Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – Enzyme component of specific activity or source
Reexamination Certificate
1999-09-23
2002-11-26
Gupta, Yogendra N. (Department: 1751)
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
Enzyme component of specific activity or source
C510S300000, C510S320000, C510S530000, C435S113000, C435S440000, C435S442000, C435S187000, C435S202000, C435S204000, C435S263000
Reexamination Certificate
active
06486113
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mutant liquefying &agr;-amylases which have the optimum pH in an alkaline range and excellent resistance to oxidizing agents and are particularly useful as enzymes for detergents comprising an oxidizing agent, and genes thereof.
BACKGROUND ART
Various enzymes are incorporated into detergents for the purpose of enhancing detergency, and it is considered to incorporate an &agr;-amylase against, for example, starch smears. However, the &agr;-amylase incorporated into a detergent must be an alkaline &agr;-amylase, since the detergent comprises a surfactant, and the pH of a detergent solution is in an alkaline range.
By the way, detergents, in which an oxidation bleaching component is incorporated to expect not only detergency against dirt, but also a bleaching action, have been recently marketed. It is considered that the &agr;-amylase is also incorporated into such an oxidation bleaching agent-containing detergent. However, the usual &agr;-amylase is easy to inactivate in the presence of an oxidizing agent and has hence been unable to be incorporated into the oxidation bleaching agent-containing detergent.
Researches have been made to impart resistance to oxidizing agents to such an &agr;-amylase. More specifically, WO 94/02597 has provided oxidizing agent-resistant mutant proteins by substituting a non-oxidizing amino acid, particularly, leucine (Leu), threonine (Thr) or glycine (Gly) for a methionine residue of an &agr;-amylase derived from
B. licheniformis.
WO 94/18314 and WO 96/30481 have reported that when Met residues corresponding to the 197-position and 15-position among methionine residues in the same enzyme as described above are replaced by, in particular, Ala, Ile or Thr and Leu, Thr, Asp, Ser, Val or Ile, respectively, the resistance to oxidizing agents and heat stability of the enzyme are enhanced. However, these mutant &agr;-amylases are all enzymes having the optimum pH in a neutral to acidic range, and there has thus been a demand for development of an alkaline &agr;-amylase having a higher optimum pH for the purpose of using in detergents.
On the other hand, as a technique for imparting resistance to oxidizing agents to an alkaline &agr;-amylase, WO 96/23873 has only reported oxidizing agent-resistant mutant &agr;-amylases obtained by modifying SEQ ID NO:1 (NCIB 12512) encoding an &agr;-amylase. According to WO 96/23873, taking into account the results of the &agr;-amylase derived from
B. licheniformis
(WO 94/18314), and the like, it is described that when Met corresponding to the 202-position in
FIG. 2
of NCIB 12512 is replaced by Leu, Phe, Ala, Thr, Val or Ser, the resultant &agr;-amylase mutant becomes oxidizing agent-resistant to a treatment with 200 mM H
2
O
2
. It has however been reported that since its resistance to oxidation is insufficient, Arg at the 181-position and Gly at the 182-position are deleted in addition to this mutation (Suzuki et al., J. Biol. Chem., 264, 18933-18938, 1989) to more stabilize the mutant. However, the half-lives (t
½
) of enzymatic activity of the mutant &agr;-amylases obtained in such a manner are only in ranges of 10 to 20 minutes for the former and 10 to 30 minutes for the latter, and so they are not satisfactory as enzymes for incorporating into detergents for both resistance to oxidizing agents and lastingness thereof.
Accordingly, it is an object of the present invention to provide an &agr;-amylase which has the optimum pH in an alkaline range and lasting and strong resistance to oxidizing agents, a gene thereof, and a detergent composition comprising such an &agr;-amylase.
DISCLOSURE OF THE INVENTION
In view of the foregoing circumstances, the present inventors have paid attention to an enzyme produced by Bacillus sp. KSM-AP 1378 (WO 94/26881), which is a sort of liquefying alkaline &agr;-amylase, and carried out various investigations. As a result, it has been found that when at least one methionine residue at the 202-position or a position homologous thereto in the amino acid sequence set forth in SEQ ID NO:1 is deleted or replaced by another arbitrary amino acid residue, the resultant &agr;-amylase mutant comes to have strong and lasting resistance to oxidizing agents and has an excellent amylase activity in an alkaline pH range, thus leading to completion of the present invention.
According to the present invention, there is thus provided a mutant &agr;-amylase having an amino acid sequence obtained by making deletion or replacement by another arbitrary amino acid residue of a methionine residue at the 202-position or a position homologous thereto in the amino acid sequence set forth in SEQ ID NO:1, which constitutes a liquefying alkaline &agr;-amylase, or in an amino acid sequence having a homology of at least 95.2% to said amino acid sequence.
According to the present invention, there is also provided a gene encoding the mutant &agr;-amylase.
According to the present invention, there is further provided a detergent composition comprising the mutant &agr;-amylase.
REFERENCES:
patent: 6093562 (2000-07-01), Bisgard-Frantzen et al.
patent: WO 96/23873 (1996-08-01), None
patent: 8506491 (1996-07-01), None
patent: 8336392 (1996-12-01), None
patent: WO 9418314 (1994-08-01), None
patent: 94 26881 (1994-11-01), None
patent: 95 26397 (1995-10-01), None
patent: 96 23873 (1996-08-01), None
patent: WO 9630481 (1996-10-01), None
patent: 94 02597 (1997-02-01), None
The Journal of Biological Chemistry, vol. 264, No. 32, Issue of Nov. 15, pp. 18933-18938, 1989, Suzuki et al, Jul. 5, 1989.
A. Tsukamoto et al., Biochem. Biophys. Res. Commun., vol. 151 (1), Jan. 1988 pp. 25-31.
P.L. Jorgensen et al., FEMS Microbiology Letters, vol. 77 (2&3), Jan. 1991, pp. 271-275.
G.L. Gray et al., J. Bacteriol., vol. 166(2), May 1986, pp. 635-643.
Araki Hiroyuki
Hagihara Hiroshi
Hatada Yuji
Hayashi Yasuhiro
Igarashi Kazuaki
Birch & Stewart Kolasch & Birch, LLP
Gupta Yogendra N.
Kao Corporation
Mruk Brian P.
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