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  Thermostable alpha-amylasesUSPTO Application #: 20070190608Title: Thermostable alpha-amylases Abstract: b) a polypeptide comprising an amino acid sequence which has at least 70% identity with the polypeptide encoded by the amylase encoding part of the polynucleotide inserted into a plasmid present in the E. coli host deposited under the Budapest Treaty with DSMZ under accession number DSM 15334; c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which has at least 70% identity with the sequence shown from position 68 to 1417 in SEQ ID NO:3; and d) a fragment of (a), (b) or (c) that has alpha-amylase activity. An isolated polynucleotide comprising an open reading frame encoding a polypeptide having alpha-amylase activity, the polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence which has at least 70% identity with amino acids 22 to 450 of SEQ ID NO:4; (end of abstract) Agent: Novozymes North America, Inc. - New York, NY, US Inventors: Lan Tang, Wenping Wu, Junxin Duan, Pia Francke Johannesen USPTO Applicaton #: 20070190608 - Class: 435069100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide The Patent Description & Claims data below is from USPTO Patent Application 20070190608. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 10/539,396 (now allowed), filed Jun. 16, 2005, which is a national application under 35 U.S.C. 371 of PCT/DK2003/00882, filed Dec. 16, 2003, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2002 01928, filed Dec. 17, 2002, and U.S. provisional application No. 60/435,483, filed Dec. 20, 2002, the contents of which are fully incorporated herein by reference. FIELD OF INVENTION [0002] The present invention relates to thermostable alpha-amylases, in particular with improved thermal stability at acidic pH. The invention also relates to the use of such alpha-amylases. BACKGROUND [0003] Alpha-Amylases (alpha-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. [0004] There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of alpha-amylases referred to as "Termamyl.RTM.-like alpha-amylases" and variants thereof are known from, e.g., WO 90/11352, WO 95/10603. WO 95/26397, WO 96/23873 and WO 96/23874. Termamyl.RTM.-like alpha-amylases are very thermostable and therefore suitable for processes carried out at high temperatures such as starch liquefaction in dextrose production processes. [0005] Another group of alpha-amylases are referred to as "Fungamyl.TM.-like alpha-amylases", which are alpha-amylases related or homologous to the alpha-amylase derived from Aspergillus oryzae. The Fungamyl-like alpha-amylases have a relatively low thermostability the commercial product sold under the tradename FUNGAMYL.TM. by Novozymes A/S, Denmark, has a optimum around 55.degree. C., and is not suitable for processes carried out at high temperatures. Fungamyl.TM.-like alpha-amylases are today used for making syrups for, e.g., the brewing industry. [0006] Clearly, it would be advantageous to provide an alpha-amylase with increased thermostability preferably at an acidic pH. This is no new realization, but actually a very long-felt need in the art. As far back as in 1980, Somkuti and Steinberg described a thermoacidophilic extracellular alpha-amylase of Rhizomucor pusillus (Mucor pusillus), that they managed to isolate and characterize. They state that: "Since high temp and acidic pH are optimum conditions for the economic hydrolysis of starch, the use of thermostable and acid-stable amylases of microbial origin for industrial purposes has been recommended", and go on to conclude about the Rhizomucor amylase that: "It is apparently the first exam pie of fungal alpha-amylase exhibiting both acidophily and thermophily simultaneously. Consequently, the alpha-amylase of M. pusillus should be of economic importance." (Somkuti G A, Steinberg D H (1980) Thermoacidophilic extracellular amylase of Mucor pusillus. Dev Indust Microbiot 21:327-337). [0007] However, despite the very clear conclusions by Somkuti and Steinberg back in 1980, the gene encoding the Rhizomucor pusillus alpha-amylase had until today not been cloned or sequenced and the amylase had until today not been produced recombinantly in industrially relevant amounts. In 1987 an improved purification method was reported, but stilt only for enzyme produced by the wildtype Rhizomucor pusillus (Turchi S L and Becker T (1987) Curr Microbiot 15:203-205). SUMMARY OF THE INVENTION [0008] A problem to be solved by this invention is how to provide a recombinant thermoacidophilic alpha-amylase. The present inventors have successfully isolated a gene from Rhizomucor pusillus encoding an alpha-amylase which they have denoted AM782, they have successfully introduced the encoding gene into a recombinant industrial filamentous fungal expression system, and produced the alpha-amylase. Characterization of the amylase has shown it to be a highly thermoacidophilic alpha-amylase which has a highly interesting activity as demonstrated by the sugar profile from maltodextrin hydrolysis by amylase AM782. [0009] The amylase AM782 can work at a very high temperature, at least up to 70.degree. C. The amylase AM782 has a very fast reaction speed; when compared at the same dosage with Fungamyl.TM. 800 L, the amylase AM782 can achieve in about 3 hours, what takes Fungamyyl.TM. 24 to 48 hours. Furthermore, the amylase AM782 can degrade DP3 into DP2 and DP1, so it gives a higher DP1 result. [0010] Accordingly, in a first aspect the invention relates to an isolated polynucleotide comprising an open reading frame encoding a polypeptide having alpha-amylase activity, the polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence which has at least 70% identity with amino acids 22 to 450 of SEQ ID NO:4, preferably 75%, more preferably 80%, even more preferably 85%, still more preferably 90%, more preferably 95%, and most preferably at least 97% identity with amino acids 22 to 450 of SEQ ID NO:4; b) a polypeptide comprising an amino acid sequence which has at least 70% identity with the polypeptide encoded by the amylase encoding part of the polynucleotide inserted into a plasmid present in the E. coli host deposited under the Budapest Treaty with DSMZ under accession number DSM 15334, preferably 75%, more preferably 80%, even more preferably 85%, still more preferably 90%, more preferably 95%, and most preferably at least 97% identity with the polypeptide encoded by the amylase encoding part of the polynucleotide inserted into a plasmid present in the E. coli host deposited under the Budapest Treaty with DSMZ under accession number DSM 15334; c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which has at least 70% identity with the sequence shown from position 68 to 1417 in SEQ ID NO:3, preferably 75%, more preferably 80%, even more preferably 85%, still more preferably 90%, more preferably 95%, and most preferably at least 97% identity with the sequence shown from position 68 to 1417 in SEQ ID NO:3; and d) a fragment of (a), (b) or (c) that has alpha-amylase activity. [0011] In a second aspect the invention relates to a nucleic acid construct comprising a polynucleotide as defined in the first aspect operably linked to one or more control sequences that direct the production of the polypeptide in a suitable host. [0012] A third aspect relates to a recombinant expression vector comprising a nucleic acid construct as defined in the second aspect. [0013] In a fourth aspect the invention relates to a recombinant host cell comprising a nucleic acid construct as defined the second aspect, or at least one copy of an expression vector as defined in the third aspect. [0014] Industrial production of the amylase AM782 along with homologues and variants is of course highly interesting. [0015] Accordingly, in a fifth aspect the invention relates to a method for producing a polypeptide having alpha-amylase activity encoded by a polynucleotide as defined in the first aspect, the method comprising: (a) cultivating a recombinant host cell as defined in any of claims 12-16 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. [0016] There are quite a few applications for an amylase such as the amylase AM782, an overview of some of the major ones is given herein, and it includes but is not limited to: the starch industry, the food processing industry, the textile industry, and the detergent industry. [0017] Consequently, additional aspects of the invention relate to a method of producing an enzymatically modified starch derivative, wherein a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is used for liquefying and/or saccharifying starch; and to a method of producing high maltose syrups, wherein a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is used for liquefying starch; a method for desizing textile, wherein a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is used for treating the textile; and to a brewing process, wherein a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is added during fermentation of wort; and to an alcohol production process, wherein a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is used for liquefaction starch in a distillery mash; and to a process, wherein a dough product comprising a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect is baked. [0018] Various uses of amylase AM782 along with homologues and variants are also contemplated in the present invention. [0019] Accordingly, a number of non-limiting aspects of the invention relate to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect in a starch conversion process for liquefaction and/or saccharification; to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect for liquefying starch in a high maltose syrup production process; to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect for textile desizing; to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect for producing alcohol; to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect for brewing; and to the use of a polypeptide having alpha-amylase activity produced according to a method as defined in the fifth aspect for baking. 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