EP2655611A1 - Procédé pour l'immobilisation covalente d'enzymes sur des supports polymères solides fonctionnalisés - Google Patents

Procédé pour l'immobilisation covalente d'enzymes sur des supports polymères solides fonctionnalisés

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Publication number
EP2655611A1
EP2655611A1 EP11811024.6A EP11811024A EP2655611A1 EP 2655611 A1 EP2655611 A1 EP 2655611A1 EP 11811024 A EP11811024 A EP 11811024A EP 2655611 A1 EP2655611 A1 EP 2655611A1
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EP
European Patent Office
Prior art keywords
enzyme
hydrophobic
immobilization
support
polymeric support
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.)
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Application number
EP11811024.6A
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German (de)
English (en)
Inventor
Lucia Gardossi
Patrizia SPIZZO
Diana FATTOR
Loris SINIGOI
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.)
Sprin SpA
Universita degli Studi di Trieste
Original Assignee
Sprin SpA
Universita degli Studi di Trieste
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Publication of EP2655611A1 publication Critical patent/EP2655611A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N11/02—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

Definitions

  • the invention relates to a method for covalent immobilization of enzymes, especially lipases, on functionalized solid polymeric supports.
  • the protein can be detached from the support in the presence of an aqueous or hydrophilic phase in which it will tend to be distributed or on account of mechanical stresses (e.g.
  • Patent US 5,776,741 describes an alternative method of immobilization that envisages the formation of granules containing the enzyme, which is maintained around a silica support through the use of a binder, such as polyvinylpyrrolidone (PVP) or gelatin.
  • a binder such as polyvinylpyrrolidone (PVP) or gelatin.
  • PVP polyvinylpyrrolidone
  • the anchoring on the support does not, however, envisage the formation of covalent bonds, therefore the preparation is recommended for applications in the presence of at least 50% of organic phase, in order to avoid damaging the enzymatic formulation with distribution of the protein in the aqueous phase.
  • Basso A. et al. (Basso A. et al., Chem. Commun., 2000, 467-468; Basso A. et al., Tetrahedron Lett., 2000, 41 , 8627-8630) describe a method of immobilization intended for use of the enzyme in non-aqueous media. Immobilization is effected non- covalently, by adsorption of the enzymes penicillin G amidase, alpha-chymotrypsin and thermolysin on hydrophilic and hygroscopic porous supports of the silica type.
  • the method envisages the use of a water-immiscible organic solvent, in which the porous silica support is placed, to which the aqueous solution containing the enzyme will then be added.
  • aqueous solution is quickly absorbed in the hygroscopic solid support together with the enzyme, which thus remains in contact with an aqueous microphase inside the pores of the support.
  • This enzymatic preparation consisting of the hydrated support and the enzyme, can then be used directly for carrying out reactions in organic solvent without further dehydration.
  • the purpose of this method is to create a hydrated microenvironment around the enzyme within the hydrophilic and hygroscopic porous material.
  • Lipases are of enormous practical and industrial importance, both in the food sector and in the fields of pharmaceuticals, cosmetics and biofuels [Biermann U. et al., Angewandte Chemie, 2000, 39, 2206-2224; Schmid R.D., Verger, R., Angewandte Chemie International Edition, 1998, 37, 1608-1633].
  • the lipases are enzymes that are also known to be active in anhydrous reaction systems and their natural substrates are the triglycerides, molecules that are insoluble in water and are highly hydrophobic.
  • lipases do not operate in aqueous solution but at the interface with hydrophobic phases. Moreover, most lipases when put in an aqueous system assume an inactive "closed” conformation and need to come into contact with a hydrophobic phase to assume the active "open” conformation. Therefore, to ensure maximum efficiency of immobilized lipases it is necessary to induce these conformational changes and lock the lipases in this conformation. Consequently, the difficulty of developing suitable immobilization techniques for lipases, so as to keep them “active", is well known. In fact, although numerous methods are described in the literature, which envisage adsorption of lipases on solid supports [Hanefeld U. et al., 2009, ref.
  • covalent immobilization of an enzyme on solid supports it is further observed that covalent coupling of the protein to the support must be performed in such a way as not to supersaturate the functional groups present on the surface of the support, producing effective covalent immobilization of only a percentage of the protein. Hence the need to load the support with an optimum amount of protein, sufficient for reaction of all the functional groups that might interfere with reagents in the course of the chemical process, but not excessive, so as to avoid detachment of protein in excess relative to said process.
  • the patent application US 20100035312 of Basheer S. provides a method of immobilization that aims to activate lipase by vigorous mixing in a two-phase system (thus creating an emulsion). In this way the lipase modifies its conformation, making the active site accessible to the substrate.
  • the immobilization step envisages the use of a solid support treated beforehand with a surfactant dissolved in an organic solvent. This would make it possible, according to the inventor, to increase the affinity of the support for the hydrophobic zone of the lipase and would thus promote immobilization thereof.
  • one purpose of the present invention is to provide a method for immobilization of enzymes, especially those that are active at the interface with hydrophobic phase such as lipases, on solid supports, in which there is formation of a stable linkage between the support and the enzyme and in which a hydrophobic "interface" forms around the solid support and enables the enzyme to: i) pass from the closed/inactive conformation (predominant when the enzyme is in an aqueous phase) to the open/activated conformation (predominant when the enzyme comes into contact with a hydrophobic phase); ii) adopt an optimum orientation, turning the opening of the hydrophobic active site towards the hydrophobic organic phase.
  • the invention relates to a method of covalent immobilization of an enzyme on a porous solid polymeric support comprising at least the following steps:
  • hydrophobic monophase by adding a hydrophobic organic compound or a monophase mixture of hydrophobic organic compounds and liquids at temperatures from 18 to 40 °C to a porous solid polymeric support having medium or high hydrophobicity, a free reactive functional group and a water content not higher than 15% w/w;
  • the method can comprise the further steps of:
  • the polymer to which the enzyme is to be covalently bound can be undergone to suitable treatments, i.e. optional preactivation of the functional groups and/or dehydration so as to reduce the water content to a value not higher than 15% w/w; and/or
  • the enzymatic preparation obtained consisting of the enzyme covalently bound to the selected polymeric support, can be separated for example by filtration or centrifugation, washed and dehydrated.
  • Figure 1 shows titration of the enzymatic activity present in solution after 15 minutes of incubation in the reaction conditions described in example 4 and resulting from detachment of the immobilized enzyme: comparison between the preparation of lipase from Candida antarctica (CALB) covalently immobilized in toluene according to the method of the invention (example 12) and a commercial enzymatic preparation (Novozym 435 ® ) prepared by adsorption on organic polymer.
  • CALB lipase from Candida antarctica
  • Novozym 435 ® a commercial enzymatic preparation
  • Figure 2 shows the 1 H NMR spectrum of the oligomer obtained by the polycondensation reaction catalysed by lipase B from Candida antarctica prepared according to example 12 between adipic acid and 1 ,4-butanediol.
  • the presence of the signal at ⁇ 4.08 (t, 2H, -CH 2 OC(O)) in the 1 H NMR spectrum proves that the polycondensation process has taken place.
  • Figure 3 shows comparison between the amount of enzyme released in the oligomer when the following are respectively used in the synthesis thereof: lipase B from Candida antarctica immobilized covalently according to example 12 and a commercial preparation immobilized by adsorption (Novozym 435 ® ).
  • the blank corresponds to the polymer produced thermodynamically in the absence of catalyst (without enzyme at 150 ⁇ for 3h and then 180°C for 3h).
  • the evaluation was performed by titrating the hydrolytic activity against tributyrin after removing the immobilized enzyme.
  • FIG. 4b shows the 1 H NMR spectrum of the polymer obtained after removing the biocatalyst and derived from polymerization of the oligomer obtained in example 12 between adipic acid and 1 ,4-butanediol, whose 1 H NMR spectrum is given in Fig. 4a.
  • Comparison of the spectra shows that in Fig. 4b the signal of the alcoholic group ( ⁇ 3.53) decreases whereas the signal relating to the stereo group ( ⁇ 4.08) increases.
  • Figure 5 shows the 13 C NMR spectrum of the polymer obtained by polymerization of the oligomer obtained by polycondensation catalysed by lipase B from Candida antarctica prepared in example 12 between adipic acid and 1 ,4-butanediol.
  • the 13 C NMR spectrum shows the presence of a single carbonyl signal attributable to a stereo group ( ⁇ 173) whereas no signals appear that are attributable to acid carbonyl groups.
  • the absence of acid carbonyl groups indicates that all the adipic acid present in the mixture reacted with the butanediol.
  • the increase in the degree of polymerization and hence continuation of polymerization can moreover be determined from the decrease of the ratio between the signals ⁇ 62.6 (-CH 2 -CH 2 -OH) and ⁇ 63.9 (-CH 2 -CH 2 -OC(O)).
  • the technical problem that the present invention aims to solve relates to the immobilization, on solid polymeric supports, of enzymes, especially those that are active at the interface with hydrophobic phases.
  • the immobilization must be able to produce a stable linkage of said enzymes to the selected supports, correct orientation and activation of said enzyme to ensure adequate efficiency of the enzymatic system bound to the solid support.
  • the method must be easy to apply from the industrial standpoint, in connection with the wide use of these enzymatic systems in various economically important industrial processes.
  • the method of immobilization of enzymes on solid polymeric supports according to the present invention solves the technical problem and fulfils the purpose, since it is able, in a single step, to produce a stable bond of the covalent type of the enzyme to the support, at the same time ensuring activation and correct orientation thereof.
  • This result is achieved by creating a hydrophobic phase around the solid polymeric support in such a way that the enzyme turns the active site not towards the support but towards the reaction mixture.
  • the presence of the organic phase further ensures that the enzyme dissolved in a minimum aqueous liquid phase undergoes the necessary conformational changes in order to be "activated".
  • the method according to the invention makes use of porous solid polymeric supports of medium or high hydrophobicity but able to absorb water and immersed in a highly hydrophobic phase, because in this way the hydrated and dissolved (and hence hydrophilic) enzyme will be distributed on the support. Moreover, treatment of the support with surfactant is made completely superfluous, because the hydrated and dissolved enzyme tends inevitably to avoid the highly hydrophobic phase that surrounds the support.
  • this method can be applied to the covalent immobilization of various types of enzymes besides lipases, such as hydrolases, oxidoreductases, lyases, transferases, isomerases and ligases.
  • immobilized enzymes can be used both for applications in a hydrophobic organic environment and in an aqueous or two-phase environment, since the covalent linkage of the enzyme to the support imparts stability to the immobilized enzyme.
  • the method has been investigated and developed so as to maximize the efficiency of covalently immobilized lipases as these enzymes have structural and conformational peculiarities that make their covalent immobilization difficult.
  • the external surface of lipases is divided into two zones: a hydrophobic zone around the active site that is to interact with the triglyceride natural substrate and a hydrophilic zone that is exposed and solvated by water.
  • lipases moreover, have a mechanism of "interfacial activation" by which the enzyme modifies its conformation, allowing the substrate to enter the active site [Hanefeld U. et al., 2009, ref. cit; Schmid R.D. et al., 1998, ref. cit.].
  • the hydrophobic phase of the substrate for example a triglyceride
  • a surface domain of the protein, which in the inactive conformation covers the hydrophobic active site is rearranged to expose more the hydrophobic zone of the active site, with which the substrate can interacts.
  • the polymer must have properties that are sufficiently hydrophobic to favour the use of the supported enzyme in systems employing oily or at any rate lipophilic substrates, but at the same time the polymer must have the capacity to absorb water, promoting diffusion thereof towards the enzyme, which is poor in the case of hydrophilic supports.
  • the polymer must not alter its structural properties and must not swell by more than 30% of its volume in contact with the usual organic solvents or aqueous buffers.
  • the polymer has to be able to preserve its own structural properties even after dehydration, as well as having functional groups capable of covalently binding the enzyme, reacting with functional residues thereof.
  • the method of covalent immobilization of enzymes on solid polymeric supports envisages the use of a porous solid support of medium or high hydrophobicity, having functional groups selected from epoxide, aldehyde, amino or carboxyl groups able to react directly with the functional groups present on the surface of the enzyme or to react in the presence of a suitable activating agent.
  • concentration of said functional groups of the solid polymeric supports usable for the method according to the invention is in the range from 0.025 to 4.5 mmol per gram of dry polymer and preferably is 2.5 mmol/g.
  • the hydrophobic porous solid polymeric support on which the enzyme is to be covalently bound is selected from acrylic, methacrylic, polyacrylamide, styrene/divinylbenzene, propylene, polyethylene, polyvinyl based organic polymers mixed copolymers thereof, such as acrylic/styrene copolymers, or mixtures thereof. More preferably they are selected from methacrylic and styrene/divinylbenzene organic polymers, and copolymers thereof.
  • the polymer support usable for the method of immobilization object of the invention has a porosity such that it is able to absorb water in an amount from 40 to 95% (w/w), preferably from 55 to 65% (w/w) and, therefore, the polymer has pores with a diameter of at least 20 nm; preferably the pore size of the polymer is in the range from 20 to 300 nm and more preferably is from 30 to 40 nm.
  • Polymers in the form of beads are preferred and in this case the particle diameter is from 50 to 1000 ⁇ , and is preferably from 100 to 300 ⁇ .
  • the polymer when the solid support is in hydrated form, before immobilization of the enzyme, the polymer must be submitted to dehydration, which can be carried out in a manner known by a person skilled in the art, and for example: i) by washing with a solvent miscible or partially miscible with water having a value of LogP in the range from +0.8 to -1 .1 , and preferably equal to -0.2, selected from acetone, isopropanol, tert-butanol, ethyl acetate, methyl acetate, tetrahydrofuran, dioxane, acetonitrile, ethanol; preferably the solvent is acetone; ii) by a stream of air; iii) by application of reduced pressure between 600 and 880 mbar.
  • the amount of residual water must be in the range from 2 to 15% and preferably equal to 5 % w/w. If organic solvent is used for dehydration, the residue of solvent is removed by application of reduced pressure. If the polymer of the support has amino functional groups, they must be preactivated before dehydration by means of a bifunctional aldehyde agent; this is preferably glutaraldehyde and the process of preactivation is carried out as described in the literature [Basso A. et al., 2007, ref. cit.].
  • the solution of glutaraldehyde in phosphate buffer must have a concentration between 0.5 and 5.0% v/v, preferably 2.0%, and a pH between 8 and 8.5.
  • the preactivation is carried out by treatment with glutaraldehyde for a time between 30 and 120 minutes, preferably 60 minutes, at a temperature in the range 18-35°C, preferably at 20-25 °C. After preactivation the support is washed 2 to 6 times, preferably 4 times, with H 2 O in volumes equal to 2-6 imL per gram of support. Then dehydration is carried out as described above.
  • commercial polymers functionalized both with epoxide and amino groups with these characteristics are: Eupergit ® C; Lewatit ® TP 260; Sepabeads ® ; ImmobeadsTM; ES-1 Resin (Hencheng, China); AM-Resin (Hecheng, China); Dilbeads.
  • the formation of a hydrophobic monophase around the selected polymeric support which constitutes the characterizing step of the method according to the invention, can be obtained by adding a volume not less than 2 imL, and preferably between 4 and 20ml_, per gram of the dry solid support, of an organic compound or of a monophase mixture of compounds, which are liquid at the temperature at which immobilization is carried out, i.e. between 18 and 40 °C, and preferably at 25 °C.
  • Said organic compounds can be i) organic solvents with LogP in the range from 0.8 to 6.0, preferably equal to 2.5; ii) an oily organic mixture.
  • the organic solvents can be selected from toluene, hexane, tert-butanol, diisopropyl ether, heptane, pentane, cyclohexane.
  • organic phase is obtained with an oily monophase mixture of hydrophobic organic compounds these are preferably a mixture of triglycerides or of fatty acids or of high molecular weight alcohols, vegetable oils such as colza, soya, palm, canola or sunflower oil, inedible vegetable oils (oil from jatropha, oil from Sapium sebiferum), oils extracted from algae, industrial, food and agricultural waste oils, such as for example spent oils, frying oils, oils extracted from olive residues or coffee residues.
  • aqueous solution containing the dissolved enzyme to be supported is then added to the organic phase containing the polymeric support.
  • the pH of the solution containing the enzyme is from 7.0 to 8.5, preferably 8.0, in the case of linkage on epoxide groups, whilst it is in the range from 5.5 to 8.5 in the case of linkage on amino groups preactivated with glutaraldehyde.
  • this aqueous solution has an ionic strength in the range from 0.01 to 0.5M; in the case of immobilization on supports bearing preactivated amino groups preferably between 0.01 and 0.04 M, and more preferably is between 0.02 M and 0.03 M.
  • this aqueous solution is a buffer consisting of a pair of organic or inorganic salts. More preferably this aqueous solution is a sodium phosphate or potassium phosphate buffer, Tris, glycine-NaOH.
  • the aqueous solution of the enzyme can also contain salts, stabilizers (polyols, sugars), other additives and glycerol.
  • the solution has to be added in volumes between 0.5 and 5.0 mL, preferably 2.5 mL, per gram of dry support.
  • the amount of protein loaded on the support must be calculated so as to saturate the reactive functional groups, with the aim of avoiding secondary reactions when applying the immobilizate in a chemical process.
  • the amount of protein must not be in excess so as to allow all the enzymatic molecules to react covalently with the reactive groups on the polymer surface and in the final analysis avoid possible subsequent detachment of the enzyme at the time of use thereof supported on polymer.
  • the amount of protein to be added to the support can be in the range from 15 to 200 mg per g of dry support, preferably using an amount from 20 to 60 mg of protein per g of polymer support.
  • the system thus, obtained is submitted to mixing by for example orbital or mechanical stirring depending on the viscosity of the system so as to promote uniform absorption of the aqueous phase by the solid support and hence of the enzyme, and consequently to enable the formation of covalent bonds between the enzyme and the support.
  • the mixture is reacted for a time for at least 12 hours and up to 72 hours, more preferably for 48 hours.
  • the system can be stirred throughout immobilization.
  • the mixture is kept at a temperature between 18 and 40 °C and preferably at 25 °C in order to allow covalent linkages to form between the enzyme and the functional groups present on the solid support.
  • the epoxide functional groups react with amino acid residues that have nucleophilic reactivity (for example residues of lysine and/or of cysteine), while the aldehyde groups react, as well as with the aforementioned amino acid residues, also with the vicinal alcoholic groups of any glycans (for example mannose units) present on the protein.
  • the supported enzyme is intended for applications in two-phase systems (for example water/solvent or water/oil) or emulsions
  • the supported enzyme can be used directly in this form without removing the organic phase.
  • hydrophobic phase for immobilization the same hydrophobic substrate consisting of a solvent or an oily mixture that is to be used in the final process of use of said enzyme, then adding the components of the reaction in the required proportions.
  • the immobilized enzyme thus obtained can optionally be submitted to washing and/or dehydration.
  • Removal of the residual water present in the pores of the support can be carried out as described above and in particular by: i) washing with solvent that has a value of LogP in the range from +0.8 to -1 .1 , preferably equal to -0.2, and more preferably acetone; ii) application of reduced pressure; or iii) a stream of air.
  • the final residual water content can be in the range from 2 to 15% w/w, preferably equal to 5%. It is then checked that the enzyme is covalently bound to the support by titrating the hydrolytic activity of the enzyme released in solution according to methods known by a person skilled in the art.
  • the method of immobilization of the enzyme on a functionalized porous polymeric support essentially comprising the addition of a suitable aqueous solution of the enzyme to be supported to a hydrophobic organic phase containing the solid polymeric support, has been shown to be suitable for:
  • esters aromas, emulsifiers, pharmaceuticals, chiral intermediates for drugs
  • emulsifiers that are aliphatic esters of fatty acids or esters derived from reaction between sugars and fatty acids; c) transformation or hydrolysis of triglycerides in aqueous or two-phase media;
  • the method of immobilization by covalent linkage of enzymes to functionalized solid polymeric supports according to the invention was tested on various lipases: from Rhizopus oryzae, from Pseudomonas cepacia and from Candida antarctica.
  • This method of immobilization was compared with a similar method, but with the steps reversed, that is, the aqueous solution of the enzyme was added to the polymer in the dry state and then a hydrophobic solvent was added to this heterogeneous system, as well as with methods of immobilization described in the literature, i.e. in phosphate buffer [Basso A. et al., Adv. Synth. Catal., 2007, ref. cit] or by adsorption on Celite R- 640 [Basso A. et al. 2000, ref. cit.].
  • the lipases from Rhizopus have, as is well known, a domain that covers the active site when put in a hydrophilic environment [Kohno M. et al., Biosci. Biotech. Biochem., 1994, 58, 1007-1012]. This domain can be lifted by interfacial activation in the presence of a hydrophobic phase.
  • the structure of the lipase from Rhizopus oryzae is very similar to that of the lipase from Rhizopus niveus (differing by 2 amino acids) and that from Rhizomucor miehei, enzymes used in the transesterification of triglycerides for use in foods (analogues of cocoa butter, triglycerides enriched with unsaturated fatty acids etc.) [Higashiyama K. et al. WO 04/024930]. Therefore the behaviour of the three lipases is expected to be similar. Regarding the lipase from Pseudomonas cepacia, this is a bacterial enzyme that has a broad hydrophobic zone corresponding to the active site.
  • Lipase B from Candida antarctica is a fungal lipase that has a surface on which there is a highly hydrophobic portion, corresponding to the zone of the active site, and an opposite face that is found to be mainly hydrophilic, also owing to the presence of glycans with numerous sugar residues.
  • it does not have interfacial activation as it does not undergo significant conformational changes on coming into contact with a hydrophobic interface.
  • the active site is covered by a domain of much smaller dimensions and so is always accessible [Schmid R.D. et al. 1998, ref. cit; Basso A. et al., 2007, ref. cit.].
  • the method of immobilization in organic solvent aims to promote correct orientation of the protein during attachment to the support, in order to induce the hydrophobic zone of the active site to turn towards the organic solvent.
  • Lipase from Rhizopus oryzae used in the examples below reported is Lipase DF (Amano) in the form of dry powder with protein content of 1 %.
  • the enzymatic preparation used in these examples had hydrolytic activity, defined with the tributyrin assay, of 15 U/mg.
  • Example 1 immobilization of the lipase from Rhizopus oryzae on epoxide support in organic phase according to the method of the invention
  • Example A immobilization of the lipase from Rhizopus oryzae on epoxide support by reversing the order of addition of the organic and aqueous phases.
  • Example B immobilization of the lipase from Rhizopus oryzae in phosphate buffer
  • TBU 26500U
  • a second experiment corresponding to 175000U per dry gram of support of Lipase DF (Amano) previously diluted in phosphate buffer Kpi 0.5M pH 8.0 is added to the same polymeric support as in example 1 and 2 (methacrylic polymer in the form of beads - particle diameter 150-300 ⁇ ; water absorption capacity 40-60% by weight, average pore diameter 30- 40 nm and functionalized with epoxide groups), to obtain a support/buffer ratio equal to 1 g for 9 imL.
  • Immobilization is carried out at 20-25 °C for 24h. After immobilization the supported enzymatic preparation is filtered on a Buchner filter and washed with acetone (3x2 imL). The excess acetone is removed under reduced pressure.
  • the procedure is repeated using a different preparation of native lipase from Rhizopus oryzae having a higher protein content (40%) and activity (350U/mg), reducing the support/buffer ratio to 1 g for 4 imL, immobilizing 15000 enzyme units and continuing immobilization for 38h.
  • the hydrolytic and synthetic activity of this immobilized enzyme are presented below.
  • Example 2 hydrolysis of triglycerides in aqueous media containing emulsifiers with the enzymatic preparations according to examples 1, A and B and evaluation of covalent coupling of the enzyme to the support
  • the hydrolytic activity is determined according to the following assay that uses a pH-stat system (Mettler-Toledo T50) and it is found to be 1 0900U/g of dry preparation.
  • One unit corresponds to the amount of enzyme that hydrolyses 1 micromole of tributyrin in one minute at 30 °C.
  • the immobilization yields are calculated on the basis of the ratio of enzyme units expressed by the enzyme after immobilization to those used in the immobilization process.
  • the hydrolytic activity of the immobilized enzyme according to example 1 is found to be 4800 and 10900U/g of preparation respectively, corresponding to immobilization yields equal to 18 and 41 %.
  • the immobilized enzyme according to example A showed a hydrolytic activity evaluated with respect to tributyrin equal to 2841 U/g of preparation.
  • the preparation assayed also indicates release of enzymatic protein around 40%.
  • the data thus, show that the immobilization procedure should envisage prior formation of a hydrophobic phase around the support in order to obtain efficient covalent immobilization of the enzyme with the correct orientation.
  • the enzyme immobilized in phosphate buffer according to example B did not demonstrate any hydrolytic activity in either of the two conditions of immobilization tested. The results obtained are shown for purposes of comparison in Table 1 .
  • the enzymatic preparation is filtered and the enzymatic activity present in the solution is evaluated.
  • the preparations of example 1 assayed indicate a release of enzymatic protein of less than 2%, demonstrating the applicability of the immobilized enzyme of example 1 in aqueous environments and in the presence of emulsions.
  • Example 3 synthesis of propyl laurate in non-aqueous medium - comparison between the enzymatic preparations of lipase from Rhizopus oryzae of example 1 and of example B
  • the synthesis reaction is carried out at 55 °C with magnetic stirring (250rpm) in a 20 imL vial using equimolar amounts of lauric acid and 1 -propanol (1 .2g and 0.36g respectively).
  • An amount equal to 30-40 mg of enzyme immobilized in examples 1 and B is added to the substrates and formation of the ester is monitored by HPLC in the first 15% of conversion (RP-HPLC, C-18 column, mobile phase 100% AcN + 0.05% TFA, flow 1 ml/min, UV-VIS detector, 210nm).
  • the immobilized lipase of example 1 the synthetic activity is found to be 441 U/g of dry preparation.
  • the immobilizate in phosphate buffer of example B no reaction product was observed within 48h, indicating that the preparation does not have any synthetic activity.
  • the lipase is immobilized with the method according to the invention (example 4) and with phosphate buffer for comparison (example C); the evaluation of enzymatic activity is reported in examples 5-8.
  • the lipase from Pseudomonas cepacia used in the examples presented below is Lipase PS (Amano) in the form of dry powder with a protein content of 1 %.
  • the hydrolytic activity of the batch used, determined with the tributyrin assay, is 14 U/mg.
  • Example 4 immobilization of the lipase from Pseudomonas cepacia (lipase PS) on epoxide support in the presence of organic phase according to the method of the invention
  • TBU 30000 units
  • enzymatic preparation lipase PS
  • buffer Kpi 0.05M pH 8.0 1 g
  • porous methacrylic polymer in beads, functionalized with epoxide groups, Sepabeads ® EC (particle diameter 150-300 ⁇ ; water absorption capacity 40-60%, average pore diameter 30-40 nm) is added to 5 imL of toluene.
  • the enzyme solution is added to the organic phase maintaining mechanical stirring of the system for 30 minutes and it is then kept at rest for 24 hours at 20-25 °C.
  • the enzymatic preparation was washed with acetone as in example 1 .
  • the hydrolytic activity and the immobilization yields are described below.
  • Example C immobilization of lipase from Pseudomonas cepacia in phosphate buffer a) 30000 units (TBU) of the enzymatic preparation is dissolved in buffer Kpi 0.05M pH 8.0 and added to 1 g of support in the form of beads Sepabeads ® EC (methacrylic polymer functionalized with epoxide groups; particle diameter 150-300 micrometers; water absorption capacity 40-60%, average pore diameter 30-40 nm). Mechanical stirring of the system is maintained for 24h at 25 °C. At the end of immobilization the enzymatic preparation was washed with acetone as in example B. The hydrolytic activity and immobilization yield are described below.
  • Example 5 hydrolysis of triglycerides in aqueous media containing emulsifiers with the enzymatic preparations according to examples 4 and C
  • the hydrolytic activity of the immobilized enzyme of example 4 was found to be equal to 6155U/g of enzymatic preparation, equal to an immobilization yield of 20.5%.
  • the amount of protein not bound covalently was always ⁇ 5%, demonstrating that covalent coupling had taken place and the possibility of using the preparation in aqueous media containing emulsifiers.
  • the hydrolytic activity of the immobilized enzyme according to example C was found to be equal to 463U/g of enzymatic preparation, equal to an immobilization yield of 1 .5%.
  • Example 6 application of the lipase from Pseudomonas cepacia immobilized according to example 4 for the synthesis of (Rj-1-phenylethyl acetate 0.6 ml_ of (R,S)-1 -phenylethanol (4.97 mmol) and 100 mg of enzymatic preparation (activity 6155U/g of enzymatic preparation) are added to 2.4 imL of vinyl acetate (26 mmol). The reaction is incubated at 25 °C with orbital stirring. The reaction was monitored by HPLC and the initial rate of formation of (R)-l -phenylethyl acetate was found to be 97 micromol/min/g of enzymatic preparation. The data demonstrate the applicability of the enzymatic preparation in non-aqueous environments.
  • Example 7 application of immobilized lipase from Pseudomonas cepacia of example 4 to the synthesis of biodiesel
  • Example 8 transesterification of triolein with stearic acid catalysed by immobilized lipase from Rhizopus oryzae from example 1 and analysis of the triglyceride by hydrolysis with immobilized lipase from Pseudomonas cepacia (lipase PS) from example 4
  • the reaction of transesterification between triolein and stearic acid in 1 :2 ratio was carried out at a temperature of 70 °C for 70h. 443 mg of triglyceride and 284 mg of stearic acid are used, which are put in a reaction flask immersed in a heated oil bath, with continuous magnetic stirring. The reaction begins on adding the enzymatic preparation of example 1 (10% w/w). The flask was equipped with a trap with molecular sieves. The reaction is stopped by filtering the mixture and washing with n- hexane, which is removed at reduced pressure.
  • the residue is then washed with cold methanol in order to remove the unreacted stearic acid and the oleic acid that formed as a result of transesterification.
  • the triglyceride was analysed by hydrolysing the triglycerides that formed in the transesterification reaction by enzymatic hydrolysis in a two-phase system of H 2 O and dichloromethane with addition of 40 mg of enzymatic preparation (immobilized lipase PS according to example 4), under magnetic stirring, for 18h.
  • the organic phase containing the free fatty acids was separated, and the solvent was removed at reduced pressure.
  • the lipase is immobilized with the method according to the invention (examples 9 and 15) and with different methods for comparison (examples D and E); the evaluation of enzymatic activity is reported in examples 10-14.
  • the lipase B from Candida antarctica used in the examples is the lipase Lipozyme CALB L (Novozymes) in liquid solution containing 44% water, 25% sorbitol, 25% glycerol, 6% enzyme, 0.20% sodium benzoate, 0.10% potassium sorbate.
  • the hydrolytic activity, determined by tributyrin assay, is >5000U/g (determined by the manufacturer).
  • Example 9 immobilization of lipase B from Candida antarctica (Lipozyme CALB L) on epoxide support in the presence of organic phase according to the method of the invention
  • the enzyme solution is then added to the organic phase and the system is stirred continuously (mechanical stirring) for 24 or 48 hours at a temperature of 20-25 °C.
  • the immobilized enzyme is filtered on a Buchner filter and washed with acetone (3x 2 mL) and the excess acetone is removed under reduced pressure.
  • acetone 3x 2 mL
  • the same immobilization of the enzyme Lipozyme CALB L was performed using colza oil, instead of toluene as medium for the hydrophobic phase for dispersion of the polymer, and stirring the mixture, consisting of the enzyme solution and the added organic phase, for 48 hours at a temperature of 20-25 °C.
  • Example D covalent immobilization of lipase B from Candida antarctica on epoxide support in phosphate buffer
  • Immobilization is carried out at 25 °C for 24 or 48h. After immobilization the immobilized enzyme is filtered on a Buchner filter and washed with acetone (3x2 imL) and the excess acetone is removed under reduced pressure.
  • Example E immobilization by adsorption of lipase B from Candida antarctica on Celite R-640
  • lipase B from Candida antarctica was immobilized on Celite R-640 by applying the method of immobilization described by Basso A. et al. [Basso A. et al., 2000, ref. cit.].
  • Example 10 hydrolysis of triglycerides in aqueous media containing emulsifiers and synthetic activities with the enzymatic preparations according to examples 9, D and E
  • the hydrolytic activity and the immobilization yields were determined as described in example 2 above and the synthetic activity (synthesis of propyl laurate) was performed as described in example 3.
  • Example 11 Application of lipase B from Candida antarctica (Lipozyme CALB L) immobilized in the presence of organic phase (example 9) and on Celite R-640 (example E) in the synthesis of biodiesel
  • Example 12 application of lipase B from Candida antarctica immobilized in the presence of organic phase (example 9) in the synthesis of a surfactant (octyl laurate) 1 .2g of lauric acid and 0.94 imL of 1 -octanol are added to 50 mg of the enzymatic preparation described in example 9 and maintained at 55 °C with orbital stirring (80rpm). The reaction is monitored by HPLC and the initial rate of formation of octanoyl laurate is 6.6 micromol/min/mg enzymatic protein, showing that the preparation can be used for the synthesis of surfactant esters in solvent-free systems.
  • Example 13 application of lipase B from Candida antarctica immobilized in the presence of organic phase of example 9 in the synthesis of polyesters by polycondensation (adipic acid and 1 ,4-butanediol)
  • the patent application WO 94/12652 describe the synthesis of polyesters by enzyme- catalysed polycondensation. It should be noted that a first step in formation of the oligomer only takes place in the presence of the enzyme, as described in the patent application cited, the polymerization reaction can be carried out starting from the oligomer, following removal of the enzyme, taking advantage of the shift of thermodynamic equilibrium of the reaction resulting from removal of the water produced during esterification (polycondensation).
  • the example presented here describes both steps of the process and it is also demonstrated that the enzyme produced in example 9 gives a dramatic reduction in release of enzymatic protein in the oligomeric product, an essential factor for preventing protein contamination. In fact, the presence of enzymatic protein in the finished product can mean lower quality of the product, or that it cannot be used in pharmaceutical applications (for example controlled release of drugs).
  • oligomerization step adipic acid (9.85g, 67 mmol) was added to 1 ,4-butanediol (6.35g, 70 mmol) (molar ratio 1 :1 .1 ), the resultant dispersion is homogenized in a glass vial, stirring the system at 50 °C using a magnetic stirring bar. The product thus obtained is transferred to a reaction syringe, addition of the enzymatic preparation of example 9 (approx. 1 % by weight relative to the total of the monomers used) starts the polycondensation reaction.
  • the product obtained (oligomer) is a clear solution, liquid at 25°C, which can be separated from the immobilized enzyme by filtration.
  • the product obtained was analysed by 1 H NMR spectroscopy:
  • the residual enzyme present in the final product (oligomer) was evaluated by a hydrolytic activity assay.
  • the immobilized enzyme was removed from the samples of oligomer by filtration and the product (oligomer) was used in the hydrolytic activity assay with respect to tributyrin.
  • the profiles in Fig. 3 indicate the enzymatic activity titrated after removal of the immobilizate, due to the native enzyme released from the enzymatic preparation during the oligomerization process (protein contamination).
  • the data given in Fig. 3 show that when the adsorbed enzymatic preparation (Novozym 435 ® ) is used, the amount of active enzyme present in the oligomer is significantly higher.
  • the synthesis then continues with the polymerization step as described below.
  • Example 14 application of the immobilized lipase B from Candida antarctica (example 9) to the synthesis of food-grade emulsifying esters (glucose+fatty acid)
  • the publications [Cao L. et al., Fett/Lipid 1996, 98, 332-335; Cao L.
  • the reaction mixture is as follows.: 1 eq. of a mixture of alpha- and beta-glucose, 1 eq. of fatty acid or ester thereof, 2 eq. (w/w) t-butanol (as adjuvant), 0.2 eq. (w/w) molecular sieves; 10 wt.% of enzymatic preparation prepared in example 9.
  • the mixture is put in a heated stirrer at 55 °C, 250rpm, or in a reaction flask immersed in a heated oil bath, with magnetic stirring.
  • the reaction takes place for a minimum time of 5h, which is increased even to 72h.
  • the reaction is stopped with syringe filtration, carrying out a first washing with n-hexane, and a second washing with THF.
  • the solvent is removed at reduced pressure.
  • TLC is performed with the following mobile phase: CHCI 3 : MeOH : CH 3 COOH : H 2 O in ratio 70 : 20 : 8 : 2.
  • the product has an R f in the range from 0.65 to 0.70, depending on the fatty acid used.
  • Example 15 immobilization of lipase B from Candida antarctica according to the method of the invention on support functionalized with amino groups
  • the AM-Resin amino support (Hecheng, China) is preactivated with a solution of glutaraldehyde at 2% v/v in phosphate buffer 0.02M pH 8.0 and is stirred at room temperature for 60 minutes.
  • the unbound glutaraldehyde is then removed by carrying out washings with distilled water and with phosphate buffer 0.02M pH 8.0, maintaining the ratio of 1 gram of wet polymer/4 imL of solvent.
  • the polymer functionalized with amino groups is dehydrated according to the procedure reported above in example 9.

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Abstract

La présente invention concerne un procédé d'immobilisation d'enzymes, en particulier d'enzymes qui sont actives à l'interface avec des phases hydrophobes, en tant que lipases, sur des supports polymères solides poreux, dans lequel il se forme une liaison covalente stable entre le support et l'enzyme, et dans lequel une « interface » hydrophobe se forme autour du support solide qui permet à l'enzyme, dissoute dans une phase aqueuse minimale, de passer de la conformation fermée/inactive (qui domine lorsque l'enzyme est en phase aqueuse) à la conformation ouverte/activée (qui domine lorsque l'enzyme entre en contact avec une phase hydrophobe) et d'adopter une orientation optimale, tournant l'ouverture du site actif hydrophobe vers la phase organique hydrophobe.
EP11811024.6A 2010-12-23 2011-12-22 Procédé pour l'immobilisation covalente d'enzymes sur des supports polymères solides fonctionnalisés Withdrawn EP2655611A1 (fr)

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ITPD2010A000392A IT1403355B1 (it) 2010-12-23 2010-12-23 Metodo per l'immobilizzazione covalente di enzimi su supporti polimerici solidi funzionalizzati
PCT/EP2011/073794 WO2012085206A1 (fr) 2010-12-23 2011-12-22 Procédé pour l'immobilisation covalente d'enzymes sur des supports polymères solides fonctionnalisés

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US5776741A (en) 1994-02-21 1998-07-07 Novo Nordisk A/S Method of enzyme immobilization on a particulate silica carrier for synthesis inorganic media
EP1537221B1 (fr) 2002-09-13 2016-02-03 Suntory Holdings Limited Procede de production d'huiles/de graisses ou de triglycerides transesterifies
ES2214136B1 (es) 2003-02-21 2007-05-16 Consejo Sup. De Invest. Cientificas. Nuevo metodo de inmovilizacion de enzimas y otras bio-macromoleculas sobre soportes activados con grupos epoxido conteniendo grupos ionizados en el brazo espaciador que une cada grupo epoxido a la superficie del soporte.
DE102005030435A1 (de) 2005-06-30 2007-01-04 William Prym Gmbh & Co. Kg Befestigungshülse
WO2007080197A2 (fr) 2006-01-16 2007-07-19 Novozymes A/S Enzymes immobilisees
IL180598A0 (en) * 2007-01-08 2007-07-04 Basheer Sobhi Immobilized interfacial enzymes of improved and stabilized activity
US7790429B2 (en) * 2007-11-28 2010-09-07 Transbiodiesel Ltd. Robust multi-enzyme preparation for the synthesis of fatty acid alkyl esters

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