WO2016193516A1 - Procédé général d'obtention de biocatalyseurs comprenant l'immobilisation d'enzymes au cours de la synthèse de matériaux métallo-organiques - Google Patents

Procédé général d'obtention de biocatalyseurs comprenant l'immobilisation d'enzymes au cours de la synthèse de matériaux métallo-organiques Download PDF

Info

Publication number
WO2016193516A1
WO2016193516A1 PCT/ES2016/070397 ES2016070397W WO2016193516A1 WO 2016193516 A1 WO2016193516 A1 WO 2016193516A1 ES 2016070397 W ES2016070397 W ES 2016070397W WO 2016193516 A1 WO2016193516 A1 WO 2016193516A1
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
solution
mof
biocatalyst
glu
Prior art date
Application number
PCT/ES2016/070397
Other languages
English (en)
Spanish (es)
Inventor
Elsa CASTRO MIGUEL
Victoria GASCÓN PÉREZ
Manuel SÁNCHEZ SÁNCHEZ
Rosa María BLANCO MARTÍN
Manuel DÍAZ GARCÍA
Original Assignee
Consejo Superior De Investigaciones Científicas (Csic)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Superior De Investigaciones Científicas (Csic) filed Critical Consejo Superior De Investigaciones Científicas (Csic)
Publication of WO2016193516A1 publication Critical patent/WO2016193516A1/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
    • 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
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2235/00—Indexing scheme associated with group B01J35/00, related to the analysis techniques used to determine the catalysts form or properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2235/00—Indexing scheme associated with group B01J35/00, related to the analysis techniques used to determine the catalysts form or properties
    • B01J2235/15—X-ray diffraction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J35/00—Catalysts, in general, characterised by their form or physical properties
    • B01J35/70—Catalysts, in general, characterised by their form or physical properties characterised by their crystalline properties, e.g. semi-crystalline

Definitions

  • the present invention falls within the scope of the development of catalysts that can be applied in very different fields, such as chemical, pharmaceutical, agricultural, energy and biotechnology. Specifically, it refers to a procedure for obtaining biocatalysts that is general, and that allows the immobilization of any enzyme during the synthesis of nanocrystalline metallo-organic materials (MOFs), taking advantage of intercrystalline mesoporosity, generated by agglomeration or aggregation of nanocrystals.
  • MOFs nanocrystalline metallo-organic materials
  • the immobilization on solid supports of enzymes allows their heterogeneization, that is, having the biomolecule supported in solid phase.
  • the obtaining of solid biocatalysts favors, among other aspects, the separation of said enzymes from the reaction medium and their reuse in successive reaction cycles.
  • the chemical nature and textural properties of the materials used as supports give different properties to the final catalyst.
  • the binding of an enzyme on a preexisting support (hereinafter post-synthesis immobilization) can be carried out through covalent or non-covalent bonds, and in any case a high chemical affinity between both species is convenient.
  • the support must offer a high specific surface area, so materials that have a high porosity with a pore diameter greater than the molecular dimensions of the enzyme to be immobilized are desirable.
  • in-situ immobilization As an alternative to post-synthesis immobilization, the possibility of entrapment of the enzyme during the support synthesis process, hereinafter in-situ immobilization (thus the synthesis of siliceous materials in the presence of enzymes is uniquely known) has been explored by several tracks.
  • porous materials with more emergent projection stand out the metal-organic materials (MOFs).
  • MOFs metal-organic materials
  • these materials comply with the two main premises indicated above (specific surface area and pore diameter).
  • its structural versatility is such that, unlike other conventional microporous materials, some of its structures have pores that go deep into the mesopore range (Deng et al., Science, 2012, 336, 1018 ).
  • MOF materials are microporous so they do not have pores large enough to immobilize enzymes inside, and those that possess them require a great effort in their preparation, because they are constituted by non-commercial organic ligands , expensive and difficult to synthesize, necessarily in steps prior to the formation of MOF itself (Deng et al., Science, 2012, 336, 1018).
  • the pores offered by these MOFs are not large enough for many biomolecules to diffuse through them, and therefore, the method is far from being considered universal.
  • the in-situ immobilization of enzymes on MOFs only the document of Shieh et al. (J. Am. Chem. Soc.
  • the catalyst thus obtained is characterized by being formed by microcrystals; a very reduced encapsulation efficiency that is 5% by weight of the enzyme (which would correspond approximately to a percentage of incorporation of the enzyme below 64%, considering a yield below 100% and a complete formation of the MOF ); and also a very reduced catalytic efficiency of the encapsulated enzyme (33 times lower than that of the free enzyme itself).
  • the invention relates to a process for obtaining a biocatalyst because it comprises: a) partially or totally synthesizing a metallo-organic material (MOF) in the presence of at least one enzyme, until obtaining a solid and, b) isolating the solid synthesized according to step a), preferably by centrifugation or filtration, and where the synthesized MOF immobilizes the enzyme in the intercrystalline mesoporosity formed between crystals or between agglomerated or aggregated domains of homogeneous sized nanocrystalline particles.
  • MOF metallo-organic material
  • the intercrystalline mesoporosity comprises at least one, and preferably more than one, hollow volume between 2 and 50 nm.
  • step (a) the synthesis of the MOF comprises contacting two solutions in the presence of at least one enzyme, where said solutions can be aqueous, organic or an aqueous-organic mixture, and where preferably the first solution is a solution of a metallic source and the second, an organic ligand solution.
  • the second organic ligand solution is chosen from an aqueous solution of an organic ligand salt or an aqueous solution of protonated organic ligand in the presence of a deprotonating agent.
  • the process of the invention is carried out at temperatures between 4 and 70 ° C and more preferably between 20 and 30 ° C.
  • the enzymes that are used are ⁇ -glucosidase and lipase.
  • the invention relates to the biocatalyst directly obtained by the process of the first aspect of the invention.
  • the technical problem that solves the present invention is the development of a general or universal, simple, fast and economical procedure, which can be used extensively to obtain biocatalysts comprising an MOF support and any enzyme that is immobilized therein during its synthesis, and that solves the problems detected in the state of the art for other biocatalysts that are based on in-situ immobilization, singularly the reduced capacity of enzymatic load, the limited retention capacity of the immobilized enzymes, and an excessive reduction of the catalytic efficiency with respect to that of the immobilized biomolecule.
  • the present invention is based on a method of in-situ immobilization of the enzyme Aspergillus niger ⁇ -glucosidase ( ⁇ -Glu) and Candida Antarctic lipase (CalB) on different MOFs, which allows to obtain biocatalysts characterized by comprising polycrystalline particles formed by nanocrystals (about 40 nm) of homogeneous size, which in their aggregation generate hollow, stable and ordered mesopore volumes (between 2 and 50 nm in diameter), and that allow them to immobilize a significant amount of the enzyme inside, which reaches at least 15% by weight of the enzyme and more than 86% of the enzyme exposed in the reaction medium see Examples 1 to 5 and 7) and additionally, reduces the leachate losses thereof (see Example 6 ).
  • reaction conditions used in this invention to obtain the biocatalysts favor the maintenance of the catalytic activity of the enzymes conferring them a high catalytic efficiency (see Examples 1 to 5 and 7), obtaining values much higher than indicated by Shieh et al. (J. Am. Chem. Soc.
  • the biocatalyst obtained in the present invention it maintains without any fall the catalytic efficiency, which is only 3% lower than the activity of the free enzyme. It is noteworthy that also in this case the soluble enzyme is tested only in the presence of a pH buffer optimized to enhance its activity, while the aliquot of the biocatalyst is tested by taking it directly from the reaction medium and therefore in the presence of all the components of the same.
  • the procedure for obtaining biocatalysts that is included in the invention, allows the stabilization of the structure and activity of the enzyme for at least 48 hours in a priori means not very favorable for it, such as ⁇ , ⁇ - dimethylformamide, where the free enzyme is completely inactivated after a few minutes (see Examples 4 and 5).
  • the invention relates to a method of obtaining a biocatalyst, hereinafter method of obtaining the invention, comprising the following steps: a) partially or totally synthesizing a metallo-organic material (MOF) in the presence of at least one enzyme, until a solid is obtained, and b) isolate the synthesized solid according to step a), and where the synthesized MOF immobilizes the enzyme, in the intercrystalline mesoporosity formed between nanocrystals or between agglomerated or aggregated nanodominiums in micrometric particles.
  • MOF metallo-organic material
  • MOFs which are those that are used as the basis in the present invention, are characterized by lacking intercrystalline pores large enough to encapsulate biomolecules the size of enzymes.
  • the crystals that form these materials are nanocrystalline with a great tendency to agglomerate or aggregate with each other in the reaction medium, giving rise to particles compact enough to be considered non-disintegrable in their crystals or crystalline domains, and containing mesopores between the crystals, which potentially covers the sizes required to house a wide range of enzymes inside.
  • intercrystalline mesoporosity means the presence in the MOF of at least one, and preferably more than one, intercrystalline hollow volume, stable, orderly and in the range of mesopores, which is established between particles formed by agglomeration or aggregation of crystals or domains of nanoporous particles of homogeneous size, compact enough to be considered non-disintegrable in their crystals or crystalline domains.
  • mesoporo is understood as any hollow volume in a material whose diameter is between 2 and 50 nm, which preferably will have a pore distribution that conforms to the dimensions of the enzyme to be immobilized (in the case of ⁇ -glucosidase with values close to 10 nm and in the case of 5 nm lipase). Pores below 2 nm are called micropores.
  • the intercrystalline mesoporosity in solid particles is that generated by the agglomeration or aggregation of crystals. Therefore, it has a much wider pore distribution than the intrinsic one and, more importantly, it is under experimental control because it depends on the modification of the MOF preparation conditions, and therefore, it can cover all the sizes required for harbor any type of enzyme inside.
  • biocatalyst is meant the solid that is synthesized in stage a) and that is isolated in stage b) of the process for obtaining the invention, and which is a composite material comprising, a MOF support and at least one enzyme , which is immobilized in the intercrystalline mesoporosity of the MOF during its total or partial synthesis.
  • MOF or metallo-organic material is meant an organic-inorganic hybrid material in which metal atoms or metalloids or clusters of those atoms are linked to each other through organic ligands, at least bidentate, to give rise to three-dimensional crystalline networks porous
  • the method is applicable to any MOF prepared with organic ligands that preferably contain carboxylic groups through which metals are coordinated.
  • the MOF materials used are MIL-53 (AI), its structural counterpart NH 2 -MIL-53 (AI) and Mg-MOF-74, as well as MOF Fe-BTC, all of them of small particle size .
  • the metal atoms that form in MOF are Al (like Al 3+ ) and the organic ligands are terephthalate or 2-amino-terephthalate, respectively.
  • the metal is Mg (with formal charge 2+) and the ligand is 2,5-dihydroxyterephthalate.
  • the MOF Fe-BTC material marketed as Basolite F300, is composed of clusters of three Fe 3+ atoms linked together through benzene-1, 3,5-tricarboxylates.
  • the Fe-BTC material is semi-morph, although it has a high specific surface area (approximately 1000 m 2 / g) for containing some cavities and pores similar to those of MIL-100 (Fe).
  • immobilization is meant, in this document, the attachment of at least one enzyme, and preferably more than one, preferably in the intercrystalline mesoporosity of the MOF material, and which is favored over others such as embedding.
  • embedding is meant the immobilization of the enzyme when trapped by MOF crystals in the crystalline growth process, so that in the final biocatalyst it is accessible only through the intrinsic (micro) pores of the MOF.
  • the MOF synthesis of step a) of the process for obtaining the invention comprises mixing, in the presence of at least one enzyme, of two solutions, a first solution or solution of a metallic source, which preferably has an acidic pH, and another second solution or solution of the organic ligand.
  • first solution or solution of metallic source means a solution of a metal salt, preferably in a polar solvent such as water.
  • metal source solution examples include an aqueous solution of an aluminum salt, or a solution of a magnesium salt in ⁇ , dime-dimethylformamide, and preferably solutions of aluminum nitrate nonahydrate or Mg acetate tetrahydrate or Fe (lll) chloride hexahydrate.
  • second solution or solution of organic ligand is meant a solution of the source of organic ligand, either a salt or the acid, in a solvent preferably in a polar solvent such as water. If the acid is used as an organic source in water, a deprotonant agent is needed.
  • organic ligand solution that are included in the scope of the invention are aqueous solution of organic ligands containing at least two groups of carboxylic acids per molecule and preferably 2-aminoterephthalic acid or 2,5-dihydroxyterephthalic acid.
  • the process for obtaining the invention can be carried out using aqueous solutions, organic solutions or solutions that are aqueous-organic mixtures.
  • the second solution is selected from an aqueous solution of an organic ligand salt or a protonated organic ligand in the presence of a deprotonation agent, with a pH (preferably neutral or basic) that at least guarantees the deprotonation or activation of two functional groups. which have to be subsequently coordinated to metals to form MOF materials.
  • the deprotonant agent is preferably selected from a strong base, a medium base or a weak base.
  • strong base that are included in the scope of the invention are alkali hydroxides and preferably sodium hydroxide.
  • Middle-base examples that are included in the scope of the invention are the amines, preferably triethylamine.
  • weak base that are included in the scope of the invention is ammonium hydroxide or ammonia.
  • enzyme any molecule of a protein nature that catalyzes chemical reactions. It can be immobilized, and the biocatalyst can be used in the field of industry (chemical, pharmaceutical or food, among others), preferably with respect to the soluble enzyme.
  • examples of enzymes that can be used in the invention are any of the enzymes existing in nature, with very diverse catalytic activities.
  • glucosidases, peroxidases, lacases, amylases, or lipases and more preferably ⁇ -glucosidases and lipases.
  • the process of the invention prefers temperature between 4 and 70 ° C, and preferably between 20 and 30 ° C, that is to say those demanded by enzymes to preserve their structure and, therefore, their catalytic activity.
  • the enzyme is added in the course of the synthesis of the MOF according to step a) of the process for obtaining the invention, once the two solutions have been mixed, to avoid conditions that may be adverse.
  • the enzyme is added in any of the formats known in the state of the art, and preferably as an aqueous solution from an extract, which may comprise other chemical substances. These chemicals or the enzyme itself can affect different aspects of MOF synthesis, such as particle size (and, therefore, intercrystalline pores), the kinetics of the process or the presence or abundance of impurities formed, or catalytic activity of the immobilized enzyme.
  • one of the solutions used for the synthesis of the MOF according to step a) of the process for obtaining the invention is added dropwise onto the other, keeping the system under stirring.
  • This addition entails the formation of a solid either during the course of the addition or after a time that can reach from 1 second to 350 h.
  • the solid formed is the biocatalyst, which comprises part or all of the enzyme, and is separated from the reaction mixture in step b) of the process for obtaining the invention preferably by centrifugation or by filtration after a time, between 1 second and 200 hours after finishing the addition of one solution over the other.
  • the process for obtaining the invention includes the possibility of simultaneous use of different enzymes. Thus, for example, it is feasible to employ more than one different class of enzyme than those mentioned herein.
  • the process for obtaining the invention comprises an additional stage or stage c) comprising drying the isolated solid according to stage b) under conditions compatible with the preservation of the structure and properties of the enzyme including the catalytic activity.
  • the solution of the metallic source is an aqueous solution of aluminum nitrate nonahydrate
  • the solution of the organic ligand is an aqueous solution of 2- aminoterephthalic acid
  • the deprotonant agent is selected from triethylamine , sodium hydroxide and ammonia
  • the enzyme is Aspergillus niger ⁇ -glucosidase which is added on the organic ligand solution and the reaction temperature is 25 ° C.
  • the solution of the metal source is a solution of Mg acetate tetrahydrate on ⁇ , dime-dimethylformamide
  • the solution of the organic ligand is a solution of 2,5-dihydroxyterephthalic acid on ⁇ , ⁇ -dimethylformamide
  • the enzyme is Aspergillus niger ⁇ -glucosidase which is added on the solution of the metal source and the reaction temperature is 25 ° C.
  • the stabilization of the enzyme structure and the maintenance of its catalytic activity is achieved for 48 hours, while the same enzyme in the free state in the same reagent is completely inactive after a few minutes.
  • the solution of the metallic source is preferably iron chloride (lll) hexahydrate (FeCI 3 -6H 2 0)
  • the Organic ligand solution is a solution of benzene-1,3,5-tricarboxylic acid (BTC) in the presence of a deprotonating agent, preferably sodium hydroxide
  • the enzyme is Candida Antarctic Lipase B which is preferably added over the organic ligand solution and the reaction temperature is 25 ° C.
  • the process for obtaining the invention may comprise another stage in which the solid isolated according to stage b) or dried according to stage c) is again exposed to at least one enzyme, to increase the enzyme load thereof using a method of post-synthesis immobilization, since in the process of immobilization in-situ the enzymes could act as a mesoporgenic agent, creating larger mesopores.
  • the technical effect achieved in the biocatalysts obtained by the method of obtaining the invention is evident through the techniques of physical-chemical and biochemical characterization that are collected in Examples 1 to 7, particularly in relation to the high amount of immobilized enzyme, at the high retention capacity of the enzyme and the reduction of the catalytic efficiency reduction with respect to that of the immobilized biomolecule.
  • the high efficiency of enzymatic immobilization is based on the fact that the formation of the MOF material is preferably due to the instantaneous formation of a colloidal solution when the metallic solution and the organic ligand solution are mixed, as a consequence of the large number of MOF nuclei. that precipitate or crystallize when the metal cation and organic anion are found. Precisely because nucleation is so favored, the nuclei are abundant and barely grow because the metal and the organic ligand are depleted in the formation of the rest of the nuclei, thus forming suspended nanoparticles with a colloid appearance.
  • the subsequent isolation of the solid from the solution preferably by centrifugation, causes the enzyme to also recover in the solid, which together with the affinity between the biomolecule and the MOF material can give high loads of immobilized biomolecules, up to 100% of the enzyme present in the medium.
  • the invention relates to the biocatalyst directly obtained by the process of the invention.
  • Figure 1 Standardized powder X-ray diffractograms of the sample of NH 2 - MIL-53 (AI) (gray) and of the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) -NaOH-24h (black).
  • Figure 2 Thermograms (solid lines) and their derivatives (dashed lines) of the ⁇ -Glu soluble enzyme extract (light gray), of MOF NH 2 -MIL-53 (AI) material prepared in the absence of enzyme (gray), and of the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) - NaOH-24h (black).
  • Figure 3 Standardized powder X-ray diffractograms of the sample of NH 2 - MIL-53 (AI) (gray) and the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) -TEA-48h (black).
  • Figure 4. Thermograms (solid lines) and their derivatives (broken lines) of the soluble enzyme extract of ⁇ -Glu (gray) and of the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) - TEA-48h (black) .
  • FIG. 7 Standardized powder X-ray diffractograms of the Mg-MOF-74 material (gray) and the p-Glu @ Mg-MOF-74-24h biocatalyst (black).
  • Figure 8. Electrophoresis gel of: 1) high molecular weight reference; 2) soluble extract of the ⁇ -Glu enzyme; 3) p-Glu @ NH 2 -MIL-53 (AI) -NaOH-1 h biocatalysts; 4) ⁇ - Glu @ NH 2 -MIL-53 (AI) -NaOH-24h; 5) p-Glu @ NH 2 -MIL-53 (AI) -TEA-2h; 6) p-Glu @ NH 2 - MIL-53 (AI) -TEA-48h; 7) p-Glu @ Mg-MOF-74-2h; 8) p-Glu @ Mg-MOF-74-24h; 9) p- Glu @ NH 2 -MIL-53 (
  • Figure 10 Scheme of the catalytic test reaction of pNPG hydrolysis in the presence of the enzyme p-glucosidase.
  • Figure 11 Schematic of the catalytic test reaction of a) hydrolysis of pNPA towards p-nitrophenol and b) hydrolysis of tributirin towards butyric acid in the presence of the enzyme lipase.
  • the Aspergillus niger ⁇ -Glucosidase enzyme has approximate dimensions of -12.3 nm x -10.7 nm x -8.1 nm, a molecular weight -240 KDa, and an isoelectric point 4.2.
  • the first solution was prepared with 2.005 g of aluminum nitrate nonahydrate (AI (N0 3 ) 3-9H 2 0) (metal source) and 6.012 g of deionized water, giving a pH of 2.0.
  • the second solution was prepared with 0.483 g of the organic ligand 2-aminoterephthalic acid NH 2 -H 2 BDC, 5.206 g of 1 M NaOH solution and 10.462 g of deionized water, giving a clear solution in a few minutes with a pH 6, 1.
  • 2.75 ml of Novozymes ⁇ -Glucosidase enzyme extract (EC 3.2.1.21) from Novozymes, supplied as a liquid enzyme preparation (Novozym 188) was added to the second solution with a concentration of the extract of ⁇ -Glucosidase measured by Bradford analysis of 14.54 mg / ml, which modified the pH to 5.6.
  • the first solution was added to the second solution, with stirring, which caused the formation of a yellowish solid in suspension practically immediately and at room temperature (25 ° C), giving a pH of 3.1, after a time ranging from 1 to 24 hours, different aliquots (suspension) were taken, from which the biocatalyst called p-Glu @ NH 2 -MIL- 53 (AI) -NaOH-nh, was removed from the supernatant (solution) by centrifugation (13,400 rpm for 90 seconds).
  • the catalytic activity of both the suspension and the supernatant in hydrolysis of 10 mM para-nitrophenyl-beta-D-glucopyranoside (pNPG) dissolved in 0.1 M phosphoric acid / trisodium citrate buffer pH 5.0 was measured to give p -nitrophenol and beta-D-glucose.
  • the catalytic activity was measured by spectrophotometry at 405 nm, with an array diode spectrophotometer (Agilent 8453 UV-Vis) provided with thermostatting and a magnetic stirring device to keep the samples in homogeneous suspension during the tests.
  • the enzymatic activity was measured in any case by the increase of absorbance per minute at 405 nm produced by the release of p-nitrophenol due to the action of the enzyme ⁇ -glucosidase ( Figure 10) at 35 ° C and at a pH of 5, 0.
  • the enzyme ⁇ -glucosidase Figure 10
  • 100 ⁇ of soluble enzyme or 100 ⁇ o of the suspension was added, or 100 ⁇ of the supernatant or 100 ⁇ of a resuspension of immobilized ⁇ -glucosidase.
  • the molar extinction coefficient of p-nitrophenol measured under these conditions was 240 M "1 cnT 1 .
  • Table 1 shows the percentage of immobilized ⁇ -Glu enzyme, determined by Bradford analysis by measuring the initial protein concentration in the enzyme solution and protein in the supernatant after 1 and 24 hours. After 1 h, 33% of the enzyme present in the medium was immobilized, while after 24 h 96% of the enzyme was immobilized.
  • These percentages of enzymatic immobilization evolved in parallel with the presence of the NH 2 -MIL-53 (AI) phase in the recovered solid, which over time grew progressively to the detriment of the phase corresponding to the protonated organic ligand (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500), which evidenced that it is the MOF phase and not the purely organic phase that contributes to the immobilization of the enzyme.
  • Enzymatic load (mg / g) c catalytic 6 catalytic '
  • nh number of hours of synthesis
  • AI ⁇ -Glu @ NH 2 -MIL-53
  • NaOH-nh biocatalyst The sample is designated as nh (number of hours of synthesis) of the ⁇ -Glu @ NH 2 -MIL-53 (AI) -NaOH-nh biocatalyst.
  • Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.
  • the enzyme load (Table 1) was determined from the milligrams of protein in the synthesis medium (calculated from Bradford analysis) and the grams of biocatalyst obtained after filtering the final synthesis solution. It is expressed as mg of protein / g of recovered biocatalyst.
  • the presence of enzyme in the biocatalyst was also detected qualitatively by elementary chemical analysis CHNS (Table 1), which gives the contents of carbon, hydrogen, nitrogen and sulfur. These analyzes were carried out on a LECO CHNS-932 Elemental Analyzer device.
  • the presence / absence of sulfur in the biocatalyst is particularly informative because it is part of the enzyme and not the MOF material studied. In good agreement with the estimate from the Bradford method, in the biocatalyst ⁇ -Glu @ NH 2 -MIL-53 (AI) -NaOH-24h the sulfur content was 0.14%.
  • Table 1 also shows the catalytic activity (expressed in U / g biocatalyst) and the catalytic efficiency (in U / mg protein) of the biocatalyst obtained after being resuspended and calculated from equations 2 and 3 below, respectively.
  • U are the units of enzyme catalytic activity defined as transformation of 1 ⁇ of substrate per minute.
  • Figure 1 compares the powder X-ray diffractograms of the p-Glu @ NH 2 -MIL-53 (AI) -NaOH-24h biocatalyst and the homologous MOF in the absence of enzyme.
  • the diffractogram of the enzyme-free material is typical of a nanocrystalline NH 2 -MIL-53 (AI) prepared at room temperature (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500).
  • the addition of the enzyme extract caused some narrow and intense peaks to appear, which have been attributed to the protonated NH 2 -H 2 BDC ligand, in line with the drop in pH caused by this addition in the overall mixture.
  • Nanocrystallinity is an indispensable condition for particles formed by their aggregation to contain intercrystalline porosity in the range of mesopores.
  • thermogram TGA was also recorded and its derivative (DTG) of the MOF NH 2 -MIL-53 (AI) without enzyme, from the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) -NaOH- was calculated 24h and of the ⁇ -Glu enzyme extract, in a Perkin-Elmer TGA7 device with a temperature sweep of 20-900 ° C at a heating rate of 20 ° C / min under a stream of dry air, and its derivative ( DTG) is presented in Figure 2.
  • This experiment shows how to obtain a biocatalyst by immobilizing the same Aspergillus niger ⁇ -Glucosidase enzyme used in Example 1, during the formation of a MOF NH 2 -MIL-53 (AI) system in which triethylamine base is used ( TORCH).
  • AI MOF NH 2 -MIL-53
  • the first solution was prepared with 2,000 g of aluminum nitrate nonahydrate (AI (N0 3 ) 3-9H 2 0) (metallic source) and 6.030 g of deionized water, giving a pH of 2.0.
  • the second solution was prepared with 0.483 g of the organic ligand 2-aminoterephthalic acid NH 2 -H 2 BDC, 0.538 g of TEA and 13.246 g of deionized water, giving a clear solution in a few minutes with a pH of 6, 1.
  • ⁇ -Glucosidase enzyme extract (EC 3.2.1.21) from Novozymes, supplied as a liquid enzyme preparation (Novozym 188), with a concentration of ⁇ -Glucosidase extract was added to the second solution. measured by Bradford analysis of 14.54 mg / ml, which modified the pH of the mixture to 5.5. Then, the first solution was added dropwise on the second solution, under stirring, which caused the formation of a yellowish solid in suspension almost immediately and at room temperature (25 ° C), giving a pH of 3.1.
  • the catalytic activity was measured, following the same experimental steps described in example 1 above, except that the sampling times were 2 and 48 hours, respectively, for the biocatalysts p-Glu @ NH 2 -MIL-53 (AI) -TEA-2h and - 48h.
  • the use of ASD as a deprotonator agent for the organic MOF ligand also resulted in an efficient immobilization of the ⁇ -Glu enzyme (99% of the enzyme exposed after 48 hours has been encapsulated).
  • Enzymatic load (mg / g) c catalytic 6 catalytic '
  • the sample is designated as nh (number of hours of synthesis) of the ⁇ -Glu @ NH 2 -MIL-53 (AI) -TEA-nh biocatalyst.
  • Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol f
  • Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3 of Example 1) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol
  • Figure 3 compares powder X-ray diffractograms of the ⁇ -Glu @ NH 2 -MIL-53 (AI) -TEA-48h biocatalyst and the homologous MOF obtained in the absence of enzyme in the synthesis medium, NH 2 -MIL-53 (AI).
  • the presence of the ⁇ -Glu enzyme introduced changes in the crystalline nature of the MOF phase formed, so that the diffractogram obtained is typical of a nanocrystalline NH 2 -MIL-53 (AI) (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500), with some impurity of protonated organic ligand. Since the diffractogram of the biocatalyst is very similar to that of the MOF material without enzyme, particularly in terms of bandwidth, its crystal size must also be very similar and, therefore, its intercrystalline mesoporosity.
  • thermogram (TGA) and its derivative (DTG) of the p-Glu @ NH 2 -MIL-53 (AI) -TEA-48h biocatalyst and the ⁇ -Glu extract were also determined 4.
  • TGA curve of the enzyme extract two global weight losses can be distinguished: the first between 30 and 190 ° C (87.1%), which is attributed to water, and the second between 190 and 271 ° C ( 2.9%), which is attributed to the enzyme.
  • This experiment shows how to obtain a biocatalyst by immobilizing the same Aspergillus niger ⁇ -Glucosidase enzyme from the previous examples, during the formation of an MOF NH 2 -MIL-53 (AI) system in which NH 3 is used as the base.
  • AI MOF NH 2 -MIL-53
  • the first solution was prepared with 2,041 g of aluminum nitrate nonahydrate (AI (N0 3 ) 3-9H 2 0) (metal source) and 6.023 g of deionized water.
  • the The second solution was prepared with 0.482 g of the organic 2-aminoterephthalic acid NH 2 -H 2 BDC ligand, 0.362 g of 25% NH 3 aqueous solution and 10.008 g of deionized water.
  • the mixture at room temperature (25 ° C) took several hours to reach the solution, which was finally orange.
  • Enzymatic load (mg / g) c catalytic 6 catalytic '
  • nh number of synthesis hours
  • p- Glu @ NH 2 -MIL-53 (AI) -NH 3 -nh Percentage of enzyme immobilized in the solid versus total enzyme added.
  • c mg of enzyme per g of biocatalyst Percentage of enzyme immobilized in the solid versus total enzyme added.
  • Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.
  • Figure 5 shows the X-ray diffractograms of the ⁇ -Glu @ NH 2 -MIL-53 (AI) -NH 3 -1 h and -24h biocatalysts.
  • two phases are identified: the one corresponding to a nanocrystalline NH 2 -MIL-53 (AI) with intercrystalline mesoporosity and the organic ligand NH 2 -H 2 BDC.
  • the NH 2 -MIL-53 (AI) phase was increasing to the detriment of NH 2 -MIL-53 (AI) with the synthesis time.
  • Figure 6 shows the thermogravimetric (TG) profiles of the biocatalyst p-Glu @ NH 2 -MIL-53 (AI) -NH 3 -24h and the ⁇ -glucosidase enzyme extract.
  • TG thermogravimetric
  • the sequence of experimental examples 1, 2 and 3 shows how the change in the nature of the base used to deprotonate the organic ligand 2-amino-terphthalic acid (NH 2 -H 2 BDC), which forms the MOF NH 2 -MOF-53 (AI) nanocrystalline at room temperature and in water by simple contact with an aqueous solution of Al, affects the immobilization efficiency of the Aspergillus niger ⁇ -Glucosidase enzyme.
  • NH 2 -H 2 BDC organic ligand 2-amino-terphthalic acid
  • AI MOF NH 2 -MOF-53
  • Example 4 Method of obtaining the biocatalyst p-Glu @ Mg-MOF-74-24h, comprising the synthesis of the Mg-MOF-74 system using N, N-dimethylformamide as solvent and in the presence of the enzyme ⁇ -Glucosidase.
  • the first solution 0.561 g of Mg acetate tetrahydrate was added over 10.013 g of DMF, which dissolved in a few minutes.
  • 0.202 g of 2,5-dihydroxyterephthalic acid (dhtp) was dissolved in 10,021 g of DMF.
  • dhtp 2,5-dihydroxyterephthalic acid
  • 0.5 ml of the same enzyme extract containing ⁇ -Glu was added in a concentration of 14.54 mg enzyme / ml of the previous examples, and immediately the second solution dropwise. A sample was taken at 2 hours and centrifuged for 15 seconds at 12,500 rpm thus obtaining the supernatant.
  • the solid was filtered under vacuum and labeled as p-Glu @ Mg-MOF-74-2h.
  • the reaction lasted for 24 hours, after which the solid biocatalyst p-Glu @ Mg-MOF-74-24h was recovered according to the same procedure.
  • Mg-MOF-74 material in this case, was not a simple immobilization of the ⁇ -Glu enzyme that immobilizes, but also in some way helped to preserve its enzymatic activity against the solvent inhibiting that activity, DMF
  • the efficiency of enzymatic immobilization was very high from the first minutes, probably as a result of the mixture of solutions of metal and organic ligand caused the formation of a virtually colloidal suspension, which left the enzyme little chance not to be part of the solid, once the MOF material was recovered.
  • Enzymatic load (mg / g) c catalytic 6 catalytic '
  • the sample is designated as nh (number of hours of synthesis) of the biocatalyst ⁇ -Glu @ Mg-MOF-74-nh
  • Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.
  • Figure 7 compares the diffractograms of a Mg-MOF-74 prepared at room temperature as published (D ⁇ az-Garc ⁇ a et al., Cryst. Growth Des., 2014, 14, 2479), but containing 0.5 ml of water to match the added with the enzymatic extract, and of the biocatalyst p-Glu @ Mg-MOF-74-2h.
  • the two diffractograms are typical of a MOF-74 structure of very small crystal size, and that contains an intercrystalline mesoporosity.
  • Example 5 Comparativelystograms of a Mg-MOF-74 prepared at room temperature as published (D ⁇ az-Garc ⁇ a et al., Cryst. Growth Des., 2014, 14, 2479), but containing 0.5 ml of water to match the added with the enzymatic extract, and of the biocatalyst p-Glu @ Mg-MOF-74-2h.
  • Enzymatic load (mg / g) c catalytic 6 catalytic '
  • nmin or nh number of minutes / hours of synthesis of the biocatalyst p-Glu @ Mg-MOF-74-nmin -nh.
  • Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.
  • Example 7 Procedure for obtaining a solid lip @ Fe-BTC-NaOH-nh biocatalyst, comprising the synthesis of the MOF Fe-BTC system (similar to the MOF marketed as Basolite F300) using NaOH as a deprotonation agent and in the presence of the enzyme lipase
  • This example shows how to obtain a biocatalyst by immobilizing the enzyme Candida Lipase Antarctic B (CaLB) during the formation of the MOF Fe-BTC system in which sodium hydroxide (NaOH) was used as the base.
  • the lipase enzyme of Candida Antarctica B has approximate dimensions of ⁇ 3 nm x ⁇ 4 nm x ⁇ 5 nm, a molecular weight -32 KDa, and an isoelectric point 6.0.
  • the first solution was prepared with 0.508 g of iron trichloride hexahydrate FeCI 3 -6H 2 0 (metal source) and 10.0 g of deionized water, giving a pH of 1.36.
  • the second solution was prepared with 0.263 g of the organic ligand benzene-1, 3,5-tricarboxylic acid, or trimesic acid (H 3 BTC), 3.685 g of 1.06 M NaOH solution and 1 g of water deionized, giving a clear solution in a few minutes with a pH of 8.0.
  • the first solution was added on the second solution, under stirring, which caused the formation of an orange brown solid in suspension practically immediately and at room temperature (25 ° C), giving a pH of 3.1, after a time that ranged between 1 min and 24 hours, different aliquots (suspension) were taken, from which the biocatalyst called Lip @ Fe (3) -BTC-NaOH-nh, was removed from the supernatant (solution) by centrifugation (13,400 rpm for 90 seconds) and filtration.
  • the catalytic activity was measured by spectrophotometry at a wavelength of 348 nm, with an array diode spectrophotometer (Agilent 8453 UV-Vis) provided with thermostatization and a magnetic stirring device to keep the samples in homogeneous suspension during the tests.
  • an array diode spectrophotometer (Agilent 8453 UV-Vis) provided with thermostatization and a magnetic stirring device to keep the samples in homogeneous suspension during the tests.
  • 50 ⁇ _ of soluble enzyme or 50 ⁇ _ of the suspension was added or 50 ⁇ _ of the supernatant.
  • the molar extinction coefficient of p-nitrophenol measured under these conditions was 5150 M "1 cnT 1 .
  • a Mettler Toledo DL-50 pHstate is used to monitor this reaction.
  • the experimental procedure that has been carried out in each reaction has been: 48.5 mL of phosphoric acid buffer (H 3 P0 4 ) / di-sodium hydrogen phosphate (NaHP0 4 -2H 2 0) 10 mM pH is stirred in a vessel 7.0 and 1.47 mL of 0.1 M tributyrin.
  • mB a known biocatalyst mass, mB (between 5-20 mg) is introduced.
  • the recording of the rate of addition of 0.1 M soda, v Na0H to maintain the constant pH of 7.0 gives us a line whose slope corresponds to the rate of hydrolysis, and therefore, the activity of the enzyme.
  • Tributirin units (U T B) are calculated from Equation 4:
  • the catalytic efficiency of an immobilized biocatalyst is defined as the activity / load ratio, using Equation 5:
  • Table 6 shows the percentage of immobilized lipase enzyme, determined by Bradford analysis by measuring the initial protein concentration in the enzyme solution and protein in the supernatant after 10 min (0.17 h), 1 h, 4 h and 22 hours. After 10 min, 95% of the enzyme present in the medium was immobilized, while after 22 h 87% of the enzyme was immobilized.
  • Enzymatic load (Table 6) was determined both by difference between the activity of the suspension and the activity of the supernatant measured spectrophotometrically in the p-NPA hydrolysis test ( Figure 11 a), and from the milligrams of protein in the synthesis medium (calculated from analysis Bradford) and the grams of biocatalyst obtained after filtering the final synthesis solution.
  • the sample is designated as Lip @ Fe-BTC-NaOH-nh where n is the number of hours of synthesis of the biocatalyst.
  • Catalytic activity (expressed in units of U T B activity per g of biocatalyst and calculated according to equation 4), in the hydrolysis of tributirin releasing butyric acid.
  • Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 5) in the hydrolysis of tributyrin releasing butyric acid.
  • thermogravimetric analysis Figure 12 shows the thermograms (TGA) and their derivatives (DTG) of the MOF Fe-BTC-NaOH without enzyme, the Lip @ Fe-BTC-1 h biocatalyst and the Lipase enzyme extract ( Figure 12).
  • the thermograms were recorded on a Perkin-Elmer TGA7 device with a temperature scan of 20-900 ° C at a heating rate of 20 ° C / min under a dry air flow of 40 mL / min.
  • thermogram of the Lip @ Fe-BTC-1h biocatalyst a double weight loss was detected in the temperature range between 130-342 ° C, with a pattern similar to that detected in the thermogram of the enzyme extract in temperature ranges similar while in the thermogram of the MOF without enzyme there was no appreciable weight loss in that interval, which evidences the presence of enzyme in the biocatalyst.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé général d'obtention de biocatalyseurs qui comprend l'immobilisation in situ d'enzymes, dans la mésoporosité intercristalline formée par l'agrégation de nanocristaux au cours de la synthèse d'un matériau métallo-organique (MOF). Le procédé de l'invention permet d'obtenir une haute teneur en enzyme sur le support, de réduire au minimum la réduction de l'efficacité catalytique relativement à ladite enzyme libre et de réduire les pertes dues à la lixiviation, apportant ainsi une solution aux limites identifiées dans les procédés d'obtention de biocatalyseurs connus basés sur l'immobilisation d'enzymes in situ. L'invention concerne également les biocatalyseurs obtenus selon le procédé.
PCT/ES2016/070397 2015-05-29 2016-05-26 Procédé général d'obtention de biocatalyseurs comprenant l'immobilisation d'enzymes au cours de la synthèse de matériaux métallo-organiques WO2016193516A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201530756A ES2595631B1 (es) 2015-05-29 2015-05-29 Procedimiento general de obtención de biocatalizadores que comprende la inmovilización de enzimas durante la síntesis de materiales metalo-orgánicos
ESP201530756 2015-05-29

Publications (1)

Publication Number Publication Date
WO2016193516A1 true WO2016193516A1 (fr) 2016-12-08

Family

ID=57440413

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2016/070397 WO2016193516A1 (fr) 2015-05-29 2016-05-26 Procédé général d'obtention de biocatalyseurs comprenant l'immobilisation d'enzymes au cours de la synthèse de matériaux métallo-organiques

Country Status (2)

Country Link
ES (1) ES2595631B1 (fr)
WO (1) WO2016193516A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109082420A (zh) * 2018-08-21 2018-12-25 江苏大学 金属有机框架材料固定化β-葡萄糖苷酶及其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174402A2 (fr) * 2011-06-16 2012-12-20 University Of South Florida Structures organométalliques mésoporeuses contenant une cage polyédrique comme plateforme de biocatalyse, procédés de fabrication et d'utilisation de ces structures
WO2014117225A1 (fr) * 2013-02-04 2014-08-07 Paolo Falcaro Structures organométalliques
US20140342429A1 (en) * 2013-05-16 2014-11-20 Chung Yuan Christian University Molecule immobilization method and its system
WO2016000032A1 (fr) * 2014-07-03 2016-01-07 Commonwealth Scientific And Industrial Research Organisation Systèmes de structure organométallique du type hôte-invité

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012174402A2 (fr) * 2011-06-16 2012-12-20 University Of South Florida Structures organométalliques mésoporeuses contenant une cage polyédrique comme plateforme de biocatalyse, procédés de fabrication et d'utilisation de ces structures
WO2014117225A1 (fr) * 2013-02-04 2014-08-07 Paolo Falcaro Structures organométalliques
US20140342429A1 (en) * 2013-05-16 2014-11-20 Chung Yuan Christian University Molecule immobilization method and its system
WO2016000032A1 (fr) * 2014-07-03 2016-01-07 Commonwealth Scientific And Industrial Research Organisation Systèmes de structure organométallique du type hôte-invité

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LYKOURINOU VASILIKI ET AL.: "Immobilization of MP-11 into a mesoporous metal-organic framework, MP-11 @mesoMOF: a new platform for enzymatic catalysis.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY UNITED STATES, vol. 133, no. 27, 13 July 2011 (2011-07-13), pages 10382 - 10385, XP055005190, ISSN: 1520-5126 *
SHIEH FA-KUEN ET AL.: "Imparting functionality to biocatalysts via embedding enzymes into nanoporous materials by a of novo approach: size-selective sheltering of catalase in metal-organic framework microcrystals.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY UNITED STATES, vol. 137, no. 13, 8 April 2015 (2015-04-08), pages 4276 - 4279, ISSN: 1520-5126 *
YOUNG KWAN PARK ET AL.: "Crystal Structure and Guest Uptake of a Mesoporous Metal-Organic Framework Containing Cages of 3.9 and 4.7 nm in Diameter Angewandte Chemie International Edition", 20071105 NOT VERLAG CHEMIEL, vol. 46, no. 43, 5 November 2007 (2007-11-05), pages 8230 - 8233, XP055066425, ISSN: 1433-7851 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109082420A (zh) * 2018-08-21 2018-12-25 江苏大学 金属有机框架材料固定化β-葡萄糖苷酶及其制备方法和应用
CN109082420B (zh) * 2018-08-21 2021-08-03 江苏大学 金属有机框架材料固定化β-葡萄糖苷酶及其制备方法和应用

Also Published As

Publication number Publication date
ES2595631A1 (es) 2017-01-02
ES2595631B1 (es) 2017-10-26

Similar Documents

Publication Publication Date Title
Liang et al. Metal–organic framework-based enzyme biocomposites
Ren et al. Immobilized carbonic anhydrase on mesoporous cruciate flower-like metal organic framework for promoting CO2 sequestration
Xu et al. Two-dimensional metal–organic framework nanosheets as an enzyme inhibitor: modulation of the α-chymotrypsin activity
Liu et al. User-tailored metal–organic frameworks as supports for carbonic anhydrase
Cheng et al. Hierarchical micro‐and mesoporous Zn‐based metal–organic frameworks templated by hydrogels: their use for enzyme immobilization and catalysis of Knoevenagel reaction
Troyano et al. Colloidal metal–organic framework particles: the pioneering case of ZIF-8
Cao et al. Immobilization of Bacillus subtilis lipase on a Cu-BTC based hierarchically porous metal–organic framework material: a biocatalyst for esterification
Huo et al. Magnetic MOF microreactors for recyclable size-selective biocatalysis
Gascón et al. In situ and post‐synthesis immobilization of enzymes on nanocrystalline MOF platforms to yield active biocatalysts
Gascón et al. Semi-crystalline Fe-BTC MOF material as an efficient support for enzyme immobilization
Vinoba et al. Biomimetic sequestration of CO2 and reformation to CaCO3 using bovine carbonic anhydrase immobilized on SBA-15
Hsu et al. Rapid fabrication of biocomposites by encapsulating enzymes into Zn-MOF-74 via a mild water-based approach
Zou et al. Effect of surface modification of low cost mesoporous SiO2 carriers on the properties of immobilized lipase
Pei et al. Biomimetic mineralization of nitrile hydratase into a mesoporous cobalt-based metal–organic framework for efficient biocatalysis
Fujiwara et al. Calcium carbonate microcapsules encapsulating biomacromolecules
Du et al. Enzyme@ silica nanoflower@ metal-organic framework hybrids: A novel type of integrated nanobiocatalysts with improved stability
Vinoba et al. Carbonic anhydrase conjugated to nanosilver immobilized onto mesoporous SBA-15 for sequestration of CO2
Fan et al. Sequence-dependent peptide surface functionalization of metal–organic frameworks
Ren et al. Challenges and opportunities: porous supports in carbonic anhydrase immobilization
Hwang et al. Carbonic anhydrase assisted calcium carbonate crystalline composites as a biocatalyst
JP2012016351A (ja) 蛋白質固定化用担体、固定化蛋白質及びそれらの製造方法
Zhang et al. Tris (hydroxymethyl) aminomethane-modified magnetic microspheres for rapid affinity purification of lysozyme
Vahed et al. (Fe) MIL-100-Met@ alginate: a hybrid polymer–MOF for enhancement of metformin's bioavailability and pH-controlled release
Zou et al. A dual enzyme-containing microreactor for consecutive digestion based on hydrophilic ZIF-90 with size-selective sheltering
Xu et al. Synthesis of mono-dispersed mesoporous SBA-1 nanoparticles with tunable pore size and their application in lysozyme immobilization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16802623

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16802623

Country of ref document: EP

Kind code of ref document: A1