STABILIZED AQUEOUS SOLUTIONS OF LIPASES

Abstract: The 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). More specifically, 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.

Description: STABILIZED AQUEOUS SOLUTIONS OF LIPASES. TECHNICAL FIELD 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. More specifically, 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. BACKGROUND ART Experiments with lipolytic enzymes isolated from raw milk have showed that Ca 2+ did not influence the stability of a lipase isolated from Pseudomonas fluorescens MC50 at 150 " C, whereas Ca 2+ did stabilize the enzyme at 60"C [Swaisσood, H.E. & Bozocrlu. F. (1984); J. Agric. Food Che . 32 p. 7-10]. From experiments with a lipase isolated from Pseudo¬ monas aerucfinosa EF2 it was noticed that the activity of this lipase was significantly enhanced by the presence of NaCl and/or CaCl 2 [Gilbert, E.J.. Cornish, A. & Jones, C. . (1991) ; Journal of General Microbiology 137 p. 2223-2229]. This observation was ascribed to the ability of sodium and calcium ions to react with free fatty acids adhering to oil droplets, thereby diminishing interfacial charge effects and/or increas¬ ing droplet surface area [as described by Brockerhoff. H. & Jensen, R.G. (1974) ; Lipolytic Enzymes; New York, Academic Press] . SUMMARY OF THE INVENTION It has now surprisingly been found that the thermal stability of aqueous solutions of lipases derived from members of the genus Pseudomonas becomes extremely improved in the presence of water-soluble inorganic salts in their dissolved form. It is remarkable that only lipases derived from members of the genus Pseudomonas become stabilized in this way. Accordingly, in its first aspect, 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. In another aspect, 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. In a third aspect, 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. In a fourth aspect, 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. DETAILED DISCLOSURE OF THE INVENTION 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. Several 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. Interestingly, only the 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. Lipases Derived from Pseudomonas In the context of this invention, 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. In a preferred embodiment, 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. 10 In a more specific embodiment, the lipase is derived from Ps. cepacia, Ps. fracri, Ps. alcaliqenes. Ps. pseudo- alcali enes. Ps. putida. or Ps. mendocina,, preferably Ps. cepacia. In its most specific embodiment, the lipase is 15 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 Mikroor- ganismen under the terms and conditions of the Budapest Treaty, and is now available according to EP Patent Specification No. 20214,761. Aqueous Solutions 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 25 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 30 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. Generally, 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. Generally, a concentration above 1 mM, particularly above 2 mM, is used for good stabilization. The concentration will usually be below 4 M, particularly below 2 M. Stabilization Methods 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. In a preferred embodiment, the stabilizing water- soluble divalent cation is added in a concentration at least stochiometrically equivalent to the amount of lipase. In another preferred embodiment, the stabilizing water-soluble divalent cation is calcium or magnesium, prefer¬ ably calcium. In a further preferred embodiment, 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. In a more specific aspect, 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. In its most specific aspect, the lipase is derived from the strain Ps. cepacia. DSM 3401, or a mutant or a variant thereof. Use of Stabilized Solutions of Lipases Furthermore, 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. In a preferred embodiment, the stabilized aqueous solution of lipases derived from members of the genus Pseudo¬ monas may be used in processes for degreasing hides and skins. In a more preferred embodiment, 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 following examples further illustrate the present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed. EXAMPLE 1 Preparation of Ps. cepacia Lipase A culture of the strain Pseudomonas cepacia. avail¬ able from DSM 3401, on an agar slant was transferred to five 500 ml shaking flasks, each with 100 ml of Bullion-3 medium, and shaken at 30°C for 1 day (200 rpm, amplitude 2,5 cm). The composition of Bullion-3 medium was as follows: Peptone 6 g/1 Trypsin digested casein 4 g/1 Yeast extract 3 g/1 Meat extract 1.5 g/1 Glucose 1 g/1 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. The composition of the PL-1 medium was as follows: Peptone 10 g/1 Tween ® -80 12 g/1 MgS0 4 ;7H 2 0 2 g/1 CaCl 2 ;2H 2 0 0.1 g 1 pH before autoclaving 6.0 The medium was autoclaved at 121°C for 40 minutes. Each PL-1 shake flask was inoculated with 0.5-2 ml of Bullion-3 culture broth and shaken with 200 rpm (amplitude 2.5 cm) at 30"C for 5 days. The culture broth from the shake flasks was pooled at harvest, totalling 39.51 with an enzyme yield of 53 LU/ml. 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. To the eluate ammonium acetate was added to a final concentration of 0.5 M. The enzyme was applied on a Toyopearl™- Butyl column and eluted with 0.05 M glycine buffer, pH 9.3. Assay for Lipase Activity: One Lipase Unit (LU) is the amount of enzyme which, under standard conditions (i.e. at 30.0°C; pH 7.0; and tribu- tyrine substrate) liberates 1 μmol 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. EXAMPLE 2 Storage Stability In a first experiment, the effect of Ca 2+ on the storage stability of lipase solutions was determined for 15 minutes at elevated temperatures. 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. Immediately after preparation the enzyme solutions were rapidly heated and preincubated at 30°C and 70°C, respec- tively, for 15 minutes. Subsequently, the enzyme solutions were rapidly cooled on an ice bath, diluted in desalted water, and the residual activity was determined at pH 7.0 by the LU assay described in Ex. 1. The results obtained from the analysis are summarized in Table 1 below. Table 1 * Average; n=2 ** Defined as 100% In a second experiment, the effect of Ca + on the 5 storage stability of lipase solutions was determined for 7 days at room temperature (20°C). 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. 0 The results of this analysis are presented in Table 2 below. Table 2 Storage Stability ; 7 Days/20 " C 25 Additive Relative activity 2 mM CaCl 2 100? 2 mM EDTA 0? 30 * No activity decay during the storage The data above clearly demonstrate that the presence of Ca + is essential to the thermal stability of the lipase. When Ca + is removed by EDTA, the activity decay at 35 30"C corresponds to a half life of approx. 1 hour. At 70"C the lipase is "perfectly stable" in the presence of the stabilizing inorganic salt, but becomes rapidly inactivated in the absence of this salt. After storage for 7 days at room temperature (20°C) no loss of activity was determined in the presence of the stabilizing inorganic salt, whereas the lipase was inactivated in the absence of this salt. EXAMPLE 3 Differential Scanning Calorimetry The thermal stability of the Ps. cepacia lipase obtained according to Ex. 1, was examined by means of Differen¬ tial Scanning Calorimetry (DSC) . The thermal denaturation temperature, T d , was determined by heating an enzyme solution at a constant programmed rate. Using the High Sensitivity Differential Scanning Calorimeter, MC-2D, from MicroCal Inc., buffered enzyme solutions were heated from 20°C to 100°C at a scan rate of 90°C/hr. 50 mM HEPES buffer, pH 7.0, containing either CaCl 2 or EDTA were used for the experiments. Three different Pseudo- monas-lipases were employed: A Ps. cepacia lipase obtained according to Ex. 1, A Ps. fluorescens lipase (AMANO P™) , 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. The results obtained from the analysis are shown in Table 3 below. Table 3 * The temperature at which approx. half the enzyme molecules present have been denatured thermally during heating. ** Stabilization of the Ps. cepacia lipase, i.e. change in denaturation temperature as a consequence of added CaCl 2 /EDTA. From the above results it appears that-removal of Ca 2+ by EDTA destabilizes the lipases significantly. In addition, the result indicates that the stability of the lipases is improved by increased amounts of the stabilizing inorganic " salt. International Application No: PCT/ MICROORGANISMS A. IDINTIFICATION Of Of POSIT I Further dapoaita an Identified on an additional sheet [~J • Nama ol depositary inatttuUon - DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELL- KULTUREN GmbH Addraa* ol depositary Inβtttutlon (including poatal coda and country) « Mascheroder Weg lb, D-3300 Braunschweig, Federal R e¬ public of Germany Data ol depoatt • Accaaalon Numb*** * 22 J u ly 1985 DSM 3401 B. ADDITIONAL INDICATIONS ' (leeve blank II not applicable). Thie Inlorrnation la continued on a aeparale attached sheet ~~ In respect of those designations in which a European patent is sought, a sample of the deposited micro¬ organism will be made available only by the issue o f such a sample to an expert nominated by the person requesting the sample (Rule 28 (4 ) EPC) unti l t h e publication of the mention of the grant of t h e E uro ¬ pean patent or until the date on which the app l i¬ cation has been refused or is deemed to be with d rawn. C. DISIONATf O STATIS FOR WHICH INDICATIONS AKI MADI • (II Iha Indlcatlont are not lor all designated State*) O. SIF-ARATI FURNISHINS OF INDICATIONS • (leave blank it not applicable) The indication* Dated below will be aubmined lo the International Bureau later • (Specify the general natur* ol the Indications e.g., " Accession Number ol Deposit " 1. ~~\ Thla sheet waa received oith the International application when filed (to be checked by the receiving Office) (Authorlied Officer) r~| The date ol receipt (Irom the applicant) by the Intarnsllonsl Bureau '• aat --. (Authorized Offlcsr) Form PCT/ftO/IM (January INI)

Claims: CLAIMS 1. An aqueous solution comprising a lipase derived from a member of the genus Pseudomonas. characterized by further comprising a stabilizing water-soluble divalent cation. 2. An aqueous solution according to claim 1, compris¬ ing a stabilizing water-soluble divalent cation in a con¬ centration at least stochio etrically equivalent to the amount of lipase. 3. An aqueous solution according to either of claims 1-2, in which the stabilizing water-soluble divalent cation is calcium or magnesium, preferably calcium. 4. An aqueous solution according to any of claims 1- 3, in which the lipase is derived from Ps. cepacia. Ps. aeruginosa. Ps. fragi. Ps. fluorescens. Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina. 5. An aqueous solution according to any of claims 1- 3, in which the lipase is derived from Ps. cepacia, Ps. fragi. Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina. preferably Ps. cepacia. 6. An aqueous solution according to any of claims 1- 3, in which the lipase is derived from the strain Ps. cepacia. DSM 3401, or a mutant or a variant thereof. 7. A method of stabilizing a lipase derived from a member of the genus Pseudomonas in an aqueous solution, characterized by incorporating a stabilizing water-soluble divalent cation. 8. A method according to claim 7, in which the stabilizing water-soluble divalent cation is incorporated in a concentration at least stochiometrically equivalent to the amount of lipase. 9. A method according to either of claims 7-8, in which the stabilizing water-soluble divalent cation is calcium or magnesium, preferably calcium. 10. A method according to any of claims 7-9, in which the lipase is derived from Ps. cepacia, Ps. aeruginosa, Ps. fragi. Ps. fluorescens. Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. mendocina. 11. A method according to any of claims 7-9, in which the lipase is derived from Ps. cepacia. Ps. fragi. Ps. alcaligenes. Ps. pseudoalcaligenes. Ps. putida. or Ps. men¬ docina r preferably Ps. cepacia. 12. A method according to any of claims 7-9, in which the lipase is derived from the strain Ps. cepacia. DSM 3401, or a mutant or a variant thereof. 13. Use of a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas according to any of claims 1-6, in a process for degreasing hides and skins. 14. Use of a stabilized aqueous solution of lipases derived from members of the genus Pseudomonas according to any of claims 1-6, in a process performed at elevated temperatures, e.g. hydrolysis of water-insoluble esters, particularly triglycerides, in paper pulping processes.

Inventors: BORCH KIM, PATKAR SHAMKANT ANANT, BORCH, KIM, PATKAR, SHAMKANT, ANANT
Applicants: BORCH KIM, NOVONORDISK AS, PATKAR SHAMKANT ANANT, NOVO NORDISK A/S
Application year: 1993
Main classification: C12N9
IPC codes: C12N9, C12R1, C12S3, C14C5, D21C9