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Novel lipolytic enzymes

A lipolytic enzyme, lard technology, applied in the field of lipolytic enzymes, can solve the problem of no fat dirt and high activity

Inactive Publication Date: 2010-08-04
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] A disadvantage of all detergent lipolytic enzymes described so far is that they perform best for fat removal after more than one wash cycle, presumably because known lipolytic enzymes (when deposited on the fat to be removed soil) is more active during drying than during washing (Gormsen et al., Proceedings of the 3rd World Conference on Detergents, AOCS Press, 1993, 198-203)
However, when tested under actual wash conditions, none of these enzymes were able to remove substantial amounts of fatty soil in one wash cycle (see examples below)

Method used

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Examples

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preparation example Construction

[0516] The preparation of homologous DNA fragments in step c) can be carried out by amplifying homologous DNA sequences (e.g. comprising one or more mutations in the lipolytic gene and contained in a plasmid or vector) using any suitable method, so A suitable method is such as the standard PCR amplification method described in US Patent 4,683,202 or Saiki et al., (1988), Science, 239, 487-491.

[0517] The vector can be introduced into the recombinant host cell by transformation (step d). Where the recombinant host cell is a S. cerevisiae strain such as S. cerevisiae YNG318 (described below), transformation can be performed as described in Sambrooks et al. ((1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, USA ) proceed as described.

[0518] Screening for positive lipolytic enzyme variants can be performed, for example, by the screening methods described above for random mutagenesis.

[0519] One of the cycles of steps a) to f) may be p...

Embodiment 1

[1215] Construction of random lipolytic enzyme variants

[1216] Random mutagenesis libraries of the complete H. lanuginosa lipolytic enzyme gene and its amino acids (aa) 91-97 and 206-211 were prepared as described above in Materials and Methods.

[1217] Amino acid regions 91-97 and 206-211 were selected for the first round of domain-specific mutagenesis because these regions have been found to be important for wash performance. Regions 91-97 are part of the lid region of the enzyme and regions 206-211 form part of the hydrophobic cleft of the enzyme.

[1218] An oligonucleotide containing 93% of the wild-type nucleotide and 2.33% each of the other three nucleotides at the amino acid codon to be mutagenized was synthesized for each region. Where no amino acid was likely to be changed, the third nucleotide in the codon (the wobble base) was synthesized with 50% G / 50% C to give a greater probability of changing to an amino acid containing one or two codons. The composition o...

Embodiment 2

[1456] Construction of a first wash variant of H. lanuginosa lipolytic enzyme

[1457] 1. Domain Shuffling by Recombination and Screening

[1458] Twenty H. lanuginosa lipolytic enzyme variants, some of which were constructed according to Example 1, with good wash performance (as assessed in various wash-related assays) were available as described herein in the Materials and Methods section. The method of in vivo recombination in Saccharomyces cerevisiae YNG318 is used for recombination. The lipolytic enzyme variants utilized are evident from Table 1. Most of these variants were selected for reduced calcium dependence and increased tolerance to the detergent component Dobanol 25-7 (see Materials and Methods section above) by random or localized random mutagenesis as described in the Materials and Methods section above. And build. Some variants are the result of two or more consecutive cycles of mutagenesis and selection.

[1459] The restriction enzyme-opened vector and th...

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Abstract

Novel lipolytic enzymes are disclosed which are capable of removing substantial amounts of lard from a lard stained swatch in a one cycle wash. Preferred lipolytic enzymes are variants of the Humicola lanuginosa lipase which may be prepared by recombinant DNA techniques. The enzymes are advantageously used in detergent compositions.

Description

[0001] The technical field of the invention [0002] The present invention relates to novel lipolytic enzymes capable of removing substantial amounts of fatty material in one wash cycle. Background of the invention [0003] For many years lipolytic enzymes have been used as detergent enzymes, ie to remove lipid or fatty soils from clothing and other textiles. For example, various microbial lipases have been suggested as detergent enzymes. Examples of such lipases include Humicola lanuginosa lipase (as described in EP 258 068 and EP 305 216), Rhizomucor miehei lipase (as described in EP 238 023 and Boel, et al., Lipids 23, 701-706, 1988 described in ), Absidia sp. lipolytic enzyme (WO96 / 13578), Candida lipase (such as C. antarctica lipase, for example C. antarctica lipase A or B described in EP 214 761), Pseudomonas lipases (such as Ps.alcaligenes and Ps.pseudoalcaligenes lipases, e.g. as described in EP 218 272, Burkeholm onion cepacia lipase, as described in EP 331 376), t...

Claims

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Application Information

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IPC IPC(8): C12N9/20C11D3/386C12N15/09
CPCC12N9/20C11D3/38627
Inventor J·S·奥克尔斯A·斯闻德森K·伯克M·瑟勒森S·A·帕特卡D·A·佩特森J·C·罗耶T·克里茨施马
Owner NOVO NORDISK AS
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