Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

• This invention relates to the use of a water-soluble inorganic salt as stabilizing additive in aqueous solutions of lipases derived from a member of the genus Pseudomonas.
• the invention provides a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas, and a method of stabilizing lipases derived from members of the genus Pseudomonas in an aqueous solution.
• the invention provides an aqueous solution comprising a lipase derived from a member of the genus Pseudomonas, said aqueous solution further comprising a stabilizing water-soluble divalent cation.
• the invention provides a method of stabilizing a lipase derived from a member of the genus Pseudomonas in an aqueous solution, which method comprises incorporation of a water-soluble divalent cation.
• the invention relates to the use of a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas for use in processes for degreasing of hides and skins.
• the invention relates to the use of a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas for use in processes performed at elevated temperatures, e.g. for use in hydrolysis of water-insoluble esters, particularly triglycerides, in paper pulping processes.
• the invention relates to the use of water-soluble inorganic salts as stabilizing additives in aqueous solutions of lipases derived from a member of the genus Pseudomonas.
• lipases of other origin than Pseudomonas have been tested (i.e. lipases obtained from Humicola, Candida, and Rhizomucor), but not found to be stabilized by the presence of calcium or magnesium ions.
• lipases obtainable from members of the genus Pseudomonas were found to be stabilized.
• the invention provides a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas, said aqueous solution further comprising a stabilizing water-soluble divalent cation.
• a lipase derived from a member of the genus Pseudomonas is understood as any lipase being obtainable from such a microorganism, including mutants and variants thereof.
• the lipase is derived from Ps. cepacia. Ps. aeruginosa, Ps. fragi. Ps. fluorescens. Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina.
• the lipase is derived from Ps. cepacia, Ps. fragi, Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina, preferably Ps. cepacia.
• the lipase is derived from the strain Ps. cepacia. DSM 3401, or a mutant or a variant thereof.
• the strain, Ps. cepacia DSM 3401 was deposited on 22 July 1985 at Deutsche Sammlung fur Mikroorganismen under the terms and conditions of the Budapest Treaty, and is now available according to EP Patent Specification No. 214,761.
• the invention can be applied whenever it is desired to stabilize a solution of a lipase derived from a member of the genus Pseudomonas, in order to maintain the lipolytic activity of the enzyme for an extended period.
• the invention brings about stabilization of the lipase at any temperature of the activity interval of the enzyme. Therefore, the invention may be applied to a lipase solution intended for use at room temperature, or, particularly as the activity decay is increased, lipase solutions for use in processes performed at elevated temperatures. Stabilizing Inorganic Salts
• the stabilizing water-soluble inorganic salt used in the invention contains a divalent cation, e.g. calcium or magnesium, preferably calcium.
• Calcium chloride or calcium carbonate, and magnesium chloride or magnesium carbonate, respectively, are preferred stabilizing inorganic salts, as they are inexpensive and were found to be effective stabilizers.
• the concentration of the inorganic salt depends on i.a. the pH of the solution, the concentration of the lipase to be stabilized, and the required degree of stabilization.
• the concentration of the inorganic salt should be at least stoechiometrically equivalent to the amount of lipase, i.e. at least a 1:1 molar ratio.
• the concentration of the inorganic salt should be in the range of from at least a 1:1 molar ratio up to the solubility limit in the system in question, depending on the required degree of stabilization.
• concentration above 1 mM, particularly above 2 mM, is used for good stabilization.
• concentration will usually be below 4 M, particularly below 2 M.
• the invention also provides a method of stabilizing lipases derived from members of the genus Pseudomonas in an aqueous solution, which method comprises incorporation of a stabilizing water-soluble divalent cation.
• the stabilizing water-soluble divalent cation is added in a concentration at least stochiometrically equivalent to the amount of lipase.
• the stabilizing water-soluble divalent cation is calcium or magnesium, preferably calcium.
• the lipase to be stabilized is derived from Ps. cepacia. Ps. aeruginosa. Ps. fragi, Ps. fluorescens, Ps. alcaligenes. Ps. pseudoalcaligenes, Ps. putida. or Ps. mendocina.
• the lipase to be stabilized is derived from Ps. cepacia. Ps. fragi, Ps. alcaligenes. Ps. pseudoalcaligenes, Ps. putida, or Ps. mendocina, preferably Ps. cepacia.
• the lipase is derived from the strain Ps. cepacia. DSM 3401, or a mutant or a variant thereof.
• the invention relates to the use of a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas for use in processes where it is desirable to maintain the lipolytic activity of the enzyme for an extended period.
• the stabilized aqueous solution of lipases derived from members of the genus Pseudomonas may be used in processes for degreasing hides and skins.
• the stabilized solution of lipases derived from members of the genus Pseudomonas may be used in a process performed at elevated temperatures, e.g. for use in hydrolysis of water-insoluble esters, particularly triglycerides, in paper pulping processes.
• the composition of Bullion-3 medium was as follows:
• the medium was autoclaved at 121°C for 40 minutes.
• the culture broth of these Bullion-3 shake flasks was used as a seed culture for inoculating two hundred 500 ml shake flasks, each with 200 ml of PL-1 medium.
• composition of the PL-1 medium was as follows:
• the medium was autoclaved at 121°C for 40 minutes.
• the culture broth was centrifuged for 35 minutes at 4100 xg by means of a Beckman Model J-6 centrifuge.
• the supernatant was concentrated by filtration (washed with approximately 1 volume of water) to 1.4 1 by a Pellicon ultrafiltration apparatus from Millipore with a 10.000 MW cut off filter sheet.
• the concentrate was freeze dried, and the yield was 56.2 g of powder with an enzyme activity of 21.500 LU/g.
• the concentrate was suspended in water and applied on a hydrophobic XAD-8 resin matrix, and eluted with 60% (v/v) 96% ethanol.
• ammonium acetate was added to a final concentration of 0.5 M.
• the enzyme was applied on a ToyopearlTM-Butyl column and eluted with 0.05 M glycine buffer, pH 9.3.
• One Lipase Unit is the amount of enzyme which, under standard conditions (i.e. at 30.0°C; pH 7.0; and tributyrine substrate) liberates 1 ⁇ mol of titratable butyric acid per minute.
• a folder AF 95/5 describing this analytical method is available upon request to Novo Nordisk A/S, Denmark.
• Enzyme solutions containing approx. 1 mg/ml of the Ps. cepacia lipase obtained according to Ex. 1, and containing either 2 mM CaCl 2 or 2 mM ELTA (metal chelating agent) were prepared.
• Enzyme solutions were prepared as described above. After storage the residual activity was determined at pH 7.0 by the LU assay described in Ex. 1.
• the activity decay at 30oC corresponds to a half life of approx. 1 hour.
• the lipase is "perfectly stable" in the presence of the stabilizing inorganic salt, but becomes rapidly inactivated in the absence of this salt.
• the thermal denaturation temperature, T d was determined by heating an enzyme solution at a constant programmed rate.
• a Ps. cepacia lipase obtained according to Ex. 1 A Ps. fluorescens lipase (AMANO PTM), purchased from AMANO, Japan, and a Ps. fragi lipase, also purchased from AMANO, Japan. Lipase concentrations of approx. 1 mg/ml were employed, and a total volume of approx. 1.2 ml was used for each experiment.

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Citations (2)

• 1992
  • 1992-08-07 DK DK99492A patent/DK99492D0/da not_active Application Discontinuation
• 1993
  • 1993-08-09 WO PCT/DK1993/000257 patent/WO1994003592A1/en not_active Application Discontinuation
  • 1993-08-09 EP EP93917567A patent/EP0672126A1/en not_active Withdrawn
  • 1993-08-09 JP JP6504912A patent/JPH08500013A/ja active Pending

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