such a position this is indicated as:
*36Asp or *36D
for insertion of an aspartic acid in position 36 Multiple mutations are separated by plus signs, i.e.:
Ala30Asp+Glu34Ser or A30N+E34S
representing mutations in positions 30 and 34 in which alanine and glutamic acid are changed to asparagine and serine, respectively. Multiple mutation may also be separated as follows, i.e., meaning the same as the plus sign:
Ala30Asp/Glu34Ser or A30N/E34S
When one or more alternative amino acid residues may be inserted in a given position it is indicated as
A30N, E or
A30N or A30E
Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:
R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.
REFERENCES:
patent: WO 96/23874 (1996-08-01), None
patent: WO 97/41213 (1997-11-01), None
Andersen Carsten
Bisgaard-Frantzen Henrik
Kjaerulff Sóeren
Svendsen Allan
Hutson Richard
Lambiris Esq. Elias J.
Novo-Nordisk A/S
Prouty Rebecca E.
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