Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-08-10
2002-08-27
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06440716
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel method of designing &agr;-amylase mutants with predetermined properties, which method is based on the hitherto unknown three-dimensional structure of bacterial &agr;-amylases.
BACKGROUND OF THE INVENTION
&agr;-Amylases (&agr;-1,4 glucan-4-glucanohydrolase, EC 3.2.1.1) constitute a group of enzymes which is capable of hydrolyzing starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. Almost all &agr;-amylases studied have a few conserved regions with approximately the same length and spacing. One of these regions resembles the Ca2+ binding site of calmodulin and the others are thought to be necessary for the active centre and/or binding of the substrate.
While the amino acid sequence and thus primary structure of a large number of &agr;-amylases are known, it has proved very difficult to determine the three-dimensional structure of all &agr;-amylases. The three-dimensional structure can be determined by X-ray crystallographic analysis of &agr;-amylase crystals, but it has proven difficult to obtain &agr;-amylase crystals suitable for actually solving the structure.
Until now the three-dimensional structure of only a few &agr;-amylases-have been determined at high resolution. These include the structure of the
Aspergillus oryzae
TAKA &agr;-amylase (Swift et al., 1991), the
Aspergillus niger
acid amylase (Brady et al, 1991), the structure of pig pancreatic &agr;-amylase (Qian et al., 1993), and the barley alpha-amylase (Kadziola et al. 1994, Journal of Molecular Biology 239: 104-121, A.Kadziola, Thesis, Dept of Chemistry, U. of Copenhagen, Denmark). Furthermore, the three-dimensional structure of a
Bacillus circulans
cyclodextrin glycosyltransferase (CGTase) is known (Klein et al., 1992) (Lawson et al., 1994). The CGTase catalyzes the same type of reactions as &agr;-amylases and exhibits some structural resemblance with &agr;-amylases.
Furthermore, crystallization and preliminary X-ray studies of
B. subtilis
&agr;-amylases have been described (Chang et al. (1992) and Mizuno et al. (1993)). No final
B. subtilis
structure has been reported. Analogously, the preparation of
B. licheniformis
&agr;-amylase crystals has been reported (Suzuki et al. (1990), but no subsequent report on X-ray crystallographic analysis or three-dimensional structure are available.
Several research teams have attempted to build three-dimensional structures on the basis of the above known &agr;-amylase structures. For instance, Vihinen et al. (J. Biochem. 107, 267-272, 1990), disclose the modelling (or computer simulation) of a three-dimensional structure of the
Bacillus stearothermophilus
&agr;-amylase on the basis of the TAKA amylase structure. The model was used to investigate hypothetical structural consequences of various site-directed mutations of the
B. stearothermophilus
&agr;-amylase. E. A. MacGregor (1987) predicts the presence of &agr;-helices and 3-barrels in &agr;-amylases from different sources, including barley, pig pancreas and
Bacillus amyloliquefaciens
on the basis of the known structure of the
A. oryzae
TAKA &agr;-amylase and secondary structure predicting algorithms. Furthermore, the possible loops and subsites which may be found to be present in, e.g., the
B. amyloliquefaciens
&agr;-amylase are predicted (based on a comparison with the
A. oryzae
sequence and structure).
A. E. MacGregor (Starch/Stärke 45 (1993), No. 7, p. 232-237) presents a review of the relationship between the structure and activity of &agr;-amylase related enzymes.
Hitherto, no three-dimensional structure has been available for the industrially important Bacillus &agr;-amylases (which in the present context are termed “Termamyl-like &agr;-amylases”), including the
B. licheniformis
, the
B. amyloliquefaciens
, and the
B. stearothermophilus
&agr;-amylase.
BRIEF DISCLOSURE OF THE INVENTION
The three-dimensional structure of a Termamyl-like bacterial &agr;-amylase has now been elucidated. On the basis of an analysis of said structure it is possible to identify structural parts or specific amino acid residues which from structural or functional considerations appear to be important for conferring the various properties to the Termamyl-like &agr;-amylases. Furthermore, when comparing the Termamyl-like &agr;-amylase structure with known structures of the fungal and mammalian &agr;-amylases mentioned above, it has been found that some similarities exist between the structures, but also that some striking, and not previously predicted structural differences between the &agr;-amylases exist. The present invention is based on these findings.
Accordingly, in a first aspect the invention relates to a method of constructing a variant of a parent Termamyl-like &agr;-amylase, which variant has &agr;-amylase activity and at least one altered property as compared to said parent &agr;-amylase, which method comprises
i) analysing the structure of the Termamyl-like &agr;-amylase with a view to identifying at least one amino acid residue or at least one structural part of the Termamyl-like &agr;-amylase structure, which amino acid residue or structural part is believed to be of relevance for altering said property of the parent Termamyl-like &agr;-amylase (as evaluated on the basis of structural or functional considerations),
ii) constructing a Termamyl-like &agr;-amylase variant, which as compared to the parent Termamyl-like &agr;-amylase, has been modified in the amino acid residue or structural part identified in i) so as to alter said property, and, optionally,
iii) testing the resulting Termamyl-like &agr;-amylase variant with respect to said property.
In a second aspect the present invention relates to a method of constructing a variant of a parent Termamyl-like &agr;-amylase, which variant has &agr;-amylase activity and one or more altered properties as compared to said parent &agr;-amylase, which method comprises
i) comparing the three-dimensional structure of the Termamyl-like &agr;-amylase with the structure of a non-Termamyl-like &agr;-amylase,
ii) identifying a part of the Termamyl-like &agr;-amylase structure which is different from the non-Termamyl-like &agr;-amylase structure,
iii) modifying the part of the Termamyl-like &agr;-amylase identified in ii) whereby a Termamyl-like &agr;-amylase variant is obtained, one or more properties of which differ from the parent Termamyl-like &agr;-amylase, and optionally,
iv) testing the resulting Termamyl-like &agr;-amylase variant with respect to said property or properties.
In a third aspect the invention relates to a method of constructing a variant of a parent non-Termamyl-like &agr;-amylase, which variant has &agr;-amylase activity and one or more altered properties as compared to said parent &agr;-amylase, which method comprises
i) comparing the three-dimensional structure of the non-Termamyl-like &agr;-amylase with the structure of a Termamyl-like &agr;-amylase,
ii) identifying a part of the non-Termamyl-like &agr;-amylase structure which is different from the Termamyl-like &agr;-amylase structure,
iii) modifying the part of the non-Termamyl-like &agr;-amylase identified in ii) whereby a non-Termamyl-like &agr;-amylase variant is obtained, one or more properties of which differ from the parent non-Termamyl-like &agr;-amylase, and optionally,
iv) testing the resulting non-Termamyl-like &agr;-amylase variant with respect to said property or properties.
The property which may be altered by the above methods of the present invention may, e.g., be substrate specificity, substrate binding, substrate cleavage pattern, temperature stability, pH dependent activity, pH dependent stability (especially increased stability at low (e.g. pH<6, in particular pH<5) or high (e.g. pH>9) pH values), stability towards oxidation, Ca
2+
-dependency, specific activity, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent Termamyl-like &agr;-amylase, has an increased specific activity at a given pH and/or
Bisgård-Frantzen Henrik
Borchert Torben Vedel
Svendsen Allan
Garbell Jason
Lambiris Elias
Novozymes A/S
Slobodyansky Elizabeth
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