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Mutant alpha-amylases

Inactive Publication Date: 2005-08-18
KAO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] It is an object of the present invention to provide an α-amylase which is a liquefying alkaline α-amylase that has high activity on the alkaline side and is stable to both chelating component and oxidation bleaching component, and has excellent heat resistance.
[0008] The present inventors have acquired various mutant enzymes as to liquefying alkaline α-amylases and investigated them. As a result, it has been found that when a mutation is introduced into a specified amino acid residue in the amino acid sequence (SEQ ID NO:1) of amylase derived from KSM-K38, the heat resistance of the enzyme is improved without losing its properties such as resistance to chelating agents and resistance to oxidizing agents and high specific activity in an alkaline region, and that the heat resistance can be further improved by combining such mutations.

Problems solved by technology

However, said enzymes exhibit inactivation at a temperature of 50° C. or higher, and so the heat resistance thereof have been somewhat insufficient in view of the fact that cleaning of clothing and tableware is generally conducted at about 10 to 60° C.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

referential example 1

Screening of Liquefying Alkaline Amylase:

[0036] Soil (about 0.5 g) was suspended in sterilized water and subjected to a heat treatment at 80° C. for 15 minutes. A supernatant of the heat-treated suspension was suitably diluted with sterilized water, and the resultant dilute solution was coated on an agar medium (Medium A) for separation. Culture was then conducted at 30° C. for 2 days to form colonies. Those on the peripheries of which transparent halo based on amylolysis had been formed were screened, and isolated as amylase-producing bacteria. Further, the thus-isolated bacteria were inoculated on Medium B and subjected to aerobic shaking culture at 30° C. for 2 days. After the culture, the resistance performance to a chelating agent (EDTA) of a supernatant centrifugally separated was determined, and its optimum pH was further measured to screen the liquefying alkaline α-amylase-producing bacteria.

[0037]Bacillus sp. KSM-K38 (FERM BP-6946) and Bacillus sp. KSM-K36 (FERM BP-6945)...

reference example 2

Culture of KSM-K38 and KSM-K36 Strains:

[0039] The KSM-K38 or KSM-K36 strain was inoculated on the liquid medium B used in Referential Example 1 to conduct shaking culture at 30° C. for 2 days. The amylase activity (at pH 8.5) of a supernatant centrifugally separated was determined. As a result, these strains had activities of 557 U and 1177 U per liter of the medium, respectively.

referential example 3

Purification of Liquefying Alkaline Amylase:

[0040] Ammonium sulfate was added to the resultant culture supernatant of the KSM-38 strain obtained in Referential Example 2 to 80% saturation. After stirring the resultant mixture, precipitate formed was collected and dissolved in 10 mM Tris-hydrochloride buffer (pH: 7.5) containing 2 mM CaCl2 and dialyzed overnight against the same buffer. The dialyzate thus obtained was passed through a DEAE-Toyopearl 650M column equilibrated with the same buffer and caused to be adsorbed on the column, and the intended enzyme was eluted with the same buffer by 0-1 M gradient of sodium chloride concentration. After the active fraction was dialyzed against the same buffer, an active fraction obtained by gel filtration column chromatography was dialyzed against the above-described buffer, thereby obtaining a purified enzyme which gave a single band on both polyacrylamide gel electrophoresis (gel concentration: 10%) and sodium dodecyl sulfate (SDS) elec...

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Abstract

The invention relates to a mutant α-amylase obtained by making replacement or deletion of at least one of amino acid residues such as the 167th Gln, 169th Tyr and 178th Ala in the amino acid sequence set forth in SEQ ID NO:1 in an α-amylase having said amino acid sequence, or an α-amylase having a homology of at least 70% to said amino acid sequence, a gene encoding the mutant α-amylase, a vector, transformed cells, a process for producing a mutant α-amylase, comprising culturing the transformed cells, and a detergent composition comprising the mutant α-amylase. The mutant α-amylase of the invention has excellent properties of high resistance to chelating agents, high specific activity in an alkaline region and excellent stability to heat, and is hence useful for detergents for automatic dish washer, laundry detergents and the like.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates to mutant liquefying alkaline α-amylases which have excellent heat resistance, and are particularly useful as enzymes for detergents, and genes thereof [0003] 2. Description of the Background Art [0004] When an α-amylase [EC.3.2.1.1] is used as an enzyme for detergents, it has heretofore been said that a liquefying alkaline α-amylase, which can decompose starch at random and is stable to alkali and also to both chelating component and oxidation bleaching component, is preferred. However, in liquefying amylases, a calcium ion is generally important for maintaining the structure of the enzymes, and the stability thereof is lowered in the presence of a chelating agent. Besides, most of such enzymes have had the optimum pH in a neutral to weakly acidic range. [0005] Under the foregoing circumstances, the present inventors found that enzymes produced by alkaliphilic Bacillus sp. KSM-K38 (FER...

Claims

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Application Information

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IPC IPC(8): C11D3/386C12N1/21C12N9/28C12N15/56
CPCC12N9/2417C11D3/386
Inventor ENDO, KEIJIIGARASHI, KAZUAKIHAYASHI, YASUHIROHAGIHARA, HIROSHIOZAKI, KATSUYA
Owner KAO CORP
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