Oxidativley stable alpha-amylase

• US 5,824,532 A
• Filed: 06/06/1995
• Issued: 10/20/1998
• Est. Priority Date: 02/11/1993
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Mitchinson, Colin, Requadt, Carol A., Power, Scott D., Barnett, Christopher C.
• Primary Examiner(s): Prouty, Rebecca E.
• Application Number:
  US08/468,220
• Time in Patent Office: 1,232 Days
• Field of Search: 435/201, 435/202, 435/203, 435/204, 435/172.3, 435/71.2, 435/252.3, 435/252.31, 435/320.1, 536/23.2, 536/23.7
• US Class Current: 435/202
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase

Abstract

Novel alpha-amylase mutants derived from the DNA sequences of naturally occurring or recombinant alpha-amylases are disclosed. The mutant alpha-amylases, in general, are obtained by in vitro modifications of a precursor DNA sequence encoding the naturally occurring or recombinant alpha-amylase to generate the substitution (replacement) or deletion of one or more oxidizable amino acid residues in the amino acid sequence of a precursor alpha-amylase. Such mutant alpha-amylases have altered oxidative stability and/or altered pH performance profiles and/or altered thermal stability as compared to the precursor. Also disclosed are detergent and starch liquefaction compositions comprising the mutant amylases, as well as methods of using the mutant amylases.

Claims

1. DNA encoding a mutant alpha-amylase, the mutant alpha-amylase derived from Bacillus and comprising:

(a) a substitution of an amino acid selected from the group consisting of threonine, leucine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, phenylalanine, proline, serine, valine, histidine and glutamine or a deletion of a methionine residue at a position equivalent to M197 in Bacillus licheniformis alpha-amylase; and

/or

(b) a substitution of an amino acid selected from the group consisting of leucine, threonine, asparagine, aspartate, serine, valine, and isoleucine for a methionine residue at a position equivalent to M15 in Bacillus licheniformis alpha amylase.

2. Expression vectors encoding the DNA of claim 1.

3. Host cells transformed with the expression vector of claim 2.

4. A method of producing a mutant alpha-amylase with the host cell of claim 3 comprising the steps of:

(a) preparing a culture medium useful for the growth and cultivation of said host cell;

(b) cultivating said host cell under appropriate conditions to induce production of said alpha-amylase by said host cell; and

(c) optionally separating said alpha-amylase from said host cells.

5. The DNA according to claim 1, wherein said alpha amylase further comprises a substitution at an amino acid equivalent to W138 in Bacillus licheniformis alpha-amylase.

6. DNA encoding an alpha amylase comprising an amino acid sequence corresponding to Seq. ID No. 37.

7. Expression vectors encoding the DNA of claim 6.

8. Host cells transformed with the expression vector of claim 7.

9. DNA encoding a mutant alpha amylase, wherein said alpha-amylase has an amino acid sequence of SEQ ID NO:

37 with a deletion or substitution of amino acid residue M15, M197 and/or W138.

10. Expression vectors encoding the DNA of claim 9.

11. Host cells transformed with the expression vector of claim 10.