Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – For cleaning a specific substrate or removing a specific...
Patent
1996-10-22
2000-01-25
Achutamurthy, Ponnathapu
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
For cleaning a specific substrate or removing a specific...
435198, 4352523, 4353201, 435874, 536 232, 536 237, 510305, 510226, 510326, 510392, 530350, C12N 920, C12N 120, C12N 1500, C07H 2104
Patent
active
060178663
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to methods for modifying lipases in a way that their stability is increased. It relates more specifically to lipases for use in detergent compositions with improved resistance towards surfactants.
Lipases are enzymes capable of hydrolyzing lipids. They are used in a wide range of applications, such as processing of fats and oil, detergent compositions for cleaning purposes and diagnostic reagents.
Extracellular lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are produced by a wide variety of microorganisms. Isolated microbial lipases have for example been disclosed in U.S. Pat. No. 3,950,277. These lipases were obtained from such diverse microorganisms as Pseudomonas, Aspergillus, Pneumococcus, Staphylococcus, Mycobacterium tuberculosis, Mycotorula lipolytica and Sclerotinia.
Examples of the use of lipases from various microorganisms in detergent compositions are given in for instance EP 463100 (Pseudomonas pseudoalcaligenes), EP 0218272 (Pseudomonas pseudoalcaligenes), EP 0214761 (Pseudomonas cepacia), EP 0258068 (Thermomyces) and EP 206390 (Pseudomonas chromobacter, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas nitroreductans, Pseudomonas gladioli, Chromobacter viscosum).
Especially the Pseudomonas lipases have favourable characteristics for the known desired applications of lipases. Pseudomonas species have therefore been extensively used for obtaining lipases. Several of the Pseudomonas lipase genes have been cloned, thereby enabling increased fermentation yield in production of these lipases in both homologous and heterologous host strains. Examples of Pseudomonas species from which cloning of a lipase gene has been reported are: Pseudomonas cepacia (EP 331376), Pseudomonas glumae (EP 464922), Pseudomonas alcaligenes (EP 334462) and Pseudomonas fragi (EP 318775). For use as an ingredient in detergent compositions, lipases should desirably be resistant to all other ingredients. It has for example been shown that it is possible to stabilize the lipase from Pseudomonas glumae against oxidizing components of a detergent composition by replacing methionines by one of the other 19 possible natural occurring amino acids (EP 407225).
Another document describes the stabilization of the same lipase against proteolytic cleavage by a protease component of a detergent composition (Frenken, L. G. J. et al. Protein Engineering 6 (1993) 637-642).
Another important stability problem is the sensitivity of enzymes towards denaturation by anionic, cationic or nonionic surfactant molecules. Anionic surfactant molecules (e.g. laurylsulphate, dodecylsulphate) are amphiphilic molecules having a hydrophobic aliphatic part and a negatively charged hydrophilic part, such as a sulphonate group. Cationic surfactant molecules contain a positively charged group such as a quarternary ammonium group instead of a negatively charged sulphonate group and in nonionics the hydrophilic part of the molecule is formed by a polar but uncharged group like an alcohol group.
The present invention solves the problem of lack of stability of lipases in the presence of surfactants. In particular, in one aspect, it provides a mutant lipase which is a modified functional form of a natural lipase or functional portion thereof having one or more amino acid substitutions at or near the surface compared to said natural lipase or portion thereof so as to increase surfactant resistance.
One of the mechanisms through which surfactant induced denaturation of proteins in general may be caused is by penetration of the hydrophobic part of the surfactant molecule into the hydrophobic core of the enzyme. This process leads to unfolding of the 3D-structure of the protein and thereby results in irreversible loss of catalytic activity.
We have now found that initiation sites for such a process in a lipase can be blocked or removed by one or a few amino acid substitutions, e.g. produced by site-directed mutagenesis of the corresponding native DNA coding sequence. We have found that it is thus possible to enhance the resis
Aehle Wolfgang
Gerritse Gijsbert
Lenting Hermanus B. M.
Achutamurthy Ponnathapu
Faris Susan
Genencor International Inc.
Saidha Tek Chand
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