Alkaline protease, process for the production thereof, use thereof, and microorganism producing the same

• US 5,981,255 A
• Filed: 03/25/1998
• Issued: 11/09/1999
• Est. Priority Date: 11/02/1995
• Status: Expired due to Term

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Nordisk A/S
• Inventors: Miyota, Yoshiaki, Fukuyama, Shiro, Yoneda, Tadashi
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): Monshipouri, Maryam
• Application Number:
  US09/043,722
• Time in Patent Office: 594 Days
• Field of Search: 435/221, 435/252.31, 435/252.5
• US Class Current: 435/221
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C11D 3/38609 Protease or amylase in solid compositions only
• CPC Class Codes: C11D 3/38618 Protease or amylase in liquid compositions only
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/54 bacteria being Bacillus
• CPC Class Codes: C12R 2001/07 Bacillus

Abstract

The present invention provides a protease obtained from Bacillus, which is enzymatically active at alkaline pH and high temperature in the presence of surfactants. In a preferred embodiment, the protease is isolated from Bacillus sp. SD 114 and has a molecular weight of about 29,000 and a pI of about 10.

Claims

1. An isolated protease derived from a Bacillus, wherein said protease:

(i) has an optimal pH for enzymatic activity of about 12 when reacted with casein as a substrate at 30°

C. for 10 minutes, and

(ii) has at least one of the following properties;

(a) Residual enzymatic activity of more than 60% after 30 minutes at 40°

C. in a surfactant solution comprising 50 mM borate buffer, pH 10, 0.1 mM EDTA, and 500 ppm of linear alkylbenzenesulfonate (LAS);

(b) Residual enzymatic activity of more than 40% after 15 minutes at 55°

C. in a buffer solution comprising 50 mM borate buffer, pH 10, and 0.1 mM EDTA;

or

(c) Residual enzymatic activity of more than 20% after 15 minutes at 55°

C. in a surfactant solution comprising 50 mM borate buffer, pH 10, 0.1 mM EDTA, and 500 ppm of LASD.

2. An isolated protease as defined in claim 1, wherein said protease:

(i) is stable at the pH range of 5 to 11 when it is incubated at 30°

C. for 24 hours;

(ii) has an optimal temperature for enzymatic activity of approximately 60°

C. when it is reacted with casein as a substrate at pH 10 for 10 minutes;

(iii) has a molecular weight of 29,000±

2,000 when measured by SDS-polyacrylamide gel electrophoresis; and

(iv) has an isoelectric point of 10.1±

0.5 when measured by isoelectric focusing polyacrylamide gel electrophoresis.

3. An isolated protease as defined in claim 1, wherein said Bacillus is Bacillus sp. SD 114 (Deposit No. FERM BP-5736).

4. An isolated protease which reacts by the Ouchterloney technique with a rabbit antibody raised against a protease as defined in claim 3.

5. A method for producing an alkaline protease as defined in claim 1, said method comprising (i) culturing a microorganism expressing said protease under conditions appropriate for said expression and (ii) recovering said protease from said culture.

6. A method as defined in claim 5, wherein said microorganism is Bacillus sp. SD 114 (FERM BP-5736).

7. An isolated bacterial culture comprising Bacillus sp. SD 114 (FERM BP-5736) or a bacterium derived therefrom, wherein said derived bacterium produces a protease as defined in claim 1.

8. A detergent comprising a protease as defined in claim 1.

9. A detergent as defined in claim 8, wherein said detergent is formulated for automatic dish washers.

10. A method for hydrolyzing a protein into peptides or amino acids said method comprising reacting said protein with a protease as defined in claim 1.

11. An isolated protease derived from Bacillus sp. SD 114, wherein said protease:

(a) has a molecular weight of about 29,000 when measured by SDS-polyacrylamide gel electrophoresis; and

(b) has an isoelectric point of about 10 when measured by isoelectric focusing polyacrylamide gel electrophoresis.