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 stabi ⁇ lized 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 r 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 5 mutants and variants thereof.
• the lipase is derived from Ps. cepacia. Ps. aeru inosa, Ps. fracri. Ps. fluorescens. Ps. alcaliqenes. Ps. pseudoalcali enes. Ps. putida. or Ps. men- docina.
• the lipase is derived from Ps. cepacia, Ps. fracri, Ps. alcaliqenes. Ps. pseudo- alcali enes. Ps. putida. or Ps. mendocina,, preferably Ps. cepacia.
• the lipase is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• 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
• 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
• the stabilizing water-soluble inorganic salt used in the invention contains a divalent cation, e.g. calcium or magnesium, preferably calcium.
• a divalent cation e.g. calcium or magnesium
• 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 solubi ⁇ lity 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, prefer ⁇ ably calcium.
• the lipase to be stabilized is derived from Ps. cepacia. Ps. aeruginosa. Ps. fraqi. Ps. fluorescens, Ps. alcaliqenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina.
• the lipase to be stabi ⁇ lized 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 desir ⁇ able to maintain the lipolytic activity of the enzyme for an extended period.
• the stabilized aqueous solution of lipases derived from members of the genus Pseudo ⁇ monas may be used in processes for degreasing hides and skins.
• the stabilized solution of lipases derived from members of the genus Pseudo ⁇ monas may be used in a process performed at elevated tempera- tures, 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 centrif ged for 35 minutes at 4100 xg by means of a Beck an 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 M 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 tribu- tyrine 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.
• 35 30"C 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 g/ml were employed, and a total volume of approx. 1.2 ml was used for each experiment.
• Thla sheet waa received oith the International application when filed (to be checked by the receiving Office)

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• Biomedical Technology (AREA)
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• Enzymes And Modification Thereof (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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

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