WO2009010561A1 - Immobilisation d'enzymes - Google Patents

Immobilisation d'enzymes Download PDF

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Publication number
WO2009010561A1
WO2009010561A1 PCT/EP2008/059390 EP2008059390W WO2009010561A1 WO 2009010561 A1 WO2009010561 A1 WO 2009010561A1 EP 2008059390 W EP2008059390 W EP 2008059390W WO 2009010561 A1 WO2009010561 A1 WO 2009010561A1
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Prior art keywords
carrier
enzyme
product
immobilized enzyme
lipase
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PCT/EP2008/059390
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English (en)
Inventor
Isabelle Mazeaud
Kaare Joergensen
Lars Saaby Pedersen
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Novozymes A/S
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Publication of WO2009010561A1 publication Critical patent/WO2009010561A1/fr

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    • 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
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the present invention relates to an immobilized enzyme product comprising a preformed single carrier particle and a lipolytic enzyme. It also relates to a method of producing the product and to processes using it.
  • Immobilization of lipolytic enzymes has been known for many years.
  • An immobilized enzyme product may be used in enzymatic modification of an organic compound such as in organic synthesis processes, vegetable oil interesterification, biodiesel production etc.
  • Enzyme immobilization is immobilizing of an enzyme protein on a carrier on which the enzyme is fixed and yet functional and for which the enzyme is not released to the liquid to which it is contacted.
  • the most commonly immobilized enzymes are glucose isomerase used for isomerization reactions and lipase used for e.g. interesterification of vegetable oil and organic synthesis.
  • the industrial use of enzymes is often limited by their high cost and rapid inactiva- tion. To improve their economic feasibility in industrial processes, enzymes are generally immobilized onto a particle.
  • Immobilization facilitates re-use of the enzymes, and may affect the selectivity and stability of the enzyme. Immobilization research has mainly focused upon means to enhance the transfer of enzymes onto the support, and upon means to ensure that the transferred enzymes remain active when immobilized.
  • lipolytic enzymes such as lipases can be immobi- lized on a number of different porous, inorganic carriers by absorption of an aqueous solution of lipase into the pore volume of the carrier or by adsorption to the surface of the carrier or by a combination of both adsorption and absorption followed by water removal by drying.
  • JP 5-292965A discloses an immobilized lipase and a method for preparing it.
  • WO 95/22606 (Pedersen et al.) describes an immobilization process based on a granulation process.
  • WO 99/33964 (Christensen et al.) describes an immobilization process wherein the enzyme is applied to a particulate porous carrier.
  • Immobilized enzymes are known to be used in continuous enzymatic reactions within a variety of industrial applications, including waste water treatment, production of pharmaceuticals, high fructose corn syrup production, vegetable oil processing and synthesis of chemicals.
  • an immobilized enzyme product used in said processes needs to provide a given productivity at an acceptable production rate in order to make the process economi- cally and technically feasible.
  • the enzyme activity is too low initially or is decreasing too fast during usage the amount of enzyme used becomes very high, or the enzyme product can not give the desired productivity. Therefore it is of great importance that the enzyme activity is as high as possible on start up and that the activity also is retained as high as possible during the operation, in order to obtain a high productivity and a high average production rate in the application.
  • the inventors have found that when immobilizing a lipolytic enzyme on a preformed carrier particle, the addition of a soluble polyol such as a carbohydrate or sugar alcohol significantly improves the performance of the immobilized lipolytic enzyme product with regard to initial enzyme activity, enzyme stability (half-life) and/or enzyme productivity.
  • a soluble polyol such as a carbohydrate or sugar alcohol
  • the inventors surprisingly found that the addition of a soluble polyol such as a carbohydrate or a sugar alcohol to the enzyme solution applied to the particles for immobilization is the key to activity of the final product.
  • a soluble polyol such as a carbohydrate or a sugar alcohol
  • the present invention provides an immobilized enzyme product with increased enzyme stability during application.
  • the invention also provides a simplified process for manufacturing an immobilized enzyme product with a high enzyme activity and good sta- bility.
  • the present invention provides an immobilized enzyme product comprising: a) a carrier which is a preformed single carrier particle, b) a soluble polyol selected from carbohydrates and sugar alcohols, and c) a lipolytic enzyme.
  • the present invention further relates to the manufacture of the immobilized enzyme product and its use. DETAILED DESCRIPTION OF THE INVENTION
  • the carbohydrate may act as an enzyme performance improver either alone or in combination with the selected carrier and/or other carbohydrates.
  • the carbohydrates and the carrier may give a synergistic stabilizing effect.
  • the enzyme product of the present invention is a particle and may have a particle size of 50-2000 microns, 100-1000 microns, 250-600 microns, or 300-800 microns.
  • the soluble polyol used in the invention is a carbohydrate or a sugar alcohol, typi- cally with a solubility of at least 0.1 g per 100 ml of water at ambient temperature (e.g. 2O 0 C)
  • the carbohydrate may consist of 1-20 monosaccharide units. This includes monosaccharides and oligosaccharides such as disaccharides, trisaccharides, maltodextrin and dextrin.
  • the monosaccharide may be a hexose, either a ketose or an aldose, such as glucose, mannose, galactose, fructose and combinations thereof.
  • Disaccharides may include sucrose, maltose, trehalose, isomaltose, cellubiose, melibiose, primeverose, rutinose, gen- tiobiose and lactose and combinations thereof.
  • the trisaccharide may be maltotriose, raffi- nose or a combination thereof.
  • the carbohydrate may be a starch hydrolysate produced by hydrolysis, e.g. acid hydrolysis, particularly with an average of 2-20 monomer glucose units, such as dextrin with DE 6-8 or maltodextrin with DE 20-23 of starch.
  • hydrolysis e.g. acid hydrolysis, particularly with an average of 2-20 monomer glucose units, such as dextrin with DE 6-8 or maltodextrin with DE 20-23 of starch.
  • the sugar alcohol may be monomeric, e.g. sorbitol or arabitol.
  • the amount of the polyol (carbohydrate or sugar alcohol) used in the particle of the present invention may be above 2 % by weight, e.g. 2 to 75 %, 2 to 50 %, 5 to 30 %, 10 to 25 % or 7,5 to 25 % by weight of the enzyme product particle.
  • the carrier particle is in a particular embodiment of the present invention a preformed single carrier particle and is hereinafter referred to as the carrier.
  • preformed particle is meant a particle having its final form and structure before adding it to the process of producing the immobilized enzyme product of the present invention.
  • the carrier is in one embodiment of the present invention a solid carrier.
  • the immobilized enzyme product is not agglomerated (or not substantially agglomerated) during the immobilization process.
  • the carrier particle may have a particle size of 50-2000 microns, 100-1000 microns, 250-600 microns, or 300-800 microns.
  • the carrier is preferably porous.
  • the pore volume may correspond to an oil uptake of at least 0.5 g of oil per g of carrier, particularly at least 1 g/g. It may have a surface area of
  • the carrier may have a pore size of 5 nm-50 ⁇ m, such as 5 nm-1000 nm, in particular 10-500 nm, more particularly 100-500 nm.
  • the carrier may have a mean particle size of at least 100 ⁇ m, at least 150 ⁇ m. It may have a mean particle size of up to 600 ⁇ m, up to 500 ⁇ m, up to 450 ⁇ m or up to 300 ⁇ m. The mean particle size may be in the range of 50-1500 ⁇ m, 100-1000 ⁇ m, 150-900 ⁇ m, 200- 800 ⁇ m, such as 250-750 ⁇ m, 250-700 ⁇ m or 300-1200 ⁇ m.
  • the carrier particles may comprise inorganic, organic or both inorganic and organic material. Said carrier may further have a hydrophilic or hydrophobic surface.
  • the carrier particles may comprise an inorganic material with a substantially hydrophilic surface, which is essentially insoluble in hydrophilic or hydrophobic liquids or mixtures thereof.
  • Carriers may be based on silicas (e.g. Sipernat 2200 from Degussa, Germany), zeo- lites (e.g. Wessalith MS330 from Degussa, Germany), aluminas, diatomaceous earth, ceramics such as disclosed in Yoshihiko Hirose et Al., Proceedings from 3rd International Symposium on Biocatalysis and Biotransformations, La Grande Motte, France, 1997, p238) and kaolins (e.g.
  • the particulate porous carrier is selected from the group consisting of silica, zeolite, alumina, ceramic and kaolin.
  • the carrier may be metal oxides such as alumina, particularly gamma alumina, silica, zirconia, silica magnesia, silica-zirconia-alumina etc.
  • the carrier may be silica, particularly having a purity of greater than 85%, greater than 90%, greater than 95% or greater than 98%.
  • the silica may be precipitated silica. It has been found that particularly the combination of a soluble polyol such as carbohydrate and a carrier based on silica with a high degree of purity has a very good influence on the stability of the enzyme during application.
  • the carrier may be silica with a mean particle size in the range of 50-1500 ⁇ m, such as 100-1000 ⁇ m, wherein the silica has a purity of more than 90%.
  • the carrier is a silica with a mean particle size of 100-1000 ⁇ m and a purity of more than 95%.
  • the carrier is not water soluble. In a further embodiment the carrier present in the enzyme product is not water disintegrable. If the immobilized enzyme product is added to water containing liquids the enzyme and carbohy- drate may go into solution - either partly or completely, whereby the immobilization effect will be destroyed, but the carrier will keep its structure and not go into solution.
  • the carrier particles comprise a hy- drophilic inorganic material coated with organic moieties, thus having a substantially hydrophobic surface, e.g. as described in JP 09000257-A, wherein an acid treated kaolin carrier is coated with N-phenyl-gamma-aminopropyltrimethoxysilane.
  • the carrier particles comprise an organic polymer resin.
  • the resin may be an adsorbent resin, preferably a polyacry- late, a polymethacrylate (e.g. polymethyl methacrylate), polystyrene cross-linked with divinyl- benzene, polyurethane or polypropylene or the resin may be an ion exchange resin, prefera- bly an anion exchange resin, e.g. a weakly basic anion exchange resin.
  • a preferred anion exchange resin is a phenolic type Duolite resin from Rohm & Haas.
  • the carrier may be made from regenerated chitosan as disclosed in DE 4429018-A.
  • the enzyme to be immobilized according to the invention is a lipolytic enzyme, i.e. an enzyme which is capable of hydrolyzing carboxylic ester bonds to release carboxylate (EC 3.1.1 ).
  • the lipolytic enzyme is an enzyme classified under the Enzyme Classification number E. C. 3.1.1.- (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).
  • the lipolytic enzyme may exhibit hydrolytic activity, typically at a water/lipid interface, towards carboxylic ester bonds in substrates such as mono-, di- and triglycerides, phospholipids, thioesters, cholesterol esters, wax-esters, cutin, suberin, synthetic esters or other lipids mentioned in the context of E. C. 3.1.1.
  • the lipolytic enzyme may, e.g., have triacylglycerol lipase activity (EC 3.1.1.3, 1 ,3-positionally specific or non-specific), phospholipase activity (A1 or A2, EC 3.1.1.32 or EC 3.1.1.4), esterase activity (EC 3.1.1.1 ) or cutinase activity (EC 3.1.1.74).
  • Suitable lipolytic enzymes include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples include lipases from Candida, C. Antarctica (e.g. lipases A and B described in WO 88/02775), C. rugosa (C. cyl- indracea), Rhizomucor, R. miehei, Hyphozyma, Humicola, Thermomyces, T. lanuginosus ⁇ H. lanuginosa lipase) as described in EP 258 068 and EP 305 216, a Pseudomonas lipase, e.g. from P. alcaligenes or P.
  • lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples include lipases from Candida, C. Antarctica (e.g. lipases A and B described in WO 88/02775), C. rugosa (C. cyl-
  • pseudoalcaligenes EP 218 272
  • P. cepacia EP 331 376
  • P. glumae P. stutzeri
  • P. fluorescens Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002)
  • P. wisconsinensis WO 96/12012
  • Bacillus lipase e.g. from B. subtilis (Dar- tois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360
  • B. stearothermophilus JP 64/744992
  • the lipase may be positionally site specific (i.e. 1 ,3 specific) or non-specific, upon interaction with triglycerides as substrates.
  • cloned lipases may be useful, including the Penicillium camembertii lipase described by Yamaguchi et al., (1991 ), Gene 103, 61-67), the Geotricum candidum lipase (Shimada, Y. et al., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipases such as a R. delemar lipase (Hass, MJ et al., (1991 ), Gene 109, 1 17-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Biochem. 56, 716-719) and a R. oryzae lipase.
  • Rhizopus lipases such as a R. delemar lipase (Hass, MJ et al., (1991 ), Gene 109, 1 17-113), a R.
  • cutinases Other types of lipolytic enzymes such as cutinases may also be useful, e.g. cutinase from Pseudomonas mendocina (WO 88/09367), Fusarium solani pisi (WO 90/09446) or H. insolens (US 5,827,719).
  • the enzyme may be an enzyme variant produced, for example, by recombinant techniques.
  • lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • lipases examples include LipexTM, LipoprimeTM, Lipo- laseTM, LipolaseTM Ultra, LipozymeTM, PalataseTM, NovozymTM 435 and LecitaseTM (all available from Novozymes A/S).
  • Other commercially available lipases include LumafastTM (Pseudomonas mendocina lipase from Genencor International Inc.); LipomaxTM (Ps. pseudoalcali- genes lipase from DSM/Genencor Int. Inc.; and Bacillus sp. lipase from Genencor enzymes. Further lipases are available from other suppliers.
  • the enzyme may be added to the immobilization process in liquid form, such as an enzyme containing liquid medium.
  • the enzyme containing liquid medium is in a particular embodiment of the present invention a hydrophilic medium.
  • the liquid medium is aque- ous. It may contain other organic or biological matter.
  • it may be a fermentation broth or an enzyme concentrate solution obtainable by purifying a fermentation broth by e.g. ultra filtration or by protein precipitation, separation and re-dissolution in another aqueous medium. It may further be substantially pure enzyme dissolved in an aqueous medium.
  • the enzyme containing aqueous liquid has not been subjected to costly processing steps prior to immobilization to remove water such as evaporation nor has it been subjected to addition of non aqueous solvents, e.g.
  • the enzyme protein load of the enzyme product particle is more that 10 mg/g carrier. In another embodiment of the present invention the enzyme protein load of the enzyme product particle is more that 15 mg/g carrier. In a further embodiment of the present invention the enzyme protein load of the enzyme product particle is more that 20 mg/g carrier
  • the enzyme product of the present invention may besides an enzyme, a carrier and one or more carbohydrates comprise additional components.
  • the carrier is particulate, consisting of preformed single particles, and the immobili- zation process is preferably done at conditions where no agglomeration or substantially no agglomeration occurs.
  • the immobilization process is preferably not a granulation process
  • a liquid or several liquids comprising the enzyme and the soluble polyol (carbohydrate) are applied to the carrier particles.
  • the carrier particles adsorb and/or absorb the liquid comprising enzyme and polyol (e.g. carbohydrate) in which process the enzyme is immobilized.
  • the carrier particles should not agglomerate during the process and should therefore not change size significantly during the process.
  • the liquid medium may comprise a volatile component such as water.
  • the process may comprise the following steps: a) introducing a liquid comprising an enzyme and a polyol (e.g. carbohydrate) by atomi- zation onto a carrier, so as to adsorb or absorb the enzyme and polyol (e.g. carbohydrate) on/in the carrier, b) removing volatile components of the liquid medium from said product .
  • a liquid comprising an enzyme and a polyol e.g. carbohydrate
  • a carrier so as to adsorb or absorb the enzyme and polyol (e.g. carbohydrate) on/in the carrier
  • the liquid is preferably introduced in an amount such that substantially no agglom- eration of the carrier particles occurs. All liquid is accumulated in the particles, so the absorption capacity of the particles is not exceeded.
  • the process may comprise the following steps: a) fluidizing the particulate, porous carrier in a fluid bed, b) introducing an enzyme containing liquid medium by atomization into the fluid bed, so as to absorb the enzyme into the carrier, and c) introducing a polyol (e.g. carbohydrate) containing liquid medium by atomization into the fluid bed, so as to absorb the polyol (e.g. carbohydrate) into the carrier, and d) removing volatile components of the liquid medium from the carrier in the fluidized bed.
  • a polyol e.g. carbohydrate
  • the liquid in b) and c) may be the same.
  • the process comprises the following steps: a) fluidizing a particulate porous carrier in a fluid bed, b) introducing a liquid comprising an enzyme and a polyol (e.g. carbohydrate) by atomi- zation into the fluid bed, so as to absorb the enzyme and the polyol (e.g. polyol (e.g. carbohydrate)) in the carrier.
  • the immobilization process is in one embodiment of the present invention a process for producing an immobilized enzyme product comprising the following steps: a) preparing a liquid comprising an enzyme; b) preparing a liquid comprising a polyol (e.g.
  • liquids of a) and b) onto a carrier particle wherein the introducing of the liquids of a) and b) onto the carrier may be in any order or simultaneously and wherein the absorption capacity of the carrier is not exceeded.
  • the liquids of a) and b) may be the same.
  • the immobilization process is in one embodiment of the present invention a process for producing an immobilized enzyme product comprising the following steps: a) preparing a liquid comprising an enzyme and a polyol (e.g. carbohydrate); b) introducing the liquid of a) onto a carrier particle
  • the immobilization process may comprise a further step of removing volatile components from the carrier.
  • the term “absorption capacity of the carrier” is meant the amount of liquid the carrier is able to absorb.
  • One way to determine the absorption capacity is to dry a sample of carrier at 105 0 C for 24 hrs., cool the carrier down to ambient temperature and place 1 g of the dried carrier material in 100 ml liquid at 20 0 C for 1 hour, then the carrier is separated from the excess liquid by drainage and the weight of the carrier comprising the absorbed liquid is determined. When the absorption capacity is exceeded the carrier is not able to absorb any more liquid, and if more liquid is present it will be called excess liquid.
  • the limit for the carriers adsorption capacity has been exceeded and excess liquid is present.
  • the amount of liquid added to the process is not resulting in the absorption capacity of the carrier being exceeded.
  • the amount of liquid added to the process should be limited so the absorption capacity of the carrier is not exceeded.
  • the liquid should be added in such amounts that substantially no agglomeration of the carrier occurs.
  • the ratio of the weight of liquid medium added to the process and the weight of the carrier is below 50.
  • the ratio of the weight of liquid medium added to the process and the weight of the carrier is below 25.
  • the ratio of liquid medium to carrier is below 10. In a further embodiment the ratio is below 5.
  • the removal of volatile components may be performed by, but is not limited to, various methods such as filtration, centrifugation, spray-drying, air-drying, and freeze-drying.
  • the removal of volatile components is conducted in a fluidized bed. Suitable temperatures of the inlet air for removing volatile components will primarily depend of the thermal stability of the enzyme (the inactivation temperature).
  • the hot inlet air temperature will result in a product temperature, which at the end of the drying process may be 30-130 0 C, 30-90 0 C, such as 50-70°C, e.g. 60 0 C.
  • a higher inlet air temperature provides shorter drying times.
  • the immobilization process may be performed in any apparatus suitable for said process.
  • the apparatus is selected from the group consisting of mixers, fluid beds and pan coaters.
  • the immobilization process may be performed in a mixer apparatus, a fluid bed or a pan coater.
  • the mixer apparatus of the present invention may be any mixer apparatus, e.g. a Lodige Mixer, Germany. Immobilizing the enzyme on the carrier in a mixer may suitably be conducted at ambient temperature. Mixing times may suitably be 5-60 minutes, preferably 10-30 minutes. If the enzyme product is produced in a mixer, the mixer is in a particular em- bodiment operated in a manner to avoid or minimize agglomeration and granulation.
  • the fluid bed apparatus may be any apparatus principally working as a fluid bed.
  • the liquid media of the present invention may be introduced onto the carrier by atomization.
  • a suitable air inlet flow in the fluid bed equipment will depend on the size and density of the immobilized enzyme product, the amount of carrier and the fluid bed equipment. Further the air inlet flow has an upper limit, as the flow should be sufficient to keep the immobilized enzyme product fluidized, but not so powerful as to "blow off" the immobilized enzyme product.
  • the drying process will occur for as long as the liquid media are atomized into the fluid bed, and may suitably be extended for 5-30 minutes after spraying of the liquid media has ended.
  • An important aspect of the invention is that the immobilization processes can easily be scaled up by applying other larger standard equipment.
  • the equipment setting ranges given vide supra may be adjusted to optimize larger scale equipment.
  • the carrier has a substantially hydrophilic surface.
  • the immobilization process may be con- ducted in a standard mixing equipment (e.g. Lodige, Germany), wherein the liquid media of step a) and b) are introduced by atomization to the dry porous and particulate carrier during mixing, e.g. using an atomizer connected to a pump (e.g. a standard peristaltic Watson- Marlow pump).
  • the immobilization of enzyme on a carrier having a substantially hydrophilic surface may alternatively be conducted in a standard fluid bed equipment, e.g.
  • a Uni-Glatt fluidized bed apparatus (Glatt, Germany), wherein the carrier is fluidized and the liquid media of step a) and b) are introduced by spraying to the fluidized carrier, e.g. using an spray nozzle connected to a pump.
  • immobilization and drying may be conducted simultaneously.
  • the immobilization of enzyme on a carrier having a substantially hydrophobic surface may be conducted in a standard mixing equipment, wherein the liquid media of step a) and b) are introduced to the carrier.
  • the immobilization of enzyme on a carrier having a substantially hydrophobic surface may alternatively be conducted in a standard fluid bed equipment, e.g. a Uni-Glatt fluidized bed apparatus (Glatt, Germany), wherein the carrier is fluidized and the liquid media of step a) and b) are introduced by spraying to the fluidized carrier, e.g. using an spray nozzle connected to a pump.
  • immo- bilization and drying are conducted simultaneously.
  • the order of enzyme and polyol (e.g. carbohydrate) addition may be any of; first: addition of a liquid solution of enzyme then addition of a liquid solution of polyol (e.g. carbohydrate) onto the carrier with or without intermediate drying of the product between the additions; second: addition of a liquid solution of polyol (e.g. carbohydrate) then addition of a liquid solution of enzyme onto the carrier with or without intermediate drying of the product between the additions; third: Addition of a blend of polyol (e.g. carbohydrate) and enzyme onto the carrier.
  • the immobilization may also be carried out by suspending the carrier in an aqueous solution of enzyme and polyol (e.g. carbohydrate). Allow time for the carrier to absorb the liquid solution into the pore structure and to adsorb polyol (e.g. carbohydrate) and enzyme from the solution. Then recover the carrier from the suspension by any practical means for solid-liquid separation - centrifugation, filtration, decanting, sedimentation etc.
  • polyol e.g. carbohydrate
  • wet particles are dried by a method that can be used for removing the remaining amount of solvent, e.g. drying on trays in an oven, vacuum drying, freeze drying or fluid bed drying.
  • the polyol (e.g. carbohydrate) and enzyme might also be added in to the carrier in separate steps in any order and with or without an intermediate drying stage.
  • Immobilized enzymes prepared according to the invention have potential applica- tions in a wide range of enzymatic employed processes such as in the production of pharmaceuticals, specialty commodity chemicals, and vegetable oil processing.
  • Immobilized enzymes prepared in the context of the invention may be used for hydrolysis, synthesis or modification of organic substances.
  • the hydrolysis, synthesis or modification preferably takes place in a medium essentially devoid of free water.
  • the invention encompasses a process for enzymatic modification of an organic compound comprising contacting in a reaction medium said organic compound with an immobilized enzyme product produced according to the invention.
  • the immobilized enzyme of the present invention may be used for enzymatic modification of an organic compound comprising contacting in a reaction medium said organic compound with an immobilized enzyme produced by the process of the invention.
  • the modification is an esterifica- tion reaction comprising contacting a first reactant which is a carboxylic acid and a second reactant which is an alcohol with an immobilized lipase produced by the process of the invention.
  • the carboxylic acid may be selected from but not limited to the group consisting of fatty acids, lactic acid, benzoic acid, acrylic acid and methacrylic acid and the alcohol may be selected from but not limited to the group consisting of methanol, ethanol, isopropanol, polyols such as glycerol, sorbitol, isosorbide, xylitol, glucosides such as ethyl and methyl glucosides, neopentyl alcohol and propylene glycol.
  • the modification may be a chiral resolvation including an enantioselective synthesis or hydrolysis of carboxylic acid ester or amides; an aldol condensation reaction between two aldehydes; or an epoxidation of olefinic groups by percarboxylic acid produced in situ by the enzyme in this present invention.
  • the modification may be a polyesterification reaction wherein the organic compound to be modified is a hydroxycarboxylic acid or oligomers of such compound e.g. lactic acid or 3-hydroxypropanoic acid.
  • the carboxylic acid is a dicarboxylic acid selected from the group consisting of adipic acid, succinic acid, fumaric acid, 2,5-furandicarboxylic acid, glu- caric acid, terephthalic acid and isophthalic acid
  • the second reactant is selected from the group consisting of polyols such as (1 ,4-butanediol, 1 ,6-hexanediol, glycerol, sorbitol, isosorbide, neopentyl alcohol, propylene glycol).
  • the modification is a ring opening polymerization reaction comprising contacting a lactone with an immobilized lipase produced by the present process. Prepared polymers may be homo or hetero polymers.
  • the modification may be a transesterification reaction comprising contacting a first reactant which is a carboxylic acid ester and a second reactant which is an alcohol with an immobilized lipase produced by the present process.
  • the modification may be an interesterification reaction comprising contacting a first reactant which is a carboxylic acid ester and a second reactant which is a second carboxylic acid ester with an immobilized lipase produced by the present process.
  • the modification is an interesterification reaction comprising contacting a first reactant which is a polycarboxylic acid ester and a second reactant which is a second poly- carboxylic acid ester, with an immobilized lipase produced by the present process.
  • the carboxylic acid ester may be selected from but not limited to the group consisting of alkyl esters of fatty acids, lactic acid, glucaric acid, benzoic acid, acrylic acid, methacrylic acid and wherein the alkyl may be methyl, ethyl, butyl and the alcohol may be selected from the group consisting of but not limited to methanol, ethanol, isopropanol, poly- ols such as glycerol, alkyl glucosides, such as ethyl glucoside or methyl glucoside, sorbitol, silicone and silicone derivatives, isosorbide, neopentyl alcohol and propylene glycol.
  • alkyl esters of fatty acids lactic acid, glucaric acid, benzoic acid, acrylic acid, methacrylic acid and wherein the alkyl may be methyl, ethyl, butyl and the alcohol may be selected from the group consisting of but not limited to methanol,
  • the modification may be a hydrolysis or synthesis producing an enantiopure com- pound; an amidation reaction comprising contacting a first reactant which is a carboxylic acid and a second reactant which is an amine with an immobilized lipase produced by the present process.
  • the modification is an epoxidation reaction comprising in situ production of epoxidation agent with an immobilized enzyme produced by the present process.
  • an immobilized lipase enzyme is used for an esterification, transesterification or interesterification process in a medium essentially devoid of free water.
  • the transesterification may be used for fatty acid substitution, comprising contacting a first reactant and a second reactant with said immobilized lipase by which a substitution reaction occurs.
  • the first reactant may be a fatty acid ester, preferably a triglyceride or a mixture of triglycerides.
  • the second reactant may be another fatty acid ester different from the first reactant, preferably a triglyceride or a mixture of triglycerides. Further the second reactant may be an alcohol or a free fatty acid.
  • the medium in this preferred embodiment of the invention comprises an organic solvent, or it may consist essentially of triglycerides. Said use of the invention may be applied in production of food products e.g. margarine or cocoa-butter substitutes.
  • the invention provides a process for conducting a reaction catalyzed by a lipolytic enzyme, comprising a) preparing a reaction mixture comprising reactants for the reaction, and b) contacting the reaction mixture with the immobilized enzyme product at conditions which are effective for conducting the reaction.
  • the contact may be done by passing the reaction mixture through a packed-bed column of the immobilized enzyme product, a continuously stirred tank reactor holding the immobilized enzyme product, a moving bed reactor where the movement of the packed bed of immobilized enzyme is either co-current or counter-current to the reaction mixture, in a batch reactor, optionally with stirring or in any other type of reactor or combination of reactor in which the desired reaction can be carrier out.
  • the lipolytic enzyme may be a lipase
  • the reactants may comprise a fatty acyl donor and an alcohol
  • the reaction may form a fatty acid alkyl ester.
  • the lipolytic enzyme may be a lipase, the reactants may comprise at least two triglycerides, and the reaction may form different triglycerides.
  • a vegetable oil blend is interesterified in a batch reaction using an immobilized lipase as catalyst. After a certain reaction time, the oil is decanted from the immobilized lipase which remains in the reactor. Next fresh vegetable oil blend is added to the immobilized enzyme in the reactor and another batch reaction is carried out.
  • the interesterification is deter- mined by measuring the solid fat content (SFC) of the oil blend.
  • SFC solid fat content
  • the average reaction rate of the enzyme is determined from each batch reaction using a first order reversible model to describe the reaction rate. Solid fat content of the oil blend is used as concentration parameter.
  • the interesterification ractions are carried out in flasks containing 0.5 g of immobilized enzyme product and 100 g of vegetable oil blend. The blend is shaking for 24 hour between oil exchange.
  • the reaction temperature is 70 0 C.
  • the average production rate is calculated using the two estimated parameters in a reaction model for interesterification of vegetable oil in a fixed bed reactor where the oil flow is adjusted in order to keep the level of interesterification constant.
  • the total production is fixed at 1500 kg of oil per kg of immobilized enzyme product.
  • the average production rate is reported as kg oil interesterified per kg of immobilized enzyme product per hour.
  • the lipase solution (according to 1 ) was then applied uniformly onto 2.425 kg of silica- based carrier (Zeofree 5170 from Huber Engineered Materials, USA) in a 20 L mixer (Lodige, Germany) using continuous mixing with a rotating speed of 150 rpm at ambient temperature. An atomizing nozzle was used to distribute the liquid over the carrier. Total spraying time was set to 12 minutes.
  • Example 2 Immobilization of lipase on a silica-based carrier with an enzyme protein load of 30 mg/g by a 1-step impregnation with maltodextrin (Maltrin M200) additive. 1. 785 g of a solution of lipase from Thermomyces lanuginosus (100 mg/g) was diluted with
  • Average production rate 3.03 kg-oil/kg-imm.prod./hr
  • the lipase solution (according to 1 ) was applied uniformly onto 2039g of silica-based carrier (Zeofree 5170 from Huber Engineered Materials, USA) in a 20 L mixer (Lodige, Germany) using continuous mixing with a rotating speed of 150 rpm at ambient temperature. An atomizing nozzle was used to distribute the liquid over the carrier. Total spraying time was set to 12 minutes.
  • Performance of the immobilized enzyme product is listed as relative average production rate and relative initial activity. The index numbers are calculated relative to the activity of the reference product made according to experiment 3.
  • Examples 3 to 7 show that when the performance of the immobilized enzyme products are quantified either by the average production rate or the initial reaction rate, then the performance is significantly improved for the products comprising a carbohydrate compared to the reference product.
  • Example 8 Immobilization of lipase on a silica-based carrier with an enzyme protein load of 55 mg/g by a 1-step impregnation without additives - the reference.
  • the lipase solution (according to 1 ) was then applied uniformly onto 1 104g of silica- based carrier (Zeofree 5170 from Huber Engineered Materials, USA) in a 10 L mixer
  • Performance of the immobilized enzyme product is listed as relative average production rate and relative initial activity. The index numbers are calculated relative to the activity of the reference product made according to experiment 8.
  • Examples 8-12 show that when the performance of the immobilized enzyme products are quantified either by the average production rate or the initial reaction rate, then the performance is significantly improved for the products comprising a carbohydrate compared to the reference product.
  • Example 13 Immobilization of various lipases
  • Thermomyces lipase, Candida antarctica B lipase and Rhizomucor miehei lipase were immobilized on a precipitated silica based carrier using either the soaking method or the spray impregnation method for the immobilization.
  • spray impregnation immobilization and the suspension, impregnation immobilization methods as well as a table giving the concentration of dextrin and enzyme concentration used in the single experiments and the performance of the resulting product measured using the multiple batch assay.
  • the suspension of the carrier in the enzyme solution was mixed for 2 hours at room temperature, using gentle mixing with a propeller type stirrer in order to avoid crushing of the particles. After 2 hours the mixing was stopped, and the particles were allowed precipitate and the free liquid was removed by decanting.
  • the wet mass of carrier particles was dried in a fluid bed. Drying conditions: Temperature of inlet air T 90 0 C-I OO 0 C. The drying was stopped when a product temperature of 63°C was reached. The air flow was kept at the minimum required for fluidization in order to avoid loosing fines. The dried product was sieved. The product fraction was the particles of size 600-710 ⁇ m.
  • the enzyme concentrate was diluted with water and the dextrin was dissolved in the diluted enzyme solution.
  • the carrier particles were loaded into a 2Ol mixer (Lodige, Ger- many).
  • the lipase solution was then applied uniformly onto the silica carrier by spraying while using continuous mixing with a rotating speed of 150 rpm. Enzyme spraying was done at ambient temperature. Liquid addition time was set to 12 minutes. Care was taken to avoid over-wetting of the carrier - meaning that the amount of liquid added was less than the liquid adsorption capacity of the carrier.
  • the wet particles were dried in a fluid bed. Drying conditions: Temperature of inlet air T 90 0 C-I OO 0 C. The drying was stopped when a product temperature of 63°C was reached.
  • the air flow was kept at the minimum required for fluidization in order to avoid loosing fines.
  • the dried product was sieved.
  • the product fraction was the particles of size 600- 710 ⁇ m
  • the dextrin used was maltrin M200.
  • the activity of the immobilized lipase was determined using the multiple batch assay. The results of the experiments are given in the table below.
  • maltrin M200 has a positive influence on the performance of all three lipases in both the immobilization methods tested.
  • the additive and the water were added to the enzyme concentrate so that a final solution in all experiments contained an enzyme amount of 33 mg/g and the amount of additive listed in the table below.
  • the silica carrier was suspended in this solution. A carrier to liquid ratio of 1 :5 (w/w) was used.
  • the carrier was incubated in the enzyme solution for 2 hours at 50 0 C - gentle mixing with a propeller type stirrer in order to avoid crushing of the particles. Then the particles were separated from the liquid phase by filtration. The wet product was dried on trays at room temperature. The dry products were sieved. The product fraction was the particles of size 300-1 OOO ⁇ m.
  • the performance of the products was determined using the multiple batch assay.
  • the performance of the product made according to the invention is given relative to the performance of a reference product, made without addition of the claimed type of additive.
  • the additive and the water were added to the enzyme concentrate to get a solution with an enzyme content of 33 mg/g of solution and a dextrin concentration of 10% (w/w).
  • the carrier particles were loaded into a 2Ol mixer (Lodige, Germany).
  • the solution of enzyme and dextrin was then applied uniformly onto the silica carrier by spraying the liquid while continuously mixing the carrier with a rotating speed of 150 rpm at ambient temperature. 1.2 kg of liquid was added per kg of carrier.
  • the wet particles were dried in a fluid bed.
  • the enzymes solution had an enzyme content of 66 mg/g and the dextrin solution had a dextrin concentration of 20% (w/w).
  • the solution of enzyme and the solution of dextrin were then applied uniformly onto the silica carrier one after the other by spraying the liquid while continuously mixing the carrier with a rotating speed of 150 rpm at ambient temperature.
  • 0.6 kg of enzyme solution and 0.6 kg of dextrin solution were added per kg of carrier.
  • the wet particles were dried and the activity of the immobilized lipase was determined using the multiple batch assay.
  • the wet particles were dried in a fluid bed.
  • the performance of the three products was determined by the multiple batch assay.
  • the performance for the single experiment is given relative the average performance of the three products.

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Abstract

Lors de l'immobilisation d'une enzyme lipolytique sur des particules de support préformées, l'addition d'un polyol soluble tel qu'un glucide ou un alcool de sucre améliore de façon significative la performance du produit enzymatique lipolytique immobilisé en ce qui concerne l'activité enzymatique initiale, la stabilité enzymatique (demi-vie) et/ou la productivité de l'enzyme.
PCT/EP2008/059390 2007-07-18 2008-07-17 Immobilisation d'enzymes WO2009010561A1 (fr)

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WO2011048083A1 (fr) 2009-10-21 2011-04-28 Novozymes A/S Procédé pour le traitement de l'huile
CN102321605A (zh) * 2011-10-21 2012-01-18 江南大学 一种以介孔分子筛-壳聚糖为载体固定化果糖基转移酶的制备方法
ES2376844A1 (es) * 2010-04-09 2012-03-20 Consejo Superior De Investigaciones Cient�?Ficas (Csic) Biocatalizadores h�?bridos.
CN102839166A (zh) * 2011-06-23 2012-12-26 丰益(上海)生物技术研发中心有限公司 Tl固定化酶及其应用
US20130183734A1 (en) * 2010-07-01 2013-07-18 Clariant Produkte (Deutschland) Gmbh Phospholipase-Carrier Complex
WO2017044545A1 (fr) * 2015-09-11 2017-03-16 Novozymes Bioag A/S Compositions d'inoculant stables et procédés de production de ces compositions
WO2017116837A1 (fr) * 2015-12-28 2017-07-06 Novozymes Bioag A/S Compositions d'inoculants stables et leurs procédés de production
CN107012136A (zh) * 2017-06-12 2017-08-04 浙江工业大学 一种固定化疏绵状嗜热丝孢菌脂肪酶的方法
CN108148827A (zh) * 2016-12-02 2018-06-12 丰益(上海)生物技术研发中心有限公司 固定化酶及其制备方法和用途
US10160997B2 (en) 2008-09-12 2018-12-25 Gentegra Llc Matrices and media for storage and stabilization of biomolecules
WO2019101951A1 (fr) * 2017-11-24 2019-05-31 Novozymes A/S Petites particules d'enzyme pour l'interestérification
EP3744838A1 (fr) * 2019-05-29 2020-12-02 Novozymes A/S Particules polymères lipolytiques pour l'estérification et l'interestérification
WO2021009550A1 (fr) * 2019-07-18 2021-01-21 Rhodia Brasil Ltda Préparation d'enzymes immobilisées
WO2022192978A1 (fr) * 2021-03-18 2022-09-22 Suzano S.A. Cellulose fonctionnalisée, procédé de fonctionnalisation enzymatique de cellulose, processus de fonctionnalisation enzymatique de cellulose à partir d'un acide organique et processus de production d'une cellulose à hydrophobie accrue, et article
CN115927288A (zh) * 2022-07-28 2023-04-07 南京林业大学 一种固定化脂肪tll酶及固体结合肽在其固定化中的应用
WO2023229519A1 (fr) * 2022-05-27 2023-11-30 Bunge Sa Procédé discontinu de modification enzymatique de lipides

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2879186A1 (fr) * 2004-12-09 2006-06-16 Total France Sa Immobilisation de proteines a l'interieur d'une matrice de silice mesoporeuse par synthese directe a partir d'alcoxysilanes
US20070087418A1 (en) * 2005-09-30 2007-04-19 Novozymes A/S Immobilization of enzymes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2879186A1 (fr) * 2004-12-09 2006-06-16 Total France Sa Immobilisation de proteines a l'interieur d'une matrice de silice mesoporeuse par synthese directe a partir d'alcoxysilanes
US20070087418A1 (en) * 2005-09-30 2007-04-19 Novozymes A/S Immobilization of enzymes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LUCKARIFT HEATHER R ET AL: "Enzyme immobilization in a biomimetic silica support", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 22, no. 2, February 2004 (2004-02-01), pages 211 - 213, XP002342765, ISSN: 1087-0156 *

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WO2011048083A1 (fr) 2009-10-21 2011-04-28 Novozymes A/S Procédé pour le traitement de l'huile
ES2376844A1 (es) * 2010-04-09 2012-03-20 Consejo Superior De Investigaciones Cient�?Ficas (Csic) Biocatalizadores h�?bridos.
US20130183734A1 (en) * 2010-07-01 2013-07-18 Clariant Produkte (Deutschland) Gmbh Phospholipase-Carrier Complex
CN102839166A (zh) * 2011-06-23 2012-12-26 丰益(上海)生物技术研发中心有限公司 Tl固定化酶及其应用
CN102321605A (zh) * 2011-10-21 2012-01-18 江南大学 一种以介孔分子筛-壳聚糖为载体固定化果糖基转移酶的制备方法
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