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Compositions and methods comprising a subtilisin variant

a technology of subtilisin and composition, applied in the field of subtilisin variants of bacillus sp., can solve the problems of difficult formulation of liquid or gel forms commonly preferred by consumers for dishwashing detergents, difficult to remove protein-containing soils that are often present on dishware in homes, hospitals, catering industries,

Inactive Publication Date: 2010-06-17
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new version of a protease called subtilisin, which is commonly used in dishwashing detergents. This new version has been designed to improve its performance in the dishwashing process. The invention also includes automatic dishwashing compositions that contain this new subtilisin variant, as well as methods for cleaning dishware using these compositions. The invention also provides a nucleic acid and an expression vector that can be used to produce the new subtilisin variant in large quantities. Overall, the invention provides a more effective and efficient protease for dishwashing detergents.

Problems solved by technology

However, removal of protein-containing soils that are often present on dishware in homes, hospitals, cafeterias, and catering industries is problematic.
In addition, very highly alkaline and chlorine-containing compositions are not considered to be consumer nor environmentally friendly.
However, such compositions have significant drawbacks in that they are difficult to formulate in the liquid or gel forms commonly preferred by consumers for dishwashing detergents.
In addition, alkaline dishwashing compositions are often considered to be irritants.
However, current low pH dishwashing compositions with have proven to be very ineffective at removing proteinaceous soils, even when high concentrations of enzymes (e.g., proteases) are formulated within the dishwashing compositions.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Subtilisin Variants

[0109]Subtilisin variants were prepared by fusion PCR as known in the art (See e.g., US Publn. No. 2006 / 0252155 incorporated herein by reference). Table 1-1 provides the sequences of the primers used for fusion PCR.

TABLE 1-1Primers Used In Fusion PCR*Primer SequencePrimer NameCGCGCTTGAGCTCGATCCAGCGATTTCSacI-Fw(SEQ ID NO: 1)GTCTCCAAGCTTTAACGAGTTGCAGHindIII-Rv(SEQ ID NO: 2)GTTAAAGTATTAGGGGCGAGCGGTNNSGGTTCGGTCS101X-FwAGCTCG(SEQ ID NO: 3)CGAGCTGACCGAACCSNNACCGCTCGCCCCTAATACS101X-RvTTTAAC(SEQ ID NO: 4)GCAATTCAGATCTTCCTTCAGGTTATGACCpHPLT-BglII-Fw(SEQ ID NO: 5)GCATCGAAGATCTGATTGCTTAACTGCTTCpHPLT-BglII-Rv(SEQ ID NO: 6)*The codon for generation of a substitution at position 101, and the restriction enzyme sites are shown in bold.

[0110]A DNA template of a B. clausii PB92 variant (containing the following substitutions G118V+S128L+P129Q+S130A; using BPN′ numbering, and designated herein as “GCI-P040”) was used to generate a PB92 protease variant further compr...

example 2

Production of Subtilisin Variant in Bacillus subtilis

[0118]The subtilisin variant was produced by growing the B. subtilis transformants overnight at 37° C. in 10 ml TSB (tryptone and soy based broth) medium. A 250 μl aliquot of the overnight culture was transferred into 25 ml of a MOPS based defined medium in a 100 ml shake flask and grown at 37° C. for 68 hours. The defined medium was made essentially as known in the art (See, Neidhardt et al., J Bacteriol, 119: 736-747, 1974), except that NH4Cl2, FeSO4, and CaCl2 were left out of the base medium, 3 mM K2HPO4 was used, and the base medium was supplemented with 60 mM urea, 75 g / L glucose, and 1% soytone. Also the micronutrients were made up as a 100× stock containing in one liter, 400 mg FeSO4.7H2O, 100 mg MnSO4.H2O, 100 mg ZnSO4.7H2O, 50 mg CuCl2.2H2O, 100 mg CoCl2.6H2O, 100 mg NaMoO4.2H2O, 100 mg Na2B4O7.10H2O, 10 ml of 1M CaCl2, and 10 ml of 0.5 M sodium citrate. The proteases of interest were isolated from the culture medium.

example 3

Analytical Methods to Determine the Purity of Subtilisin Sample

[0119]In this Example, methods used to determine the purity of the recombinant subtilisin obtained from B. subtilis cultures are described. The protease was considered pure when a single band or peak was found by gel electrophoresis and high performance liquid chromatography (HPLC), respectively.

[0120]Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) was conducted as known in the art (Laemmli, Nature, 227:680-685, 1970). However, prior to denaturation of the protein samples (e.g., 10 min in SDS-containing sample buffer at 100° C.), inactivation of the protease activity was required in order to prevent auto-degradation. Protease inactivation was accomplished by incubating the protein sample with 1 mM PMSF for 30 min at room temperature or by precipitation of the protein with 8% trichloroacetic acid (TCA) for 30 min on ice. Protein samples were subjected to native PAGE carried out a...

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Abstract

The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides automatic dishwashing compositions comprising this serine protease variant.

Description

[0001]The present application claims priority to U.S. Provisional Patent Appln. Ser. No. 61 / 113,561, filed on Nov. 11, 2008, and herein incorporated by reference.FIELD OF THE INVENTION[0002]The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides automatic dishwashing compositions comprising this serine protease variant.BACKGROUND OF THE INVENTION[0003]Typically, traditional domestic and industrial dishwashing compositions rely on a combination of high alkalinity detergent washes and chlorine bleach for cleaning and sanitizing dishware. Such systems generally perform well on bleachable stains. However, removal of protein-containing soils that are often present on dishware in homes, hospitals, cafeterias, and catering industries is problematic. In addition, very highly alkaline and chlorine-containing compositions are not considered to be consumer nor environmentally friendly.[0004]Various attempts have been made to produce dishwas...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C11D3/386C12N9/56
CPCC11D3/38609C11D3/38618C11D3/386C11D3/3942C12N9/54C11D3/38681C11D3/38627C11D3/38654C12Y304/21062
Inventor ESTELL, DAVID A.GOEDEGEBUUR, FRITSPOULOSE, AYROOKARAN J.
Owner DANISCO US INC
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