# US 5,352,603 A · Highly alkaline proteases > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,352,603 A - Document ID: US5352603A - Status: Granted - Kind: A - Application number: 07/661,378 - Filed: 1991-02-28 - Granted: 1994-10-04 - Earliest claimed: 1989-08-31 - Assignees: Kali-Chemie Aktiengesellschaft; Gesellschaft fuer Biotechnologische Forschung mbH - Inventors: Roman Vetter; Detlef Wilke; Antoine Amory; Andre Clippe; Dietmar Schomburg; Wolfgang Aehle - Web page: https://patentgrep.com/patent/US5352603A ## CPC - C12N 9/54: bacteria being Bacillus - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Novel, optimized highly alkaline proteases which are suitable for use in detergent formulations are prepared by employing microorganisms transformed by mutated DNA sequences. The mutated sequences are obtained starting from DNA sequences which code for highly alkaline protease usually produced by Bacillus species by altering these DNA sequences in defined positions by directed mutagenesis (point mutation) in such a way that the codon in which the point mutation is located now codes for an amino acid which is more strongly basic than the original amino acid. The result is highly alkaline proteases in which original amino acids have been replaced by more strongly basic amino acids, preferably by the amino acids lysine or arginine. Synthetic oligonucleotides, DNA sequences, vectors and transformed microorganisms which are used for generating and obtaining the optimized highly alkaline protease are also described. ## Claims ### Claim 1 Highly alkaline protease, which has an amino-acid sequence having at least 80% sequence similarity with the protease amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2) commencing at residue number 1 at the `protease` mark and differing from said FIG. 1 (SEQ. ID. NO. 2) sequence in from one to three of the positions 18, 42, 49, 57, 96, 107, 114, 115, 135, 138, 166, 176, 179, 188, 189, 198, 238, 255, or 266 in FIG. 1 (SEQ. ID. NO. 2), or sequence positions corresponding thereto, in that the amino acid located in the differing position has been replaced by a more strongly basic amino acid selected from the group consisting of lysine and arginine. ### Claim 2 (depends on 1) Highly alkaline protease according to claim 1, in which an amino acid in 1 or 2 of the positions indicated in claim 1, has been replaced by a more strongly basic amino acid. ### Claim 3 (depends on 1) Highly alkaline protease according to claim 1, which has a pH optimum from 10 to 12.5 and a molecular weight of 26,000 to 28,000 g/mole. ### Claim 4 (depends on 1) Highly alkaline protease according to claim 1, which has an amino-acid sequence having at least 90% sequence similarity with the amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2). ### Claim 5 (depends on 4) Highly alkaline protease according to claim 4, which has an amino-acid sequence having at least 95% sequence similarity with the amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2). ### Claim 6 (depends on 1) A detergent composition comprising a highly alkaline protease according to claim 1, and at least one conventional detergent ingredient. ### Claim 7 (depends on 6) A detergent composition according to claim 6, comprising at least one conventional detergent ingredient selected from the group consisting of surfactants, builders and bleaching agents. ### Claim 8 (depends on 7) A detergent composition according to claim 7, wherein said composition further comprises at least one detergent additive selected from the group consisting of enhancers, enzyme stabilizers, anti-redeposition agents, compatibility promoters, complexing and chelating agents, foam regulators, optical brighteners, opacifying agents, corrosion inhibitors, anti-electrostatic agents, dyestuffs, bactericides, bleaching agent activators, and peracid bleaching agent precursors. ### Claim 9 Highly alkaline protease, which has an amino-acid sequence having at least 80% sequence similarity with the protease amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2) commencing at residue number 1 at the `protease` mark and differing from said FIG. 1 (SEQ. ID. NO. 2) sequence in from one to three of the positions 18, 49, 57, 107, 114, 115, 135, 138, 166, 176, 179, 188, 189, 198, 238, or 255 in FIG. 1 (SEQ. ID. NO. 2), or sequence positions corresponding thereto, in that the amino acid located in the differing position has been replaced by a more strongly basic amino acid selected from the group consisting of lysine and arginine. ### Claim 10 (depends on 1, 9) Highly alkaline protease according to claim 9, in which an amino acid in 1 or 2 of the positions indicated in claim 1, has been replaced by a more strongly basic amino acid. ### Claim 11 (depends on 9) Highly alkaline protease according to claim 9, which has a pH optimum from 10 to 12.5 and a molecular weight of 26,000 to 28,000 g/mole. ### Claim 12 (depends on 9) Highly alkaline protease according to claim 9, which has an amino-acid sequence having at least 90% sequence similarity with the amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2). ### Claim 13 (depends on 12) Highly alkaline protease according to claim 12, which has an amino-acid sequence having at least 95% sequence similarity with the amino-acid sequence illustrated in FIG. 1 (SEQ. ID. NO. 2). ### Claim 14 (depends on 9) A detergent composition comprising a highly alkaline protease according to claim 9, and at least one conventional detergent ingredient. ### Claim 15 (depends on 14) A detergent composition according to claim 14, comprising at least one conventional detergent ingredient selected from the group consisting of surfactants, builders and bleaching agents. ### Claim 16 (depends on 15) A detergent composition according to claim 15 wherein said composition further comprises at least one detergent additive selected from the group consisting of enhancers, enzyme stabilizers, anti-redeposition agents, compatibility promoters, complexing and chelating agents, foam regulators, optical brighteners, opacifying agents, corrosion inhibitors, anti-electrostatic agents, dyestuffs, bactericides, bleaching agent activators, and peracid bleaching agent precursors.