Alpha-amylase

Abstract

This invention relates to a novel alpha-amylase, a process for its preparation and the use of the amylase. The invention relates to a newly identified polynucleotide sequence from Alicyclobacillus pohliae comprising a gene that encodes the novel alpha-amylase enzyme. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein of the gene. The invention also relates to methods of using these proteins in industrial processes, for example in food industry, such as the baking industry. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins and cells.

1 . A polynucleotide encoding for a polypeptide having alpha-amylase activity comprising:
 (a) a polynucleotide sequence encoding a polypeptide having an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or   (b) a polynucleotide sequence encoding a polypeptide having at least 99.5% identity to a polypeptide having an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or   (c) a polynucleotide sequence as set out in nucleotides 100 to 2157 of SEQ ID NO: 1 or SEQ ID NO: 3; or   (d) a polynucleotide sequence as set out in SEQ ID NO: 1 or SEQ ID NO: 3.   
     
     
         2 . The polynucleotide according to  claim 1 , wherein the polynucleotide is produced by  Alicyclobacillus pohliae  NCIMB14276. 
     
     
         3 . A vector comprising the polynucleotide sequence according to  claim 1 . 
     
     
         4 . The vector according to  claim 3  which is an expression vector wherein the polynucleotide sequence is operably linked with at least one regulatory sequence allowing for expression of the polynucleotide sequence in a suitable host cell. 
     
     
         5 . The vector according to  claim 4 , wherein the suitable host cell is an  Aspergillus, Bacillus, Chrysosporium, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces, Streptomyces  or  Talaromyces  species, preferably a  Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, Aspergillus Niger  or  Aspergillus oryzae  species. 
     
     
         6 . A recombinant host cell comprising the polynucleotide according to  claim 1 . 
     
     
         7 . The recombinant host cell according to  claim 6  capable of expressing or over-expressing said polynucleotide or a vector comprising said polynucleotide. 
     
     
         8 . A method for manufacturing the polynucleotide according to  claim 1  comprising culturing a host cell transformed with said polynucleotide or a vector thereof and isolating said polynucleotide or said vector from said host cell. 
     
     
         9 . An alpha-amylase polypeptide comprising:
 (a) an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or   (b) an amino acid sequence having at least 99.5% identity to an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or   (c) an amino acid sequence encoded by a polynucleotide as set out in nucleotides 100 to 2157 of SEQ ID NO: 1 or SEQ ID NO: 3; or   (d) an amino acid sequence having at least 70% identity to an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one of Asp at position 184, Ala at position 297, Thr at position 368 and Asn at position 489, said positions being defined with reference to SEQ ID NO: 2; or   (e) an amino acid sequence having at least 70% identity to an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one of Asp at position 184, Ala at position 297, Thr at position 368 and Asn at position 489, said positions being defined with reference to SEQ ID NO: 2 and said amino acid sequence characterized in that when used to prepare a baked product having a least 5 wt % sugar based on flour, said baked product has reduced hardness after storage in comparison with a baked product prepared without use of said amino acid sequence.   
     
     
         10 . The polypeptide according to  claim 9  obtainable by expressing the polynucleotide a vector comprising said polynucleotide in an appropriate host cell. 
     
     
         11 . A method for manufacturing the polypeptide according to  claim 9  comprising cultivating a recombinant host cell under condition which allow for expression of the polynucleotide or a vector comprising said polynucleotide and, optionally, recovering an encoded polypeptide from the cell or culture medium. 
     
     
         12 . A polypeptide according to  claim 9  capable of being used in food manufacturing. 
     
     
         13 . A polypeptide according to  claim 12  capable of being used in manufacture of a baked product, optionally a bread or a cake. 
     
     
         14 . Enzyme composition comprising the polypeptide according to  claim 9  and one or more components selected from the group consisting of milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, an oxidant such as ascorbic acid, bromate and azodicarbonamide, a reducing agent such as L-cysteine, an emulsifier such as mono-glycerides, di-glycerides, glycerol monostearate, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, gums such as guargum and xanthangum, flavours, acids such as citric acid and propionic acid, starch, modified starch, gluten, humectants such as glycerol, and preservatives. 
     
     
         15 . Enzyme composition according to  claim 14 , wherein the additional enzyme is a lipolytic enzyme, optionally a phospholipase. 
     
     
         16 . Method to prepare a dough comprising the step of combining the polypeptide according to  claim 9  and at least one dough ingredient. 
     
     
         17 . A dough comprising the polypeptide according to  claim 9 . 
     
     
         18 . Method to prepare a baked product comprising baking the dough according to  claim 17 . 
     
     
         19 . Baked product obtainable by the method according to  claim 18 . 
     
     
         20 . A method to produce a polypeptide having at least 60% identity to
 (a) an amino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2 or an amino acid sequence having at least 99.5% identity to amino acids 34 to 719 of the amino acid sequence of SEQ ID NO: 2; or   (b) an amino acid sequence encoded by the polynucleotide according to  claim 1 , comprising using  Alicyclobacillus pohliae  NCIMB14276.   
     
     
         21 . The polypeptide according to  claim 9  capable of being used to reduce hardness after storage of a baked product comprising at least 5 wt % sugar based on flour.