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Use and production of storage-stable neutral metalloprotease

a neutral metalloprotease and production technology, applied in the field of use and production of storage-stable neutral metalloprotease, can solve the problems of difficult formulation and use of cleaning compositions, difficult to completely remove stains, and difficult to achieve the effect of improving storage stability

Inactive Publication Date: 2008-11-27
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]In some particularly preferred embodiments, these variants have improved performance as compared to wild-type B. amyloliquefaciens neutral metalloprotease. The present invention also provides neutral metalloprotease variants having at least one improved property as compared to the wild-type neutral metalloprotease. In some additional particularly preferred embodiments, these variants have improved stability as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In some further preferred embodiments, these variants have improved thermostability as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In yet additional preferred embodiments, these variants have improved performance under lower or higher pH conditions, as compared to wild-type B. amyloliquefaciens neutral metalloprotease.
[0017]The present invention also provides variant neutral metalloproteases, wherein the neutral metalloproteases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO:18, and wherein variant metalloproteases have better performance in at least one property, as compared to wild-type B. amyloliquefaciens metalloprotease.
[0051]The present invention also provides variant neutral metalloproteases having improved performance as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In some preferred embodiments, the improved performance comprises improved thermostability, as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In alternative preferred embodiments, the improved performance comprises improved performance under lower or higher pH conditions, as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In additional preferred embodiments, the improved performance comprises improved autolytic stability, as compared to wild-type B. amyloliquefaciens neutral metalloprotease. In some particularly preferred embodiments, the enzyme compositions of the present invention have comparable or improved wash performance, as compared to presently used neutral metalloproteases. Other objects and advantages of the present invention are apparent herein.

Problems solved by technology

Despite the complexity of current detergents, there are many stains that are difficult to completely remove.
Furthermore, there is often residue build-up, which results in discoloration (e.g., yellowing) and diminished aesthetics due to incomplete cleaning.
These problems are compounded by the increased use of low (e.g., cold water) wash temperatures and shorter washing cycles.
These stains present difficult challenges to the formulation and use of cleaning compositions.
In addition, colored garments tend to wear and show appearance losses.
A portion of this color loss is due to abrasion in the laundering process, particularly in automated washing and drying machines.
Moreover, tensile strength loss of fabric appears to be an unavoidable result of mechanical and chemical action due to use, wearing, and / or washing and drying.

Method used

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  • Use and production of storage-stable neutral metalloprotease
  • Use and production of storage-stable neutral metalloprotease
  • Use and production of storage-stable neutral metalloprotease

Examples

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example 1

Cloning of the Neutral Metalloprotease Gene from B. amyloliquefaciens

[0277]In this Example, methods used to clone the B. amyloliquefaciens neutral metalloprotease gene are described. The gene-encoding neutral metalloprotease was cloned from B. amyloliquefaciens using well-established methods in this art. The non-exempt (i.e., the strain carries extrageneric DNA (besides the chloramphenicol selectable marker which is allowed in an exempt strain), specifically the plasmid pJM102 sequences) strain BC91504 (aprE / nprE-pJM102 in BG3594::comK) carries the B. subtilis aprE promoter and signal sequence fused to B. amyloliquefaciens nprE propeptide / mature gene in integrating plasmid pJM102.

[0278]The following two sequences (SEQ ID NO:1 and SEQ ID NO:2) of B. subtilis and B. amyloliquefaciens were generated via PCR with the oligonucleotide primers corresponding to the underlined sequences.

[0279]B subtilis chromosomal EcoRI restriction site (GAATTC) and aprE start codon (GTG) and B. amylolique...

example 2

Expression and Fermentation of the Purified MULTIFECT® Neutral and Recombinant Neutral Metalloprotease (nprE)

[0294]The recombinant Bacillus subtilis produced as described in Example I was cultivated by conventional batch fermentation in a nutrient medium as described below. One glycerol vial (prepared as described in Example 1) of B. subtilis culture containing the B. amyloliquefaciens neutral metalloprotease was used to inoculate 600 ml of SBG1% medium containing 200 mg / L chloramphenicol. The cultures were grown for 48 hours at 37° C., after which time, the culture fluid was recovered by centrifugation at 12,000 rpm, as known in the art. This procedure was done in duplicate. The final enzyme concentrations obtained were in the range of about 1.4 and 2 g / L.

example 3

Purification and Characterization of Neutral Metalloprotease

[0295]This Example describes the methods used to purify the neutral metalloprotease expressed by the organisms described in Example 2. After 36 hours of incubation at 37° C., the fermentation broth was recovered and centrifuged at 12 000 rpm (SORVALL® centrifuge model RC5B). The secreted neutral metalloproteases were isolated from the culture fluid and concentrated approximately 10-fold using an Amicon filter system 8400 with a BES (polyethersulfone) 10 kDa cutoff.

[0296]The concentrated supernatant was dialyzed overnight at 4° C. against 25 mM MES buffer, pH 5.4, containing 10 mM NaCl. The dialysate was then loaded onto a cation-exchange column Porous HS20 (total volume ˜83 mL; binding capacity ˜4.5 g protein / mL column; Waters) as described below. The column was pre-equilibrated with 25 mM MES buffer, pH 5.4, containing 10 mM NaCl. Then, approximately 200-300 mL of sample was loaded onto the column. The bound protein was el...

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Abstract

The present invention provides methods and compositions comprising at least one neutral metalloprotease enzyme that has improved storage stability. In some embodiments, the neutral metalloprotease finds use in cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions comprising neutral metalloprotease(s) obtained from Bacillus sp. In some more particularly preferred embodiments, the neutral metalloprotease is obtained from B. amyloliquefaciens. In still further preferred embodiments, the neutral metalloprotease is a variant of the B. amyloliquefaciens neutral metalloprotease. In yet additional embodiments, the neutral metalloprotease is a homolog of the B. amyloliquefaciens neutral metalloprotease. The present invention finds particular use in applications including, but not limited to cleaning, bleaching and disinfecting.

Description

[0001]The present application claims priority to pending U.S. Provisional Patent Application Ser. No. 60 / 726,448, filed Oct. 12, 2005.FIELD OF THE INVENTION[0002]The present invention provides methods and compositions comprising at least one neutral metalloprotease enzyme that has improved storage stability. In some embodiments, the neutral metalloprotease finds use in cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions comprising neutral metalloprotease(s) obtained from Bacillus sp. In some more particularly preferred embodiments, the neutral metalloprotease is obtained from B. amyloliquefaciens. In still further preferred embodiments, the neutral metalloprotease is a variant of the B. amyloliquefaciens neutral metalloprotease. In yet additional embodiments, the neutral metalloprotease is a homolog of the B. amyloliquefaciens neutral metalloprotease. The present invention finds particular use in applic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C11D7/42C12N9/54C07H21/00C12N15/63C12S9/00C12S11/00C12N1/21C07K14/00C12N15/00A23K10/16A23K20/189D06L4/40
CPCC11D3/386C11D3/38663C12N9/54C12N9/96C12Q1/6876C14C11/00D06M16/003C12Y304/24C11D1/02
Inventor SHAW, ANDREWWALLACE, LOUISEESTELL, DAVID A.HOMMES, RONALDUS W.J.LEE, SANG-KYUOH, HIROSHISADLOWSKI, EUGENE S.
Owner DANISCO US INC
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