Detergent Formulations Containing Alkaline Lipase Derived From Pseudomonas Plantarii

In U.S. Pat. No. 4,707,291 there is disclosed a detergent composition comprising a mixture of an anionic and a nonionic detergent-active compound in combination with a lipase which shows a positive immunological cross-reaction with the antibody of the lipase produced by Pseudomonas fluorescens IAM 1057, specifically those produced by a microorganism of the species Pseudomonas fluorescens , P. gladioli or Chromobacter viscosum. While these organisms were known to have lipolytic activity at the time the application which matured into the 4,707,291 patent was filed patentability predicated on the stability of these enzymes in the detergent containing formulation.

In European published application 0 271 153 there is disclosed a composition comprising a nonionic detergent, a protease and a lipase which shows a positive immunological response to the antibody of the lipase produced by Chromobacter viscosum, var. lipolyticum NRRL-B 3673. Lipases derived from Pseudomonas species P. fluorescens, P. fragi, P. nitroreduscens var. lipolyticum, P. cepacia and P. gladioli are specifically disclosed.

The bacterial genus Pseudomonas is actually comprised of four sub-genera. P. cepacia and P. gladioli belong to Pseudomonas subgroup I whereas P. fragi and probably P. nitroreduscens belong to subgroup I.

Azegami et al report a new species of Pseudomonas, P. plantarii, in Int. Journal of Systematic Bacteriology, Apr. 1987, p. 144-152. This article indicates a positive response for lipase, using the Tween 80 hydrolysis method, for lipase from the species P. plantarii as well as that from P. gladioli. All other strains of P. plantarii are reported by Azegami to behave identically in the taxonomic tests described, suggesting that this is a very tight homologous species. In addition, the lipase in all 21 tested strains are reported to catalyze both Tween 80 hydrolysis and cottonseed oil hydrolysis. The strain used in these examples, i.e. ATCC 43733, is the Type strain, a designation that means it is the most indicative representative of the new species. While the gladioli and plantarii species of Pseudomonas are related, they have definite taxonomic differences, such as, for example, P. plantarii can (whereas P. gladioli cannot) utilize L. Rhamose for growth, P. plantarii cannot (whereas P. gladioli can) utilize trehalose, adonitol, .beta.-alanine, lactose, benzoate, levulinate for growth. P. plantarii cannot grow at 40.degree. C. whereas P. gladioli can. Furthermore P. plantarii has been reported to be pathogenic to rice seedlings whereas P. gladioli has not.

The present invention is a composition comprising a nonionic and/or anionic detergent and bacterial lipase derived from an organism of the species Pseudomonas plantarii.

The present invention is predicated on the discovery that lipase from P. plantarii is unexpectedly stable in the presence of nonionic and/or anonic detergents. It is significantly more stable than lipase from P. gladioli which the prior art recognizes as being detergent stable.

A typical formulation suitable for removing fatty soils from fabrics will include one or more detergent surfactants such as nonionic surfactants [e.g. alkyl and nonylphenylpoly (ethylene glycerol) ethers]; anionic surfactants (e.g. alkylbenzene sulfonates, fatty alcohol ether sulfates or alphaolefin sulfonates) and the powdered lipase typically in an amount of from about 0.1 to 100 lipase units per milligram. Optional ingredients include a detergent builder such as potassium diphosphate, sodium tripolyphosphate, sodium citrate, sodium nitrilotriacetate or sodium silicate; foam boosters (e.g. fatty acid alkanolamides); alkalies (e.g. sodium carbonate); optical brighteners (e.g. stilbene derivatives); stabilizers (e.g. triethanolamine); fabric softeners (e.g. quaternary ammonium salts) together with bleaching agents and systems (such as sodium perborate and ethylene diaminetetraacetate). Additional ingredients may include fragrances, dyes, lather boosters, foam depressors and anticorrosion agents, formulation acids. In addition, other enzymes such as proteases, amylases or cellulases may be present.

A colony of Pseudomonas plantarii or Pseudomonas gladioli from a nutrient agar plat was used to inoculate 50 ml of the described seed medium. The seed flask was allowed to grow for 24 hours after which time it was diluted 1:1 with a sterile 20% glycerol solution, aliquoted 1.0 ml into 1.5 ml freezer vials and stored at -70.degree. C. for future use. Seed cultures of P. gladioli, ATCC 10248, and P. plantarii, ATCC 43733, were propagated by inoculating 50 ml of PY80 medium described below with 0.1 ml of a -70.degree. C. frozen stock culture.

The inoculated PY80 seed medium was incubated at 28.degree. C. for 16 hours using a New Brunswick G-25-R shaker set at 250 rpm.

The fermentation medium (FGH 80) used is described below:

Each fermentation flask was inoculated with 1 ml seed grown as described for seed preparation. The inoculated flasks were incubated at 28.degree. C. for 72 hours with stirring at 425 rpm in a New Brunswick G-25-R shaker.

Alternatively lipase was produced using 30-liter fermentation vessels (Biostat U-300, Braun Instruments, Bethlehem, Pa.). The seed medium used was as described previously with the exception that a volume of 600 ml was grown in fernbach flasks; 600 ml of 16 hour seed culture was transferred into each 30-liter fermentor. The fermentation was stopped after 72 hours incubation at 28.degree. C. with agitation at 300 rpm and aeration at 15 liters/minute with back pressure maintained at 90 Bar.

The lipase powder was obtained by initially heating the fermentor whole beer to 60.degree. C. for 10 minutes. After cooling to 25-30.degree. C., five percent w/v bentonite was added to the heat treated beer. While mixing, an equal volume of isopropanol was added to the bentonite treated beer. The isopropanol/bentonite beer had 0.75% FW-6, a filter aid, added and was then filtered through shark-skin paper using a table filter. The isopropanol filtrate was collected and the isopropanol removed using a vacuum concentrator. The isopropanol-free sample was polished by adding 1% w/v FW-6 filter aid and filtering through a fine bed of the same filter aid. The polished sample was then concentrated by ultrafiltration, using an Amicon PM-10 cartridge, to approximately 8-10X.

Complete precipitation of the proteins was accomplished by the addition of isopropanol to 80% w/v with slow mixing. Proteins were separated from the alcohol by adding 0.5% w/v FW-6 filter aid on a table filter. The dry filter cake was resuspended in water that had been previously adjusted to pH 9.3-9.5 with 1N NaOH at a ratio of water to cake of 1:2. The cake and water were mixed for 20 minutes and then refiltered. The slurry process was repeated two additional times with all of the filtrates being saved and frozen at -70.degree. C. overnight. The frozen filtrate was then lyophilized to obtain a powdered lipase preparation.

Detergent formulations containing powdered lipase prepared as described above were formulated and tested for stability. These experiments are described in the following examples:

The stability of lipase from P. plantarii and P. gladioli in a wash system was determined by adding 3,000 Esterase units of lipase per liter of standard tap water along with 1.96 ml detergent base WA.

The mixture was incubated at 45.degree. C. and then assayed at 0, 10, 20, 30, 40, 50 and 60 minutes by titrating the production of butyrate produced in gum arabic emulsions of tributyrin at pH 8.5 and 45.degree. C. to determine percent of enzyme activity remaining. A blank containing the detergent and water was also assayed. The detergent did not interfere with the assay.

From the foregoing data, it can be determined that lipase from P. plantarii is inherently more stable to simulated detergent wash conditions that contain mixtures of anionic and nonionic surfactants.

The relative stability of P. plantarii and P. gladioli lipase were also tested in a wash system containing 1 g/liter ALL.RTM. laundry detergent powder containing a nonionic detergent formulation from Lever Brothers, Inc. Each lipase, 3,000 esterase units per liter, were added to the ALL wash system at 45.degree. C. and assayed at 0, 10, 20 and 40 minutes by titrating the production of butryate produced in gum arabic emulsions of tributyrin at pH 8.5 and 45.degree. C. to determine percent of enzyme activity remaining. A blank containing the detergent and water was also assayed. The detergent did not interfere with the assay.

Improved stability of P. plantarii lipase compared to P. gladioli lipase, which has similar pH and temperature optimums, was observed under the specified conditions. This property would be advantageous in pre-soak applications or spot cleansing prior to washing, in addition to incorporation in standard detergent formulations for enhanced removal of fatty stains during the regular wash cycle.