# US 5,707,848 A · Calcium free subtilisin mutants

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 5,707,848 A
- Document ID: US5707848A
- Status: Granted
- Kind: A
- Application number: 08/562,833
- Filed: 1995-11-27
- Granted: 1998-01-13
- Earliest claimed: 1993-06-01
- Assignees: University of Maryland
- Inventors: Philip N. Bryan; Patrick A. Alexander; Susan L. Strausberg
- Web page: https://patentgrep.com/patent/US5707848A

## 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 calcium free subtilisin mutants are taught, in particular a subtilisin which has been mutated to eliminate amino acids 75-83.

## Claims

### Claim 1

A DNA comprising a nucleic acid sequence encoding a mutated subtilisin protein which has been modified to lack the ability to bind calcium at a high affinity calcium binding site, wherein said encoded subtilisin mutant comprises a deletion of amino acids corresponding to the amino acids at positions 75-83 of mature subtilisin BPN'.

### Claim 2 (depends on 1)

The DNA of claim 1, wherein the nucleic acid sequence encoding said mutant subtilisin comprises a deletion of nucleotides corresponding to the nucleotides at positions 993-1019 of SEQ ID NO:1.

### Claim 3 (depends on 1)

The DNA of claim 1 further comprising a transcriptional promoter and other regulatory DNA sequence elements suitable for expression of the encoded mutant subtilisin in a transformed host cell.

### Claim 4 (depends on 3)

The DNA of claim 3, wherein said encoded subtilisin mutant is selected from the group consisting of a subtilisin BPN' mutant, a subtilisin Carlsberg mutant, a subtilisin DY mutant, a subtilisin amylosacchariticus mutant, and a subtilisin mesentericopeptidase mutant.

### Claim 5 (depends on 1)

The DNA of claim 1 further comprising one or more substitution mutations which provide for enhanced thermal stability or which provide for the restoration of cooperativity of folding of the subtilisin mutant protein.

### Claim 6 (depends on 5)

The DNA of claim 5 which, upon expression, results in a subtilisin BPN' mutant selected from the group consisting of S15, S39 and S47.

### Claim 7 (depends on 6)

The DNA of claim 6 which, upon expression, results in the mutant subtilisin BPN' mutant S47.

### Claim 8 (depends on 1)

An expression vector comprising the DNA of claim 1.

### Claim 9 (depends on 2)

An expression vector comprising the DNA of claim 2.

### Claim 10 (depends on 3)

An expression vector comprising the DNA of claim 3.

### Claim 11 (depends on 4)

An expression vector comprising the DNA of claim 4.

### Claim 12 (depends on 5)

An expression vector comprising the DNA of claim 5.

### Claim 13 (depends on 6)

An expression vector comprising the DNA of claim 6.

### Claim 14 (depends on 7)

An expression vector comprising the DNA of claim 7.

### Claim 15 (depends on 1)

A transformed host cell comprising the DNA of claim 1.

### Claim 16 (depends on 2)

A transformed host cell comprising the DNA of claim 2.

### Claim 17 (depends on 3)

A transformed host cell comprising the DNA of claim 3.

### Claim 18 (depends on 4)

A transformed host cell comprising the DNA of claim 4.

### Claim 19 (depends on 5)

A transformed host cell comprising the DNA of claim 5.

### Claim 20 (depends on 6)

A transformed host cell comprising the DNA of claim 6.

### Claim 21 (depends on 7)

A transformed host cell comprising the DNA of claim 7.

### Claim 22 (depends on 1)

A recombinant method for the production of a subtilisin mutant which lacks the ability to bind calcium at a high affinity calcium binding site comprising:
(a) transforming microbial host cells with an expression vector comprising the DNA of claim 1 which permits the expression of a subtilisin mutant lacking the ability to bind calcium at a high affinity calcium binding site;
(b) culturing said host cells under conditions which provide for the expression of said subtilisin mutant; and,
(c) recovering the expressed subtilisin mutant from said microbial host cells.

### Claim 23 (depends on 22)

The method of claim 22, wherein said subtilisin mutant is selected from the group consisting of a subtilisin BPN' mutant, a subtilisin Carlsberg mutant, a subtilisin DY mutant, a subtilisin amylosacchariticus mutant, and a subtilisin mesentericopeptidase mutant.

### Claim 24 (depends on 23)

The method of claim 23, wherein said subtilisin mutant is the subtilisin BPN' mutant.

### Claim 25 (depends on 24)

The method of claim 24, wherein said subtilisin BPN' mutant DNA comprises one or more additional mutations which provide for enhanced thermal stability or which provide for the restoration of cooperativity of folding of the subtilisin mutant protein.

### Claim 26 (depends on 25)

The method of claim 25, wherein said subtilisin mutant is selected from S15, S39 and S47.

### Claim 27 (depends on 26)

The method of claim 26, wherein said subtilisin mutant is S47.