EP1163329A2 - Surfactant-lipase complex immobilized on insoluble matrix - Google Patents

Surfactant-lipase complex immobilized on insoluble matrix

Info

Publication number
EP1163329A2
EP1163329A2 EP00911221A EP00911221A EP1163329A2 EP 1163329 A2 EP1163329 A2 EP 1163329A2 EP 00911221 A EP00911221 A EP 00911221A EP 00911221 A EP00911221 A EP 00911221A EP 1163329 A2 EP1163329 A2 EP 1163329A2
Authority
EP
European Patent Office
Prior art keywords
lipase
surfactant
group
immobilized
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00911221A
Other languages
German (de)
English (en)
French (fr)
Inventor
Sobhi Basheer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzymotec Ltd
Original Assignee
Enzymotec Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Enzymotec Ltd filed Critical Enzymotec Ltd
Publication of EP1163329A2 publication Critical patent/EP1163329A2/en
Withdrawn legal-status Critical Current

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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • 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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6454—Glycerides by esterification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Definitions

  • the present invention relates to an insoluble matrix immobilized
  • surfactant-coated lipase complex to a method of preparing same and to the
  • procedures include two steps.
  • the enzyme is activated by
  • the enzyme is immobilized
  • alkoxide or cobalt chloride that catalyze acyl migration among triglyceride
  • Lipases with 1,3 -positional specificity principally catalyze hydrolysis of fats
  • lipases with 1,3 -positional specificity are also capable of catalyzing
  • reaction is trans-esterification in which two different triglyceride molecules
  • lipases At high concentrations of water, e.g., above 5 % of solvent weight, lipases
  • cocoa butter substitute simulated human milk fat
  • cardiovascular disease cardiovascular disease, immune disorders and inflammation, allergies,
  • diabetes diabetes, kidney diseases, depression, brain development and cancer.
  • medium-chain fatty acids incorporated into the same triglyceride molecule are of major importance in some clinical uses, especially,
  • organic media e.g., Basheer, S., Mogi, K., Nakajima, M., 1995, Process.
  • Biochemistry 30: 531-536 were included fixed- and fluidized-bed reactors,
  • inorganic matrix was used both in a batch reactor system, and in fixed-bed
  • Yet a further purpose of the invention is to provide a process for preparing
  • the invention is primarily directed to a lipase preparation comprising an
  • the immobilization of the lipase complex onto the insoluble matrix may be any immobilization of the lipase complex onto the insoluble matrix.
  • the surfactant-coated lipase complex is covalently,
  • the invention encompasses the use of many types of matrix, said matrices
  • the inorganic insoluble matrix is
  • alumina selected from the group consisting of alumina, diatomaceous earth, Celite,
  • the abovementioned ion-exchange resin may be of any suitable material, but
  • the organic insoluble matrix is selected from the
  • the content of the lipase is 2-20 weight percent of
  • the content of the lipase is 0.01-1.0 weight percent of the preparation.
  • the invention provides the above-described lipase preparation, wherein the
  • surfactant in the surfactant-coated lipase complex includes a fatty acid
  • the fatty acid conjugated to a hydrophilic moiety.
  • the fatty acid 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-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the hydrophilic moiety is selected from the group consisting of a
  • the sugar is selected from the
  • hydrophilic moiety may be linked by any suitable type of bond, in
  • the fatty acid and the hydrophilic moiety are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • lipase may be derived or obtained from any convenient source
  • the lipase is derived from a microorganism.
  • Candida selected from a species selected from the group consisting of Burkholderia sp., Candida
  • Candida rugosa Candida rugosa
  • Pseudomonas sp. Candida antractica A
  • the invention is directed to a lipase preparation
  • the organic solvent is
  • the invention is further directed to the use of said lipase preparation as a catalyst for esterification,
  • the lipase triglycerols and fatty alcohols.
  • the lipase triglycerols and fatty alcohols.
  • the invention is directed to a lipase preparation as
  • the invention also provides a lipase preparation, as described hereinabove,
  • insoluble matrix has been modified with a fatty acid derivative.
  • the invention is directed to an enzyme preparation, as
  • the invention also encompasses a method for improving the stability of a
  • surfactant-coated immobilized lipase complex comprising granulating same
  • the invention provides a method of preparing an insoluble
  • the lipase is first
  • the lipase is first contacted with the lipase
  • this method also further comprises the step of
  • the drying step may be accomplished by any convenient method, in a
  • said drying is effected by freeze drying.
  • the matrix-immobilized surfactant-coated lipase is the matrix-immobilized surfactant-coated lipase
  • lipase and surfactant are contacted in the aqueous medium by:
  • the method further comprises sonication of
  • insoluble matrix is selected from the group consisting of alumina,
  • the surfactant of the method includes a
  • said fatty acid is selected from the group consisting of monolaurate, monomyristate, monopalmitate, monostearate. dilaurate,
  • dimyristate dipalmitate, distearate, trilaurate, trimyristate, tripalmitate and
  • hydrophilic moiety is selected from the group consisting of a sugar and a
  • the sugar is selected from the
  • 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 lipase is
  • the lipase may be
  • the lipase is
  • Candida antarctica B Candida rugosa, Pseudomonas sp., Candida antractica
  • the invention is directed to a process for preparing
  • surfactant-coated lipase complex is contacted with the substrates in the
  • the oil is selected from the group consisting of:
  • the fatty acid is selected from the group consisting of
  • the fatty acid is selected from the group consisting of:
  • oleic acid consisting of oleic acid, palmitic acid, linolic acid, linolenic acid, stearic acid,
  • the invention also encompasses a triacylglycerol prepared according to the
  • FIG. la presents an inter-esterification acidolysis reaction catalyzed by
  • P represents glycerol bound palmitic
  • C glycerol bound capric acid.
  • PA and CA represent free
  • FIG. lb presents a trans-esterification reaction catalyzed by lipase with
  • FIG. 2 depicts the chemistry associated with covalent immobilization of
  • FIG. 3 presents inter-esterification reaction profiles of physically
  • reaction system was magnetically stirred and thermostated at 40 °C.
  • FIG. 4 presents an Arrhenius plot for the inter-esterification reaction of
  • FIG. 5 is a bar graph showing the functional stability of Lilipase A
  • FIG. 6 is a bar graph showing the functional stability of powdered
  • FIG. 7 is a bar graph showing the functional stability of Lilipase A
  • FIG. 8 is a bar graph showing the functional stability of Lilipase A
  • FIG. 9 is a bar graph showing the functional stability of Lilipase A
  • FIG. 10 is a bar graph showing the functional stability of Lilipase A
  • FIG. 11 is a bar graph showing the functional stability of Lilipase A
  • FIG. 12 is a bar graph showing the functional stability of Lilipase A
  • FIG. 13 is a bar graph showing the functional stability of Lilipase A
  • FIG. 14 is a bar graph showing the functional stability of Lilipase A
  • FIG. l ⁇ is a bar graph showing the functional stability of Lilipase A
  • FIG. 16 is a bar graph showing the functional stability of Lilipase A
  • the present invention relates to a surfactant-coated lipase or phospholipase
  • particulate solid support which can be used to catalyze inter and
  • the invention also makes provision for preparing the enzyme
  • the present invention can be used for preparing
  • the present invention is further directed to a method of preparing
  • surfactant-coated lipases or phospholipases are provided.
  • fatty acid sugar ester types lead to activation of the lipases for
  • inorganic matrix showed high inter/trans-esterification activity and only
  • present invention were produced by inter-esterification of long-chain
  • triglycerides such as the hard fraction of palm oil, with short-chain fatty acids
  • hydrolysis side reaction and their percentage was typically less than 7 weight
  • liquid olive oil was
  • a lipase preparation which includes an insoluble matrix and a
  • surfactant-coated lipase complex immobilized onto the insoluble matrix.
  • lipase is not limited to this specific enzyme, but is meant to embrace also
  • the complex is immobilized to the
  • insoluble matrix via hydrophobic (physical) interaction, ionic interaction or via
  • inorganic insoluble matrix inorganic insoluble matrix
  • solubility in both polar (e.g., water) and non-polar (hydrophobic) solvents e.g., water
  • polar e.g., water
  • non-polar hydrophobic
  • the inorganic insoluble matrix according to the present invention is preferably, the inorganic insoluble matrix according to the present invention
  • calcium sulfate, ion-exchange resin such as, but not limited to, Amberlite and
  • DE diatomaceous earth
  • the inorganic insoluble matrix employed is Amberlite and Dowex,
  • Suitable organic solid matrices according to the present invention include
  • the lipase represents 0.01-1 weight percent of the preparation.
  • the lipase represents 0.01-1 weight percent of the preparation.
  • lipase represents about 0.7 weight percent of the preparation.
  • lipid which includes a fatty acid conjugated to a hydrophilic moiety.
  • fatty acid is preferably monolaurate, monomyristate, monopalmitate,
  • hydrophilic moiety is preferably
  • a sugar such as, but not limited to, sorbitol, sucrose, glucose and lactose, a
  • the fatty acid and the hydrophilic moiety are conjugated via an
  • lipase extraction examples include Burkholderia sp., Candida antarctica B,
  • Humicola sp. Mucor miehei, Rhizopus javan., Pseudomonas fluor, Candida
  • Rhizopus japonicus and Candida antractica Rhizopus japonicus and Candida antractica.
  • Lipase catalytic activity include hydrolysis, esterification, inter-esterification,
  • the organic solvent is typically a
  • hydrophobic solvent such as, but not limited to, n-hexane, toluene, iso-octane,
  • n-octane benzene, cyclohexane and di-iso-propylether.
  • the method includes the following method steps, wherein in a first step a
  • lipase, an insoluble matrix and a surfactant are contacted in an aqueous
  • surfactant-coated lipase is interacted with the matrix.
  • the lipase is first interacted with the matrix and only thereafter the matrix
  • immobilized lipase is interacted with the surfactant.
  • the method further includes the step of
  • drying is preferably effected via freeze drying, fluidization or
  • complex preferably includes less than 100, more preferably less than 50, most
  • solution is effected by dissolving the surfactant in an organic solvent (e.g.,
  • the dissolved surfactant solution e.g., dropwise in the aqueous solution
  • the lipase is first interacted with the
  • hydrophobized carrier such as aluminium stearate, fatty-acid
  • At least one of the substrates is an oil, a fatty acid
  • the oil may be any of the above listed oils.
  • the fatty acid may be any of the above listed oils.
  • a medium or a short-chain fatty acid or an ester derivative thereof is a medium or a short-chain fatty acid or an ester derivative thereof.
  • suitable fatty acid is, for example, oleic acid, palmitic acid, linolic acid,
  • reaction reactor e.g., a tank reactor or a fixed-bed reactor.
  • oils/fats e.g., triacylglycerols
  • long-chain fatty alcohols (LCFAL) to produce wax esters by alcoholysis
  • surfactant-coated lipase complex represents 2-30 weight percent of the
  • oil/fat substrates are liquid
  • oils and solid fats may be any of the above listed oils in a native or
  • the triacylglycerol serves an
  • esterification (inter- and trans-esterification), acidolysis, alcoholysis and hydrolysis catalytic activities with respect to substrates, yielding esterification
  • hydrolysis products represent less
  • Sorbitan fatty acid esters including
  • Tris(Hydroxymethyl)aminomethane (tris) was from Sigma (USA). Inorganic
  • organic matrices used as supports for the modified lipases include
  • activators e.g. gum Arabic or polyethylene glycol.
  • a typical enzyme e.g. gum Arabic or polyethylene glycol.
  • the resulting enzyme solution was sonicated for 15min and then
  • polypropylene, aluminium stearate or chitin) or inorganic matrix (20 g such
  • formed powder can be directly used for batch enzymatic reactions or
  • binding reagents such as starch, methyl or ethyl cellulose,
  • resin used include: strong and week basic anion exchange resins,
  • ion-exchange resins examples include: Dowex 22, Dowex
  • the enzyme was primarily coated with a surfactant and then the
  • lipase-surfactant complex was covalently linked to an Eupergit matrix, which
  • enzyme preparations prepared according to this method contained 0.9 - 1.5 wt
  • the esterification reaction was initiated by adding 10 mg lipase preparation
  • the transesterification reaction was initiated by adding 10 mg lipase
  • the alcoholysis reaction was initiated by adding 10 mg lipase preparation to
  • immobilized lipases was determined by the microkejldahl method.
  • insoluble matrix was tested using a 1ml vials containing the substrates.
  • the vials were shaken at 40 °C and samples were analyzed after certain
  • the protein content varied from 0.05 % to 1.12
  • esterification transesterificatio ( ⁇ mol/min.mg ( ⁇ mol/min.mg n) protein) protein
  • ⁇ mol/min.mg protein transesterificatio ( ⁇ mol/min.mg ( ⁇ mol/min.mg n) protein)
  • Celite resulted in detectable levels of esterification and transesterification.
  • Figure 3 presents the conversion of tripalmitin with time when
  • the concentration of hydrolysis products did not exceed 5 wt % of
  • fatty acids having longer alkyl chains such as palmitic and
  • lipase complexes than fatty acids having shorter alkyl chains.
  • Enzyme/Insoluble ri ri (transri matrix* (esterification) esterification) (alcoholysis) ( ⁇ molVmin.mg ( ⁇ mol/min.mg ( ⁇ mol/min.m protein) protein) g protein)
  • fatty acid derivative-treated insoluble matrix (Aluminum monostearate, fatty acid derivative-treated Celite) is much greater than the activity of
  • Fig. 5 shows that the activity of Lilipase A-10FG immobilized on Celite

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Fats And Perfumes (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP00911221A 1999-03-22 2000-03-16 Surfactant-lipase complex immobilized on insoluble matrix Withdrawn EP1163329A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IL12908699 1999-03-22
IL12908699A IL129086A0 (en) 1999-03-22 1999-03-22 Surfactant-lipase complex immobilized on insoluble matrix
PCT/IL2000/000166 WO2000056869A2 (en) 1999-03-22 2000-03-16 Surfactant-lipase complex immobilized on insoluble matrix

Publications (1)

Publication Number Publication Date
EP1163329A2 true EP1163329A2 (en) 2001-12-19

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EP00911221A Withdrawn EP1163329A2 (en) 1999-03-22 2000-03-16 Surfactant-lipase complex immobilized on insoluble matrix

Country Status (7)

Country Link
EP (1) EP1163329A2 (ja)
JP (1) JP2002539782A (ja)
KR (1) KR100774281B1 (ja)
CA (1) CA2368179A1 (ja)
IL (2) IL129086A0 (ja)
NZ (1) NZ514271A (ja)
WO (1) WO2000056869A2 (ja)

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IL152290A0 (en) * 2002-10-14 2003-05-29 Enzymotec Ltd Immobilization of compounds on polymeric matrix
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US8052992B2 (en) 2003-10-22 2011-11-08 Enzymotec Ltd. Glycerophospholipids containing omega-3 and omega-6 fatty acids and their use in the treatment and improvement of cognitive functions
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