Detergents containing amylase and protease

Details Detergents containing amylase and protease Detergents containing amylase and protease

2001-02-09

2002-04-30

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

C510S320000, C510S392000, C510S530000, C435S202000, C435S203000, C435S219000, C435S220000, C435S221000

Reexamination Certificate

active

06380147

ABSTRACT:

The present invention relates to enzyme containing detergents comprising besides customary constituents an amylase from
Bacillus amyloliquefaciens
and a certain protease.
Amylases have the function of facilitating the removal of starchy stains by means of catalytic hydrolysis of the starch polysaccharide, and have been used for this purpose for a fairly long time in dishwashing detergents, but also in detergents for use in textile laundering. In by far the great majority of cases the amylase involved has comprised a heat-stable amylase from
Bacillus licheniformis
, which is customary in commerce under the designation Termamyl®, for example. More recently, there has been increased use in such compositions of genetically manipulated amylases; that is, amylases whose amino acid sequence has been altered, using gene technology methods, in comparison to naturally occurring amylases. As well as increasing their capacity to perform, the objective of genetically modifying amylases is essentially to enhance the stability of the enzyme, especially their stability to attack by oxidizing agents. One approach toward achieving this objective, which was proposed in International Patent Application WO 94/18314, consists in removing particularly oxidation-susceptible amino acids, such as methionine, tryptophan, cysteine or tyrosine, from the amino acid sequence of the amylase, or replacing them by other amino acids more stable to oxidation. A similar approach is proposed in International Patent Application WO 95/21247, which recommends replacing at least one methionine in the amylase amino acid sequence by an amino acid which is neither methionine nor cysteine.
Although such genetic modifications may lead to improved amylase stability under certain application conditions, they do not help to increase the contribution of the amylase to the wash or cleaning performance of corresponding compositions comprising the amylase.
Besides amylases, other enzymes, especially proteolytic enzymes, are being used to an increased extent in laundry detergents, laundering assistants, and cleaning products. Those used at present comprise almost exclusively proteases from the subtilisin family. This family comprises extracellular proteins having a molecular weight in the range from about 20,000 to 45,000. Subtilisins are relatively unspecific enzymes which in addition to the hydrolytic effect on peptide bonds also have esterolytic properties (M. Bahn and R. D. Schmidt, Biotec 1, 119, 1987). Many representatives of the subtilisins have been the subject of precise physical and chemical characterization. Their three-dimensional structure is often known in detail through X-ray structural analysis. This has provided the prerequisites for molecular modeling and so-called protein engineering in the form of site-directed mutagenesis (Kraut, Ann. Rev. Biochem. 46, 331-358, 1977). Genetic modifications of proteases have been described on numerous occasions; in June 1991, for instance, there were already 219 known protein variants of the subtilisin obtained by protein engineering (A. Recktenwald et al., J. Biotechnol. 28, 1-23, 1993). The majority of these variants have been produced in order to enhance the stability of the proteases.
A protease from the subtilisin family which is active and stable under strongly alkaline conditions may be produced as described in International Patent Application WO 91/02792 in
Bacillus lentus
(DSM 5483). This
Bacillus lentus
alkaline protease (BLAP) can be produced by fermenting
Bacillus licheniformis
transformed using an expression plasmid which carries the gene for BLAP under the control of the promoter from
Bacillus licheniformis
ATCC 53926. Both the composition and three-dimensional structure of BLAP are known (D. W. Godette et al., J. Mol. Biol. 228, 580-595, 1992). This protease is characterized by the 269 amino acid sequence described in the cited literature, a calculated molecular weight of 26,823 daltons, and a theoretical isoelectric point of 9.7. Variants, accessible by mutation, of this
Bacillus lentus
DSM 5483 protease are described in U.S. Pat. No. 5,340,735. They include protease enzymes which lead to particularly low substrate damage, or destruction of the fiber assemblies, when textiles comprising proteinogenic fibers—for example, sheetlike textile structures of natural silk or wool—are laundered, especially when they are laundered repeatedly, and do so with no loss in cleaning performance.
It has surprisingly now been found that the combination of an optionally genetically manipulated protease from
Bacillus lentus
as described above and a naturally occurring &agr;-amylase leads to unexpectedly synergistic performance improvements when used in detergents.
The invention accordingly provides an amylase and protease containing detergent which comprises, in addition to customary ingredients compatible with such enzymes, &agr;-amylase from
Bacillus amyloliquefaciens
and protease from
Bacillus lentus
, which protease may optionally have been genetically manipulated.
The invention further provides for the use of a combination of &agr;-amylase from
Bacillus amyloliquefaciens
and protease from
Bacillus lentus
, which protease may optionally have been genetically manipulated, for enhancing the cleaning performance of detergents, especially with respect to starchy stains, when used in detergent solutions, especially aqueous detergent solutions. &agr;-Amylase from
Bacillus amyloliquefaciens
has been known for a long time, for example, from the U.S. Pat. No. 1,227,374. It is available commercially, for example, under the designation Amylase BAN®.
A composition of the invention contains preferably from 0.001 mg to 0.5 mg, in. particular from 0.02 mg to 0.3 mg, of amylolytic protein per gram of the overall composition. The protein concentration may be determined using known methods, such as the bicinchonic acid technique (BCA technique, Pierce Chemical Co., Rockford, Ill.) or the Biuret technique (A. G. Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem. 177, 751-766, 1948).
The composition preferably has a proteolytic activity in the range from about 100 PU/g to about 10,000 PU/g, in particular from 300 PU/g to 8000 PU/g. The protease activity is determined in accordance with the standardized method described below, as described in Tenside 7 (1970), 125: a solution of 12 g/l casein and 30 mM sodium tripolyphosphate in water of hardness 15° dH [German hardness] (containing 0.058% by weight CaCl
2
.2H
2
O, 0.028% by weight MgCl
2
.6H
2
O and 0.042% by weight NaHCO
3
) is heated to 70° C. and the pH is adjusted to 8.5 at 50° C. by adding 0.1 N NaOH. 200 ml of a solution of the test enzyme in 2 percent strength by weight sodium tripolyphosphate buffer solution (pH 8.5) are added to 600 ml of the substrate solution. The reaction mixture is incubated at 50° C. for 15 minutes. The reaction is then stopped by adding 500 ml TCA solution (0.44 M trichloroacetic acid and 0.22 M sodium acetate in 3 percent strength by volume acetic acid) and cooling (ice bath at 0° C., 15 minutes). The TCA-insoluble protein is removed by centrifugation and 900 ml of the supernatant are diluted with 300 ml of 2 N NaOH. The absorbance of this solution at 290 nm is determined using an absorption spectrometer, the blank absorbance value being determined by measuring a centrifuged solution prepared by mixing 600 ml of the abovementioned TCA solution with 600 ml of the abovementioned substrate solution and then adding the enzyme solution. The proteolytic activity of a protease solution which under the stated measurement conditions brings about an absorbance of 0.500 OD is defined as 10 PU (protease units) per ml.
The proteases which can be used in accordance with the invention include not only the naturally occurring protease from
Bacillus lentus
but also genetically modified proteases of the abovementioned BLAP type in which in position 211 (BLAP numeration) the amino acid leucine (L in the common one-letter code) present at this position in the wild type protease has been replac

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