Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2002-06-28
2003-11-04
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S202000, C435S320100, C536S023200, C536S023700, C510S226000
Reexamination Certificate
active
06642044
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, inter alia, to novel variants (mutants) of parent Termamyl-like a-amylases, notably variants exhibiting alterations in one or more properties (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, and there is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes.
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 comprising the amino acid sequence shown in SEQ ID No. 4 herein and amino acids 301-483 of the C-terminal end of the
B. licheniformis
&agr;-amylase comprising the amino acid sequence shown in SEQ ID No. 2 herein (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
As indicated above, the present invention relates, inter alia, to novel &agr;-amylolytic variants(mutants) of a Termamyl-like &agr;-amylase, in particular variants exhibiting altered properties which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).
Alterations in properties which may be achieved in mutants of the invention are alterations in, e.g., substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile [such as 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 a reduced Ca
2+
dependency and/or an altered pH/activity profile.
The invention further relates, inter alia, 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, e.g. starch liquefaction.
DETAILED DISCLOSURE OF THE INVENTION
The Termamyl-like &agr;-Amylase
It is well known that a number of &agr;-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the
B. licheniformis
&agr;-amylase comprising the amino acid sequence shown in SEQ ID No. 2 (commercially available as Termamyl™) has been found to be about 89% homologous with the
B. amyloliquefaciens
&agr;-amylase comprising the amino acid sequence shown in SEQ ID No. 4 and about 79% homologous with the
B. stearothermophilus
&agr;-amylase comprising the amino acid sequence shown in SEQ ID No. 6. Further homologous &agr;-amylases include an &agr;-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the &agr;-amylase described by Tsukamoto et al.,
Biochemical and Biophysical Research Communications
, 151 (1988), pp. 25-31. Still further homologous &agr;-amylases include the &agr;-amylase produced by the
B. licheniformis
strain described in EP 0252666 (ATCC 27811), and the &agr;-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like
B. licheniformis
&agr;-amylases are Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ (available from Genencor), and Keistase™ (available from Daiwa).
Because of the substantial homology found between these &agr;-amylases, they are considered to belong to the same class of &agr;-amylases, namely the class of “Termamyl-like &agr;-amylases”.
Accordingly, in the present context, the term “Termamyl-like &agr;-amylase” is intended to indicate an &agr;-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl™, i.e. the
B. licheniformis
&agr;-amylase having the amino acid sequence shown in SEQ ID No. 2 herein. In other words, a Termamyl-like &agr;-amylase is an &agr;-amylase which has the amino acid sequence shown in SEQ ID No. 2, No. 4 or No. 6 herein, or the amino acid sequence shown in SEQ ID No. 1 of WO 95/26397 (which amino acid sequence is shown in FIG.
1
and
FIG. 2
herein) or in SEQ ID No. 2 of WO 95/26397 (which amino acid sequence is shown in
FIG. 2
herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in
FIG. 2
herein) or i) which displays at least 60%, such as at least 70%, e.g. at least 75%, or at least 80%, e.g. at least 85%, at least 90% or at least 95% homology with at least one of said amino acid sequences and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said &agr;-amylases, and/or iii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified &agr;-amylases which are apparent from SEQ ID Nos. 1, 3 and 5 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID Nos. 2, 4 and 6 herein, respectively), from SEQ ID No. 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in
FIG. 1
herein and encodes the amino acid sequence shown in
FIG. 1
herein) and from SEQ ID No. 5 of WO 95/26397, respectively.
In connection with property i), the “homology” may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG package version 7.3 (June 1993) using default values for GAP penalties [Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711].
Property ii) of the &agr;-amylase, i.e. the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the relevant Termamyl-like &agr;-amylase. The antibody, which may either be monoclonal or poly-clonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et. al., 1989. In this respect, immunological cross-reactivity between the &agr;-amylases having the amino acid sequences SEQ ID Nos. 2, 4 and 6, respectively, has been found.
The oligonucleotide probe used in the characterization of the Termamyl-like &agr;-amylase in accordance with property iii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the &agr;-amylase in question. Suitable conditions for testing hybridization involve presoaking in 5×SSC and prehybridizing for 1 h at ~40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 &mgr;g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 1000 &mgr;M ATP for 18h at ~40° C., or other methods described b
Bisg{dot over (a)}rd-Frantzen Henrik
Borchert Torben Vedel
Svendsen Allan
Garbell Jason I.
Lambiris Elias J.
Novozymes A/S
Saidha Tekchand
LandOfFree
&agr;-amylase mutants does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with &agr;-amylase mutants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and &agr;-amylase mutants will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3138714