Multiply-substituted protease variants with altered net charge for use in detergents

A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components in the wash water comprising:

• substituting an amino acid residue at one or more position in a precursor subtilisin wherein the substitution alters the overall charge of the precursor subtilisin to make a subtilisin variant having a more positive charge or less negative charge compared to the precursor;
• b) testing the variant in medium detergent system having between about 800 ppm and about 2000 ppm detergent components present in the wash water by comprising the ability of the precursor and the variant to remove a stain wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0;
• and
• c) producing said variant having improved effectiveness.

A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components present in the wash water comprising:

• a) substituting an amino acid residue at one or more position in a precursor subtilisin wherein the substitution alters the overall charge of the precursor to make a subtilisin variant having a more negative charge or less positive charge compared to the precursor;
• b) testing the variant in a medium detergent system having between about 800 ppm and about 2000 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0;
• and
• c) producing said variant having improved effectiveness.

A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in both a low detergent concentration system and a high detergent concentration system comprising:

• a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor subtilisin;
• b) substituting an amino acid residue at one or more positions in the precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor subtilisin;
• c) obtaining a subtilisin variant of step a) and step b) wherein the overall change in charge compared to the precursor is neutral or positive;
• d) testing the variant obtained in step c) in both a low detergent concentration system having less than about 800 ppm detergent components present in the wash water and a high detergent concentration system having greater than about 2000 ppm detergent components present in the wash water;
• and
• e) selecting the variant having a higher stain release effectiveness than said precursor subtilisin in both the low concentration system and the high concentration system wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.

A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water, comprising:

• a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor;
• b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative;
• c) testing the variant obtained in step b) in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water;
• and
• selecting the variant having a higher stain release effectiveness in the low detergent concentration system than said precursor or wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.

A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system comprising:

• a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor protease;
• b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative;
• c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm and about 200 ppm detergent components present in the wash water;
• and
• d) selecting the variant having a higher stain release effectiveness in the medium detergent concentration system than said precursor wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.

A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system, comprising:

• a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor;
• b) obtaining a subtilisin protease variant of step a) wherein the overall change in charge compared to the precursor is positive;
• c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm to about 2000 ppm detergent components present in the wash water;
• and
• d) selecting the variant having a stain release effectiveness in the medium detergent concentration system than said precursor wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.