Metalloprotease from kribbella aluminosa and detergent compositions comprising the metalloprotease
A composition and detergent technology, applied in the directions of detergent compositions, detergent compounding agents, biochemical equipment and methods, etc., can solve the problems of instability and non-commercialization of metalloprotease
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[0355] Materials and Methods
[0356] Test material for washing determination
[0357] Grass stain swatch 062KC was obtained from Warwick Equest Ltd (Unit 55, Consett Business Park, Consett, County Durham, DH8 6BN, UK )acquired,
[0358] The remaining small swatches were obtained from the Center For Testmaterials BV (PO Box 120, 3133 KT Vlaardingen, The Netherlands).
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[0360] standard detergent
[0361] Standard detergents B, J, M and Y were used. Standard Detergent B is similar to a European type liquid detergent composition. Standard Detergent J is similar to an American type liquid detergent composition. Standard Detergent M is similar to a Chinese type powdered detergent composition. Standard Detergent Y is similar to the developing and emerging (D&E) Asia Pacific market type powder detergent composition.
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[0368] washing assay
[0369] Automated Mechanical Stress Assay (AMSA) f...
example 1
[0404] Example 1. Cloning and expression of M4 metalloprotease from Alum korean bacteria
[0405] The metalloprotease was derived from a strain of Alumenum korea, which was isolated from a soil sample collected from China in 2009.
[0406] Chromosomal DNA from pure cultures was purified and subjected to whole genome sequencing using Illumina technology. The assembled genome sequence and subsequent analysis of the 16S ribosomal subunit gene sequence indicated correct taxonomic identification as a species of S. alumensis.
[0407] Genomic sequences were analyzed for metalloproteases from the MEROPS family M4 compared to the M4 protease NprE from Bacillus subtilis 168 (Uniprot P68736) by searching using the BLAST program. This analysis identified a gene encoding a putative M4 metalloprotease having the nucleotide sequence given in SEQ ID:1.
[0408] The gene encoding the M4 metalloprotease was amplified by PCR and fused with regulatory elements and homology regions for recombin...
example 2
[0413] Example 2. SDS-PAGE and N-terminal sequencing
[0414] When the purified protease preparation was analyzed by SDS-PAGE and the gel was stained with Coomassie, three bands at approximately 55 kDa, 47 kDa and 8 kDa were seen. Edman degradation, complete MS, and in-gel peptide map analysis showing that all three bands are derived from the M4 protease, indicating that bands 47kDa and 8kDa represent nicked molecules in which the full-length M4 protease cuts once in the loop And the two peptide chains are still associated with each other and give a fully active molecule. The relative intensities of bands 47kDa and 8kDa suggest a 1:1 molar ratio between these two bands, supporting this hypothesis.
[0415] N-terminal sequencing of the purified protein was performed and the mature enzyme was found to be initiated at GTGNSMY, confirming the identity of the enzyme and the presence of the N-terminal propeptide. Intact molecular weight analysis of purified proteins by mass spectr...
PUM
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