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    • 专利摘要:
    Hybrid material that comprises a protein matrix and copper nanoparticles in it, its preparation process and its use. The present invention refers to a hybrid material that comprises: a protein matrix comprising lipase B from Antarctic Candida and nanoparticles of copper species selected from: Cu (0), Cu2 O, Cu3 (PO4)2 or any of its combinations, having its largest dimension between 3 and 15 nm. The material was prepared by using an enzyme in a buffer solution and a copper salt at room temperature. The material has an excellent catalase-type activity, an excellent catalytic capacity in the degradation of organic pollutants, such as Bisphenol A, and in the reduction of 4-nitrophenol to 4-aminophenol. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2795844A1
    申请号:ES201930459
    申请日:2019-05-24
    公开日:2020-11-24
    发明作者:Carmona Jose Miguel Palomo；Garcia Noelia Losada；Otero Alba Rodríguez
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Samsung Electronics Co Ltd；
    IPC主号:
    专利说明:

    [0002] Hybrid material that comprises a protein matrix and copper nanoparticles in it, its preparation process and its use
    [0004] The invention relates to a hybrid material comprising a protein matrix and copper nanoparticles. The present invention also describes its synthesis by means of a unique and green technology. The hybrid material can be used as a catalyst, as it shows excellent catalytic activity, excellent catalytic performance in the degradation of the organic pollutant Bisphenol A and in the reduction of 4-nitrophenol (pNP) to 4-aminophenol (pAP).
    [0006] BACKGROUND OF THE INVENTION
    [0008] Copper is an abundant metal in the land and low cost, which has been described as very useful for different applications, as an antimicrobial agent, environmental remediation111, electronics and especially in catalysis. In fact, very extensive work has been carried out in recent years in the area of application of copper catalysts in chemical reactions, especially in C-C bonds (click chemistry, C-H activation, etc.), selective oxidation, reductions.
    [0010] In particular, Cu nanoparticles have generated great interest in recent years121. The high surface to volume ratio of nanomaterials compared to bulk materials makes them generally attractive candidates for application as catalysts.
    [0012] In fact, different practical and direct ways of preparing Cu131 nanomaterials have been described.
    [0014] However, the use of Cu nanoparticles (NPs) is restricted by the inherent instability of Cu under atmospheric conditions, which makes it prone to oxidation. Efforts to develop methods and support materials that increase the stability of Cu NPs by altering their sensitivity to oxygen, water, and other chemical entities has encouraged exploration of alternative Cu-based nanoparticles with more complex structures, such as Core / shell Cu NPs ( core / shell) or systems based on copper oxides.
    [0016] The synthesis of Cu and copper oxide NPs is essentially focused on four types of chemical reactions: reduction, oxidation, hydrolysis or condensation. Depending on the choice of end products, one or a combination of the chemicals mentioned above can be applied. The synthesis of Cu NPs often involves the reduction of Cu (l) or Cu (ll) sources. The synthesis of copper oxide NPs, on the other hand, basically requires hydrolysis of the precursors followed by a dehydration process leading to the final products. Furthermore, an oxidation process (sometimes unavoidable for Cu-based NPs) can be deployed for the preparation of Cu-based NPs with higher oxidation numbers of their respective lower oxidation state precursors. In synthetic processes, the applied techniques provide a suitable environment and energy to facilitate the selection process, while additional constraints are imposed to modulate the stability, properties and morphology of the final NPs.
    [0018] Therefore, the development of methodologies to prepare highly active, selective, stable and robust copper nanoparticles is mandatory141. Sustainable systems, cheap and high quantity CuNPs are also desirable from a more applicable point of view.
    [0020] The copper species in terms of catalysis is one of the important aspects in the final properties of nanocatalysts in a particular reaction. Therefore, the development of technologies that make it possible to specifically control the synthesis of a copper species represents a major challenge.
    [0022] An important application in which copper catalysts have been used in recent years has focused on the degradation of organic pollutants, and specifically Bisphenol A (BPA).
    [0024] BPA is an important monomer in the manufacture of polycarbonate plastics, food cans, and other everyday chemicals. The daily and worldwide use of BPA and BPA products led to their ubiquitous distribution in water, sediment / soil and atmosphere. Additionally, BPA has been identified as a disruptor endocrine disease due to its estrogenic and genotoxic activity151. Therefore, BPA contamination in the environment is a growing concern around the world, and methods to effectively remove BPA from the environment are urgently recommended. In 2018, the European Commission adopted a proposal to strengthen the regulation on the use of BPA in materials in contact with food, the new regulation introduces a new specific migration limit (SML) of 0.05 ppm of BPA.
    [0026] Therefore, a methodology to obtain a very fast, efficient and green strategy to eliminate BPA is a must. Actually, only a few examples using Cu or Cu-based materials have been applied to this reaction in many cases using homogeneous catalysts161.
    [0028] In this invention, excellent results have been achieved in the complete degradation of BPA in water with heterogeneous Cu bionanohybrides, which were reused several times without losing activity, a critical role for industrial application.
    [0030] The degradation of hydrogen peroxide (H2O2) in water and oxygen has an important reaction in biological systems. In fact, the formation of H2O2 due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. The biological catalyst involved in the removal is catalase, a copper metalloenzyme.
    [0032] The application of this enzyme could be the target of antioxidant therapy, however this is restricted by its release of a labile and inappropriate nature.
    [0034] Therefore, the development of stable and well distributed artificial systems that mimic this biological activity represents a challenge.
    [0035] References
    [0037] [1] Singh, J., Dutta, T., Kim, K.-H., Rawat, M., Samddar, P., Kumar, P. 'Green' synthesis of metais and their oxide nanoparticles: Applications for environmental remediation . J. Nanobiotechnol., 2018, 16, art. no. 84.
    [0038] [2] Gawande, A.Goswami, MB, Felpin F.-X., Asefa, T., Huang, X., Silva, R., Zou, X., Zboril, R., Varma, RS Cu and Cu- Based Nanoparticles: Synthesis and Applications in Catalysis. Chem. Rev. 2016, 116, 3722-3811.
    [0039] [3] Zaera, F. Nanostructured Materials for Applications in Heterogeneous Catalysis. Chem. Soc. Rev. 2013, 42, 2746-2762.
    [0040] [4] Ben Aissa, MA, Tremblay, B., Andrieux-Ledier, A., Maisonhaute, E., Raouafi, N., Courty, A. Copper Nanoparticles of Well-Controlled Size and Shape: A New Advance in Synthesis and Self-Organization. Nanoscale 2015, 7, 3189-3195.
    [0041] [5] Ulutao, OK, Yildiz N, Durmaz, E, Ahbab MA, Barias N, Qok Á. An in vivo assessment of the genotoxic potential of bisphenol Aand 4-tert-octylphenol in rats. Arch Toxicol 2011, 85, 995-1001.
    [0042] [6] Pachamuthu, MP, Karthikeyan, S., Maheswari, R., Lee, AF, Ramanathan, A. Fenton-like degradation of Bisphenol A catalyzed by mesoporous Cu / TUD-1. Appl. Surf. Sci., 2017, 393, 67-73.
    [0044] DESCRIPTION OF THE INVENTION
    [0046] The present invention describes a hybrid material comprising a protein matrix and nanoparticles (NPs) of a Cu species as well as the process for their preparation. The material has an excellent catalytic activity, an excellent catalytic capacity in the degradation of organic pollutants, such as Bisphenol A (BPA) and in the reduction of 4-nitrophenol (pNP) to 4-aminophenol (pAP).
    [0048] In a first aspect, the present invention refers to a hybrid material comprising:
    [0049] - a protein matrix comprising lipase B from the Antarctic Candida and
    [0050] - nanoparticles of copper species selected from: Cu (0), CU2O, Cu3 (PÜ4) 2 or any of their combinations,
    [0051] where the nanoparticles have an average diameter of between 3 and 15 nm and are homogeneously distributed within the matrix.
    [0052] Antarctic Candida lipase B (CALB) (GenBank reference number: CAA83122.1) is a non-specific Antarctic Candida lipase . CALB is stable over a wide pH range, especially the alkaline pH range. This enzyme exhibits a high degree of substrate specificity, allowing large clumps on the carboxylic acid and resulting in highly regio and enantioselective conversions.
    [0054] Lipase B from Antarctic Candida is commercially available, for example, from Novozymes under the name Lipozyme® CALB.
    [0056] The interactions between the matrix and the nanoparticles are not covalent.
    [0058] The matrix consists of lipase B from the Antarctic Candida in aggregate form. The term "aggregate" is a multiplicity of protein molecules that have been grouped together through steric or otherwise interaction with each other.
    [0060] In a preferred embodiment of the invention, the matrix consists of lipase B from Antarctic Candida.
    [0062] In a preferred embodiment, the hybrid material has between 22 and 94% by weight in Cu, obtained by ICP-OES elemental analysis (Inductively Coupled Plasma Optical Emission Spectrometry).
    [0064] A second aspect of the present invention relates to a process for preparing the hybrid material described in the first aspect of the present invention. The process comprises the following stages:
    [0065] a) Stirring addition of Antarctic Candida lipase B to a buffer solution, in which the pH of the buffer is between 7 and 10
    [0066] b) adding a copper salt to the solution obtained in step a) at room temperature (20-25 ° C)
    [0067] c) incubation of the solution obtained in step b) for a time between 16h and 3 days,
    [0068] d) collecting, washing and drying the hybrid material obtained in the previous step.
    [0070] In a preferred embodiment, the collected hybrid material (which has formed as a solid in solution) is washed with water and / or lyophilized drying in step d).
    [0071] In a preferred embodiment, the process includes an additional step c) '(after step c) and before step d)) which can be:
    [0072] - a reduction step comprising the addition of a reducing agent, preferably sodium borohydride, to the mixture obtained in step c) or
    [0073] - an oxidation stage comprising the addition of an oxidizing agent, preferably hydrogen peroxide or NaOH, to the mixture obtained in stage c).
    [0075] In a preferred embodiment, the reduction or oxidation step is carried out for approximately 30 min.
    [0077] Preferably, the buffer solution (ie, an aqueous solution) of step a) is a sodium phosphate buffer solution or a sodium bicarbonate buffer solution; more preferably a 0.1 M phosphate buffer (pH 7) or a 0.1 M sodium bicarbonate buffer (pH 10).
    [0079] In a preferred embodiment, between 0.3 and 3 mg of Antarctic Candida lipase B are added per ml of buffer solution, more preferably 18 mg per 60 mL of buffer solution in step a).
    [0081] Preferably the copper salt is CU2SO4, more preferably CU2SO45H2O.
    [0083] In a preferred embodiment, 10 mg of copper salt is added in step b) per ml of buffer solution.
    [0085] Preferably, the incubation time in step c) ranges from 16 h to 24 h, more preferably 16 h. Incubation means that the mixture is left under agitation for the specified time.
    [0087] In a preferred embodiment, in step c ') the reducing agent, preferably sodium borohydride, is added to a final concentration of at least 0.012 M in the mixture, more preferably between 0.012 and 0.12 M, even more preferably at a final concentration of 0.12.
    [0089] In one embodiment, the process for preparing the hybrid material comprises:
    [0090] a) Adding Antarctic Candida lipase B to a buffer solution with stirring of 0.1 M sodium bicarbonate (pH 10), in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    [0091] b) addition of a copper salt, preferably CU2SO45H2O, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10 mg per ml of buffer solution,
    [0092] c) incubation of the solution obtained in step b) for 16 h.
    [0093] c ') addition of sodium borohydride to a final concentration of 0.12M,
    [0094] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0096] This embodiment provides a hybrid material that has Cu (0) as the main copper species and contains about 20% by weight of Cu (I) in the form of CU2O. Nanoparticles are about 9 nm in size (in the present invention, the term "size" refers to mean diameter). The Cu content in the material is around 84% by weight.
    [0098] In another embodiment, the process for preparing the hybrid material comprises:
    [0099] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.3 mg per ml of solution tampon,
    [0100] b) addition of a copper salt, preferably CU2SO45H2O, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10 mg per ml of buffer solution,
    [0101] c) incubation of the solution obtained in step b) for 16 h.
    [0102] c ') addition of sodium borohydride to a final concentration of 0.12M,
    [0103] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0105] In this case, the main Cu species is Cu (I) in the form of CU2O around 70% by weight and also containing around 30% by weight Cu (0). This hybrid material has 81% Cu by weight and the nanoparticles are 15 nm.
    [0107] In another embodiment, the process for preparing the hybrid material comprises:
    [0108] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium bicarbonate buffer solution (pH 10), in which the protein is added in a ratio of 0.6 mg per ml of buffer solution,
    [0109] b) adding a copper salt, preferably Cu2S04-5H20, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0110] c) incubation of the solution obtained in step b) for 16 h.
    [0111] c ') addition of sodium borohydride to a final concentration of 0.12M,
    [0112] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0114] In this case, a hybrid material is also obtained which has Cu (0) as the main copper species. The nanoparticles are approximately 6 nm in size. The Cu content in the material is around 94% by weight.
    [0116] In another embodiment, the process for preparing the hybrid material comprises:
    [0117] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.6 mg per ml of solution tampon,
    [0118] b) adding a copper salt, preferably Cu2S04-5H20, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0119] c) incubation of the solution obtained in step b) for 16 h.
    [0120] c ') addition of sodium borohydride to a final concentration of 0.12M,
    [0121] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0123] In this embodiment, differences were observed in the Cu species and in the size of the nanoparticles. In this case, the hybrid material exclusively comprises Cu20 species, without traces of Cu (0). Nanoparticles are crystalline and are approximately 10 nm in size. The Cu content in the material is about 61% by weight.
    [0125] In another embodiment, the process for preparing the hybrid material comprises:
    [0126] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.3 mg per ml of solution tampon,
    [0127] b) adding a copper salt, preferably Cu2S04-5H20, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0128] c) incubation of the solution obtained in step b) for 16 h.
    [0129] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0131] In this case, the copper species obtained is Cu3 (P04) 2. The nanoparticles are approximately 3-5 nm in size. The Cu content in the material is around 32% by weight.
    [0133] In another embodiment, the process for preparing the hybrid material comprises:
    [0134] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.3 mg per ml of solution tampon,
    [0135] b) adding a copper salt, preferably Cu2S04-5H20, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0136] c) incubation of the solution obtained in step b) for 16 h.
    [0137] c ') addition of H202 to a final concentration of 0.1 M,
    [0138] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0140] In this case, the copper species obtained is Cu3 (P04) 2. The nanoparticles are approximately 5 nm in size. The Cu content in the material is around 22% by weight.
    [0142] In another embodiment, the process for preparing the hybrid material comprises:
    [0143] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.3 mg per ml of solution tampon,
    [0144] b) adding a copper salt, preferably Cu2S04-5H20, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0145] c) incubation of the solution obtained in step b) for 16 h.
    [0146] c ') addition of NaOH to a final concentration of 0.5 M,
    [0147] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0149] In this case, the copper species obtained is Cu3 (P04) 2- The nanoparticles have an approximate size of 6 nm. The Cu content in the material is around 35% by weight.
    [0151] In another embodiment, the process for preparing the hybrid material comprises:
    [0152] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 0.3 mg per ml of solution tampon,
    [0153] b) addition of a copper salt, preferably CU2SO45H2O, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0154] c) incubation of the solution obtained in step b) for 16 h.
    [0155] c ') addition of sodium borohydride to a final concentration of 0.012 M,
    [0156] d) collecting, washing with water and drying by lyophilization the hybrid material obtained in the previous step.
    [0158] Under these synthetic conditions, the hybrid material is composed of extremely crystalline Cu3 (P04) 2 nanoparticles (size approx. 10 nm). Furthermore, the Cu content in this case is around 48% by weight.
    [0160] In another embodiment, the process for preparing the hybrid material comprises:
    [0161] a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 3 mg per ml of buffer solution,
    [0162] b) addition of a copper salt, preferably CU2SO45H2O, to the solution obtained in the previous step at room temperature (20-25 ° C), where the copper salt is added in a proportion of 10mg per ml of buffer solution,
    [0163] c) incubation of the solution obtained in step b) for 3 days.
    [0164] c ') the optional addition of sodium borohydride at a final concentration of 0.12 M, d) collecting, washing with water and drying the hybrid material obtained in the previous step by lyophilization.
    [0165] In these cases, Cu3 (P04) 2 is the main copper species, also containing Cu20, especially in cases where the reduction step was carried out. A Cu content of around 50% by weight is obtained.
    [0167] The present invention also relates to the hybrid material obtained by the process described in the second aspect of the invention. The hybrid material obtained by the process of the invention has the characteristics of the hybrid material described in the first aspect of the present invention.
    [0169] Another aspect of the present invention relates to the use of the hybrid material described in the present invention as a catalyst.
    [0171] In a preferred embodiment, the hybrid material described in the present invention is used as a catalyst to degrade organic pollutants, preferably phenolic compounds; more preferably to degrade Bisphenol-A, forming C02 and H20.
    [0173] In another preferred embodiment, the hybrid material described in the present invention is used for the reduction of 4-nitrophenol (pNP) to 4-aminophenol (pAP).
    [0175] In another preferred embodiment, the hybrid material described in the present invention is used for the degradation of H2O2, forming H2O and O2, since the hybrid material has catalase-like activity.
    [0177] In the present invention, the terms "hybrid material", "biohybrid material", "bionanohybrid material" or simply "biohybrid" or "bionanohybrid" or "hybrid material comprising a protein matrix and copper nanoparticles" are used synonymously and are refer to a material that comprises a matrix of Antarctic Candida lipase B , (organic compound) and Cu species (inorganic compound) selected from: Cu (0), Cu20, Cu3 (P04) 2 or any of their combinations, in the form of nanoparticles with a mean diameter between 3 and 15 nm.
    [0179] The term "average" of a dimension of a plurality of particles means the average of that dimension for the plurality. For example, the term "mean diameter" of a plurality of nanoparticles means the average of the diameters of nanoparticles, where the diameter of a single nanoparticle is the average of the diameters of that nanoparticle. Nanoparticles are spherical or substantially spherical. The mean diameter is obtained by transmission electron microscopy (MET).
    [0181] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of those of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0182] BRIEF DESCRIPTION OF THE FIGURES
    [0184] FIG. 1: Characterization of the Cu-CALB-BIC biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images.
    [0186] FIG. 2: Characterization of the Cu-CALB-PHOS biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images.
    [0188] FIG. 3: Characterization of the Cu-CALB-BIC2 biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images.
    [0190] FIG. 4: Characterization of the Cu-CALB-PHOS2 biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images.
    [0192] FIG. 5: Characterization of the Cu-CALB-PHOS-NR biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images.
    [0194] FIG. 6: Characterization of the Cu-CALB-PHOS-NRNaOH biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images. C) C) HTEM images.
    [0196] FIG. 7: Characterization of the CU-CALB-PHOS-NRH2O2 biohybrid prepared in Example 1 of the present invention. A) XRD spectrum, B) TEM images. C) HTEM images.
    [0198] FIG. 8: Characterization of the Cu-CALB-PHOS10% biohybrid. A) XRD spectrum, B) TEM images.
    [0200] FIG. 9: Characterization of the Cu-CALB-PHOS-10 biohybrid. A) XRD spectrum, B) SEM image.
    [0202] FIG10: Characterization of the Cu-CALB-PHOS-NR-10 biohybrid. A) XRD spectrum, B) SEM image.
    [0204] FIG. 11: Representation of the degraded percentage of BPA catalyzed by different Cu-CALB nanohybrids at 100 mM phosphate buffer pH 8 in the presence of 100 mM H2O2.
    [0206] FIG. 12: Representation of the degraded percentage of BPA catalyzed by different hybrids in the presence of 100 mM H2Ü2 at different pHs. A) Cu-CALB-PHOS2 and B) Cu-CALB-PHOS.
    [0208] FIG. 13: BPA degradation by Cu-CALB-PHOS2 at 100 mM phosphate buffer pH 8 in the presence of different concentrations of H2O2.
    [0210] FIG. 14: Recycling of Cu-CALB-PHOS2 in the oxidative degradation of BPA at 100 mM phosphate buffer pH 8 in the presence of 100 mM H2O2.
    [0212] FIG. 15: A. Representation of the degraded percentage of BPA (4.6 ppm) catalyzed by different Cu-CALB-PHOS2 at 100 mM phosphate buffer pH 8 in the presence of 100 mM H2O2. B. Proposed mechanism for the degradation of BPA.
    [0214] FIG. 16: Catalase activity of Cu-CALB-PHOS2. A) degradation of 50 mM H2O2 in distilled water adjusted to different pHs. B) degradation of 50 mM H2O2 to different concentrations of phosphate buffer at pH 6.
    [0216] FIG. 17: Scheme of preparation of the bionanohybrides comprising the NPs of the copper species and a protein matrix.
    [0218] EXAMPLES
    [0220] Some examples carried out by the inventors are provided below to illustrate the present invention:
    [0222] Experimental Section
    [0224] general
    [0225] Lipase B from Antarctic Candida solution (CAL-B) (Lipozyme®CALB) (10 mg / mL) was purchased from Novozymes (Copenhagen, Denmark). Copper (II) sulfate pentahydrate [CU2SO4 x 5H2O] and hydrogen peroxide (33%) are from Panreac (Barcelona, Spain). P-nitrophenol, p-nitrophenyl propionate, sodium bicarbonate, sodium phosphate, sodium borohydride, and bisphenol A (BPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC grade acetonitrile was purchased from Scharlab (Barcelona, Spain). Inductively coupled plasma optical emission spectrometry (ICP-OES) was performed on an OPTIMA 2100 DV instrument (PerkinElmer, Waltham, MA, USA). X-ray diffraction (XRD) patterns were obtained using a D8 Advance Texture Analysis Diffractometer (Bruker, Billerica, MA, USA) with Cu Ka radiation. Transmission electron microscopy (TEM) and high resolution TEM microscopy (HRTEM) images were obtained on a 2100F microscope (JEOL, Tokyo, Japan) equipped with an EDX INCA x-sight detector (Oxford Instruments, Abingdon, UK) . The interplanar spacing in the nanostructures was calculated using the inverse Fourier transformation with the GATAN digital micrography program (Corporate Headquarters, Pleasanton, CA, USA). Scanning electron microscopy (SEM) images were made on a TM-1000 microscope (Hitachi, Tokyo, Japan). For the recovery of the biohybrids, a refrigerated Biocen 22 R centrifuge (Orto-Alresa, Ajalvir, Spain) was used. Spectrophotometric analyzes were performed with a V-730 spectrophotometer (JASCO, Tokyo, Japan). A P100 spectrum HPLC system (Thermo Scientifics, Waltham, MA, USA) was used. The analyzes were carried out at 25 ° C using an oven L-7300 column (Hitachi, Tokyo, Japan) and a UV6000LP detector (Thermo Scientifics, Waltham, MA, USA).
    [0227] Example 1: General synthesis of Cu-CALB-BIC and Cu-CALB-PHOS bionanohybrides.
    [0229] 1.8 mL (18 mg protein) of commercial Antarctic Candida lipase solution was added to a 60 mL 0.1M buffer (sodium bicarbonate pH = 10 or sodium phosphate pH 7) in a 250 mL glass bottle that it contained a small magnetic rod stirrer. Then 600 mg of CU2SO4 x 5H2O (10 mg / ml) was added to the protein solution and kept for 16 hours. After the first 30 minutes of incubation, the solution turned cloudy (turquoise) and the solution was measured for pH, indicating a decrease from 8 to 6, depending on the buffer used. After 16 h, 6 mL of aqueous NaBH4 solution (300 mg) (1.2 M) were added to the cloudy solution (in two 3 mL times) obtaining a final concentration of 0.12 M of sodium borohydride in the mixture. The solution quickly turned black and the mixture was reduced for 30 minutes. After incubation, in all cases, the mixture was centrifuged at 8000 rpm for 5 min, (10 mL per hawk-type tube). The pellet generated was re-suspended in 15 mL of water. The pH of the supernatant solution was measured at about 7 or 9. It was centrifuged again at 8000 rpm for 5 minutes and the supernatant was removed. The pH of the supernatant solution was measured again, with a pH value of 7. The process was repeated two more times. Finally, the supernatant was removed and the precipitate from each falcon was resuspended in 2 mL of water, all solutions were collected in a round bottom flask, frozen with liquid nitrogen and lyophilized for 16 hours. Then 150 mg of the so - called Cu-CALB-BIC and Cu-CALB-PHOS, respectively, were obtained.
    [0231] Different modifications of the protocol were made to obtain different species. Initially, a catalyst was prepared using double the amount of enzyme (3.6 mL of CALB solution instead of 1.8 mL), obtaining Cu-CALB-PHOS-2 and Cu-CALB-BIC-2.
    [0233] Another variation of the protocol was used, in which the reduction step was not performed, obtaining Cu-CALB-PHOS-NR. In addition to the last variation, an oxidation step was also performed instead of a reduction step by adding 6 ml of a 500 mM sodium hydroxide (NaOH) solution for 30 minutes or 6 ml of a 0.1 M hydrogen peroxide (H2O2) solution for 30 minutes (60 pL of the H202 stock solution in 6 mL of distilled water), obtaining Cu-CALB-PHOS-NRNNaOH and CU-CALB-PHOS-NRH2O2, respectively. Another variation was the reduction of sodium borohydride to 10%, adding 6 mL of water with NaBH4 (30 mg), obtaining Cu-CAL-B-PHOS10% R.
    [0235] The last variation referred to both the amount of enzymes and the incubation time, increasing them from 1.8 mL to 18 mL in enzyme volume and from 16 h to 72 h; the catalysts obtained were Cu-CALB-PHOS-10 and Cu-CALB-PHOS-NR-10.
    [0237] The characterization of the different Cu bionanohybrides was carried out by XRD, ICP-OES, TEM and SEM analysis.
    [0239] Example 2: Catalytic reduction of 4-nitrophenol (pNP) to 4-aminophenol (pAP) To an aqueous solution of p-nitrophenol (pNP) (1 mM; 2 mL), solid NaBH4 (3 mg) was added to reach a concentration final 0.04 M (The typical catalytic reaction was carried out by adding an excess of NaBH4 (0.04 M) to ensure its constant concentration throughout the entire reaction and, therefore, to apply a pseudo-first order kinetics relative to pNP to an aqueous solution of the substrate in the presence of catalysts). Under these conditions, upon the addition of NaBH4, the initial absorbance band of the pNP solution changes immediately from 317 to 400 nm due to the formation of 4-nitrophenolate ions. Immediately afterwards, 3 mg of the different Cu-CALB bionanohybrides were added, shaking gently at 25 ° C on an orbital shaker. The progress of the reaction was monitored by taking an aliquot of the solution (0.1 mL) at different times, diluting it with distilled water (2 mL) and measuring the absorption spectrum between 500 and 300 nm in a PMMA cuvette (Table 1 ).
    [0240] Table 1. Degradation of pNP by all bionanohybrids.
    [0242]
    [0244] Degradation
    [0245] Weather
    [0246] PNP bionanohybrid
    [0247] (min)
    [0249] Cu-CALB-PHOS 100 1.5
    [0250] Cu-CALB-BIC 100 0.5
    [0251] Cu-CALB-PHOS-2 100 3
    [0252] Cu-CALB-BIC-2 100 0.5
    [0253] Cu-CALB-PHOS-NR 100 4
    [0254] Cu-CALB-PHOS-NR-NaOH 100 3
    [0256] Cu-CALB-PHOS10% R 100 1.5
    [0257] Cu-CALB-PHOS-10 100 12.5
    [0258] Cu-CALB-PHOS-NR-10 100 15
    [0260] Example 3: Catalytic degradation of Bisphenol-A (BPA)
    [0261] A 10 mM solution of BPA in neat acetonitrile was prepared. 0.2 ml of this solution was dissolved in 10 ml of 100 mM or 5 mM sodium phosphate buffer at pH 6, pH 7 or pH 8 to achieve a BPA concentration of 0.2 mM. The pH of the solution was adjusted using HCl or 1M NaOH. Hydrogen peroxide was added to this BPA solution to obtain different concentrations (12, 25, 50, 100 or 150 mM). To initialize the reaction, 3 mg of the nanohybrid were added to 2 µL of this solution (BPA and H2O2) in a 7 mL glass bottle. A roller was gently agitating at room temperature. Samples (30 µl) were taken at different times and the reaction was followed by HPLC. The samples were diluted 5 times in a 50/50 distilled water / acetonitrile mixture prior to injection. The HPLC column was C8 Kromasil 150x4.6mm AV-2059. The HPLC conditions used were: a socratic mixture of 50% acetonitrile and 50% double distilled water, UV detection at 225 nm using a diode array detector, and a flow rate of 1 mL / min. Under these conditions, the retention time for BPA was 4.90 minutes, and for H202 it was 1.57 minutes.
    [0263] Example 4: Reuse of the nanohybrid Cu-CALB-PHOS2 in the degradation of Bisphenol-A (BPA)
    [0264] The Cu-CALB-PHOS2 nanohybrid was reused in five cycles for the degradation of BPA using the conditions described above. The catalyst was washed several times with water and centrifuged before the next reaction.
    [0266] Example 5: Catalytic activity of Cu nanohybrids
    [0267] A substrate solution was prepared by adding 52 pL of hydrogen peroxide to 9.8 mL of 100 mM or 5 mM phosphate buffer (pH 6, pH 7 and pH 8) or distilled water to obtain a final concentration of 50 mM. The pH of the solution was adjusted with HCI or 1M NaOH. To start the reaction, 4.5 mg of the Cu nanohybrid or 50 pL of Catazyme® 25L (1 mg / mL in distilled water) were added to 3 mL of the above solution at room temperature. The reaction was followed by measuring the degradation of hydrogen peroxide, recording the decrease in absorbance by spectrophotometry at 240 nm in quartz cuvettes with a path length of 1 cm, adding 2 mL of this solution at different times. After each measurement, the added volume was recovered and poured into the reaction solution.
    [0269] To determine the catalase activity for each catalyst, the AAbs / min value was calculated using the linear part of the curve (AAbsS).
    [0271] The specific activity (U / mg) was calculated using the following equation:
    [0272] AAbs Vf( mV)
    [0273] U ( ^ mol / min) -
    [0274] t ( min) mL
    [0275] £ ( ^ mol ■ cm) ■ b ( cm)
    [0276] where the molar extinction coefficient () used was 43.6 M'1cm-1
    [0278] Results and Discussion:
    [0280] 1) Preparation and characterization of different bionanohybrides that comprise the lipase B protein of the Antarctic Candida (CAL-B) and NPs of different Cu species:
    [0281] The synthesis of these bionanohybrids containing copper nanoparticles has been carried out in an aqueous medium by adding the commercial lipase B from Antarctic Candida (CAL-B, 33 kDa, monomeric enzyme, supplied by Novozymes) to an aqueous solution of totally soluble copper salt. in water at room temperature and under gentle agitation (Figure 14). Copper (II) sulfate pentahydrate [CU2SO4 x 5H20] was used as copper salt with a concentration of 10 mg / mL. 1.8 mL of CAL-B was added to 60 mL of copper saline at room temperature. Different buffers and different concentrations were tested and the use of 100 mM sodium bicarbonate (pH 10) and 100 mM sodium phosphate (pH 7) were the best options.
    [0283] After 16 hours of incubation, the solid was reduced using sodium borohydride, washed several times with distilled water, centrifuged and lyophilized overnight to obtain the two heterogeneous biohybrids Cu-CALB-BIC and Cu-CALB-PHOS, respectively. Both catalysts were characterized by different analysis techniques such as XRD, ICP-OES, TEM and SEM. Different species were obtained depending on the buffer used in the synthesis.
    [0285] The XRD analysis showed that the main copper species in the Cu-CALB-BIC hybrid was Cu (0), which contained about 20% by weight of Cu (l) as Cu20 (Figure 1). TEM analysis showed the formation of nanoparticles around 9 nm in size (Figure 1B). The ICP-OES showed that the Cu content in the Cu -CALB-BIC biohybrid Cu was 84% (The percentages of Cu in the hybrid material indicated in the present invention refer to percentages by weight). Using heating at 100 ° C to dry instead of lyophilization, the Cu20 increased to about 60% of the Cu species, the remainder was Cu (0) (data not shown).
    [0287] In the case of the Cu-CALB-PHOS hybrid , the XRD showed that the main Cu species was Cu (l) in the form of Cu20 around 70% and that it also contained around 30% Cu (0) (Figure 2) . This biohybrid presented 81% Cu calculated by ICP analysis. TEM experiments revealed the formation of 15 nm nanoparticles (Figure 2B), slightly larger than with the use of bicarbonate as a synthesis buffer (Figure 1). Furthermore, this method was modified using acetone in the washing step instead of water, or heating to 80 ° C instead of lyophilization to obtain the solid. Under all these conditions, the nanohybrid presented a mixture of the different Cu species and the catalytic properties and mechanical stability of the solid were worse.
    [0289] A modification in the previous protocols was to add twice the amount of enzyme in the preparation, keeping the remaining steps the same. This modification did not affect the copper species in the method that uses carbonate as a buffer, where the XRD pattern of the so - called Cu-CALB-BIC2 nanohybrid showed the characteristic peaks of Cu (0) and a minority at 37 ° of CU2O (so which was slightly lower than that of Cu-CALB-BIC) (Figure 3). However, the Cu content in this sample was 94%, about 10% more than using half the amount of protein (Cu-CALB-BIC). TEM analysis revealed the formation of Cu (0) NPs as a 6 nm nucleus (Figure 3B), a size smaller than that observed in Cu-CALB-BIC.
    [0291] In the case of using phosphate as a buffer, differences were observed in the size of the Cu species and of the nanoparticles. In this case, the XRD pattern determined that the Cu-CALB-PHOS2 biohybrid showed peaks corresponding exclusively to CU2O species, without traces of Cu (0) (Figure 4). Changes were also observed in the amount of Cu, being for this hybrid 61% determined by ICP-OES, 20% less than in Cu-CALB-PHOS. TEM analysis also showed the formation of crystalline nanoparticles with a diameter of 10nm (Figure 4).
    [0293] Therefore, using this last methodology, a new Cu bionanohybrid of controlled morphology, size and metallic species was synthesized. This could be explained by the concept that a greater amount of protein influences the control of the reduction of the copper oxide species and also influences the coalescence step, controlling the growth of the nanoparticles.
    [0295] In addition, when selecting the methodology using phosphate buffer, other types of Cu bionanohybrides were synthesized avoiding the reducing step (Cu-CALB-NR) or changing it by incubation in the presence of hydrogen peroxide (oxidative step) (CU-CALB-NRH2O2) o NaOH (Cu-CALB-NRNaOH) in the methodology. In all three cases, a light blue solid was obtained instead of the typical black color for the other biohybrids. The XRD showed that in this case the copper species were Cu3 (PÜ4) 2 in all cases (Figure 5-7) and the Cu content was 32%, 22% and 35%, respectively.
    [0296] TEM analysis revealed the slight differences in the diameter size of the nanoparticles. In all cases, crystalline spherical nanoparticles were formed, 3-5 nm in Cu-CALB-NR (Figure 5), 5.8 nm in Cu-CALB-NRNaOH (Figure 6) and 5 nm for CU-CALB-NRH2O2 ( Figure 7).
    [0298] Finally, a decrease was made in the amount of sodium borohydride used in the reduction step. The 10% (w / v) of NaBH4 used in the previous method was added in this case, keeping intact the rest of the synthetic steps in the Cu-CALB-PHOS synthesis method , in this case the biohybrid called Cu-CALB- PHOS10% R.
    [0300] Under these synthetic conditions, a biohybrid composed of extremely crystalline Cu3 (P04) 2 nanoparticles (diameter approx. 10 nm) (Figure 8) was obtained instead of the CU / CU2O NPs biohybrid synthesized in Cu-CALB-PHOS (Figure 2) . Furthermore, the Cu content in this case was 48% instead of 81% with the total reduction.
    [0302] A final test was performed using 10 times more protein and increasing the incubation time from 1 day to 3 days. These variations were used in two protocols, using phosphate buffer and with or without reduction step, being the bionanohybrides Cu-CALB-PHOS-10 and Cu-CALB-PHOS-NR-10, respectively. In this case, SEM analysis revealed the formation of well-formed nanoflowers in both cases (Figure 9-10). The XRD pattern showed the presence of Cu3 (P04) 2 as the main copper species, also containing CU2O, especially in Cu-CALB-PHOS-10 where the reduction step was used (Figure 9). The ICP-OES determined a Cu content of around 50% in each case.
    [0304] In all cases, the bionanohybrids were synthesized in a very effective, simple and sustainable way on a multigram scale, easily scalable to grams.
    [0306] 2) Determination of the reducing catalytic activity of pNP
    [0307] To evaluate the metallic activity of the new bionanohybrides prepared in Example 1, their activity was carried out in the reduction of p-nitrophenol (pNP) to p-aminophenol (pAP) in aqueous medium and at room temperature (ta) (Table 1). The differences in the degradation rate of pNP that exists between the different catalysts is due to the Cu species obtained.
    [0308] Cu-CALB-BIC and Cu-CALB-BIC2 (mainly Cu (0)) were the fastest nanocatalysts with a complete transformation from pNP to pAP (150 ppm) in 30 seconds. However, the Cu nanohybrids containing Cu (l) species showed lower catalytic efficiency, Cu-CALB-PHOS2 was more active than Cu-CALB-PHOS, which takes twice as long to complete the transformation, 3 minutes instead of 1.5 minutes (Table 1).
    [0310] The catalyst prepared without reduction steps or with a very high amount of proteins showed a lower catalytic activity, being necessary between 3 and 15 minutes for complete degradation (Table 1).
    [0312] 3) Degradation of bisphenol A by Bionanohybrides
    [0314] The catalytic capacity of the bionanohybrides prepared in Example 1 was evaluated in the degradation of Bisphenol A (BPA) in aqueous medium at room temperature. The experimental conditions selected were 100 mM phosphate buffer in the range of pH 6-8 and the amount of BPA was 46 ppm using H2O2 as oxidant. All Cu nanocatalysts were initially tested using 100 mM H2O2 in a 100 mM phosphate buffer at pH 8. Of them, the results obtained for the best four are represented in Figure 11. The best results were obtained using Cu- CALB-PHOS and Cu-CALB-PHOS2 where> 95% of BPA was degraded in 20 minutes. This represents, as far as we know, the fastest degradation process to remove BPA in aqueous media. The Cu-CALB-BIC hybrid degraded 68% of BPA, while the Cu-CALB-NR only removed 47% at 20 minutes reaching> 90% degraded BPA after 4 hours of incubation (Figure 11). These results showed that the species of CU2O NPs showed better catalytic activity than those of Cu (0).
    [0316] The reaction was also tested with these two excellent catalysts at different pHs (from 6 to 8) (Figure 12). In both cases, Cu nanohybrids were better catalysts at pH 8, although surprisingly the efficiency of Cu-CALB-PHOS2 decreased at pH 7 more than at pH 6 compared to Cu-CALB-PHOS where the results were quite similar to these two pHs.
    [0318] The amount of H2O2 was also evaluated. The reaction was carried out using Cu-CALB- PHOS2 as a catalyst at 100 mM phosphate pH 8 in the presence of different concentrations of H202 from 12 to 150 mM (Figure 13). The results showed that 100 mM appears to be the optimal amount to run the reaction, because using a larger amount did not speed up the reaction, yet adding less H2O2 slowed down the reaction.
    [0320] Once the optimal conditions were obtained, a recycling experiment was carried out using the best Cu bionanohybrid, Cu-CALB-PHOS2 (Figure 14). The catalysts showed excellent stability maintaining 95% of the catalytic efficiency after six cycles of use.
    [0322] Considering the very fast degradation process by this catalyst and in order to elucidate a possible BPA degradation mechanism, the reaction was carried out reducing the ratio mg catalyst / mL of reaction volume from 1.5 (the previous result) to 0 ,3. Under these conditions, the catalyst still worked well, degrading more than 95% of BPA in 60 minutes (Figure 15A). By evaluating the intermediate points, it was possible to observe the formation of a peak mainly in HPLC corresponding to benzoquinone. Thus, a possible mechanism of the process may be the formation of a hydroxyl radical that is rapidly transformed mainly into benzoquinone that, by opening the ring, follows a degradation path common to other intermediate peaks to finally degrade into CO2 and H2O (Figure 15B) , mechanism also described by Giu et al. Giu et al. (Gui, L., Jin, H., Zheng, Y., Peng, R., Luo, Y., Yu, P. Electrochemical Degradation of Bisphenol A Using Different Modified Anodes Based on Titanium in Aqueous Solution. Int. J. Electmchem. Sci., 2018, 13, 7141 7156).
    [0324] 4) Catalytic activity of Cu-nanoparticle biohybrid
    [0325] The catalase activity (degradation of H2O2) of the different Cu bionanohybrides synthesized in water, 5 mM buffer and 100 mM buffer at different pHs was evaluated (Table 2). Interesting differences were found between the different Cu bionanohybrides and especially in comparison with the native catalase from Novozymes (Catazyme).
    [0326] Table 2. Catalase activities of the different Cu bionanohybrides (catalysts).
    [0327]
    [0330] The Cu-CALB-PHOS2 biohybrid exhibited the highest catalytic activity in the degradation of hydrogen peroxide, showing 2.49 U / mg of specific activity in distilled water and 2.11 U / mg in water adjusted to pH 6. In the latter In this condition, this catalyst showed an activity 6 times higher compared to Cu-CALB-BIC, 4 times more than Cu-CALB-BIC2 and 2 times more than Cu-CALB-PHOS (Table 2). In simply distilled water, Cu-CALB-PHOS2 showed almost six times more activity than Cu-CALB-PHOS, 2 times more than Cu-CALB-BIC and 4 times more than Cu-CALB-BIC2.
    [0332] In particular, for Cu-CALB-PHOS2, the evaluation of catalase activity was measured in distilled water in a pH range of 4 to 9 (Figure 16, Table 2). The best result was obtained at pH 6, and good values were also obtained at pH 4 or pH 9 (Figure 16A) although the catalyst was unstable under these conditions. Also, Figure 13B showed the negative influence of the presence of buffer in the solution, clearly decreasing the catalase activity of this Cu biohybrid under these conditions.
    [0334] In the case of the other Cu bionanohybrides, for example, Cu-CALB-PHOS showed the highest activity value in distilled water adjusted to pH 6, while the best catalytic activity at higher pH was achieved in the presence of a buffer. 5 mM (Table 2).
    [0336] For Cu-CALB-BIC, the best results were obtained in distilled water, also adjusted to pH 6 and 7, while for Cu-CALB-BIC2 they were obtained in distilled water at H 7 and 8.
    [0338] Compared with natural catalase, the most interesting results were found in distilled water conditions (without adjusting the pH) where Cu-CALB-PHOS2 showed an activity similar to that of the natural enzyme.
    [0340] Conclusions from the examples
    [0341] New biohybrids of Cu nanoparticles have been synthesized, where the control of the Cu species, size, crystallinity and morphology of the nanoparticles is possible depending on the methodology used, for example, being possible to obtain bionanohybrides that contain Cu (0) nanoparticles very small in size, exclusively Cu20 NPs or crystalline Cu3 (P04) 2 NPs.
    [0343] Copper bionanohybrides showed excellent catalytic activity in reducing p-nitrophenol. These new catalysts showed excellent catalytic performance in the degradation of a toxic and polluting compound such as BPA, where the best, the Cu-CALB-PHOS2 biohybrid, was able to eliminate more than 95% BPA (46 ppm) in 20 minutes in the presence of hydrogen peroxide at pH 8 using 1.5 g / L of catalyst.
    [0345] In addition, here it has been shown for the first time that these novel Cu bionanohybrides showed catalase activity, even in one case similar values of specific activity to natural Catalase (Catazyme from Novozymes) in distilled water, which shows that they are artificial metallo-enzymes. stable with possible interesting applications. This can also be extended to the comparison with other catalases from different sources or even pseudocatalases.
    权利要求:
    Claims (24)
    [1]
    1. A hybrid material comprising:
    - a protein matrix comprising lipase B from the Antarctic Candida and
    - nanoparticles of copper species selected from: Cu (0), Cu20, Cu3 (P04) 2 or any of their combinations,
    where the nanoparticles have an average diameter of between 3 and 15 nm and are homogeneously distributed within the matrix.
    [2]
    Hybrid material according to claim 1, in which the protein matrix is formed by lipase B from the Antarctic Candida.
    [3]
    3. A hybrid material according to claim 1 or 2, wherein the hybrid material has between 22 and 94% by weight in Cu.
    [4]
    4. A process to prepare the hybrid material described in claims 1-3, the process comprises the following stages:
    a) Stirring addition of Antarctic Candida lipase B to a buffer solution, in which the pH of the buffer is between 7 and 10
    b) adding a copper salt to the solution obtained in step a) at room temperature (20-25 ° C)
    c) incubation of the solution obtained in step b) for a time between 16h and 3 days,
    d) collecting, washing and drying the hybrid material obtained in the previous step.
    [5]
    A process according to claim 4, in which the collected hybrid material is washed with water and / or dried by lyophilization in step d).
    [6]
    6. A process according to any of claims 4-5, where the process includes an additional stage c) ', after stage c and before stage d), which will be:
    - a reduction step comprising the addition of a reducing agent to the mixture obtained in step c) or
    - an oxidation stage comprising the addition of an oxidizing agent to the mixture obtained in stage c).
    [7]
    7. A process according to any of claims 4-6, wherein the buffer solution of step a) is a sodium phosphate buffer or a sodium bicarbonate buffer.
    [8]
    8. A process according to any of claims 4-7, in which between 0.3 and 3 mg of lipase B from Antarctic Candida are added per ml of buffer solution in step a).
    [9]
    9. A process according to any of claims 4-8, wherein the copper salt is CU2SO45H2O.
    [10]
    10. A process according to any of claims 4-9, wherein 10 mg of copper salt is added in step b) per ml of buffer solution.
    [11]
    11. A process according to any of claims 4-10, wherein the incubation time in step c) ranges from 16h to 24h.
    [12]
    12. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium bicarbonate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of CU2SO45H2O to the solution obtained in the previous step at 20-25 ° C, in which the CU2SO45H2O is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in step b) during 16 h.
    c ') addition of sodium borohydride to its final concentration of 0.12M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [13]
    13. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in which Cu2S04-5H20 is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in step b) for 16 h.
    c ') addition of sodium borohydride to its final concentration of 0.12M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [14]
    14. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium bicarbonate buffer solution, in which the protein is added in a proportion of 0.6 mg per ml of buffer solution,
    b) addition of CU2SO45H2O to the solution obtained in the previous step at 20-25 ° C, in which the CU2SO45H2O is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in step b) during 16 h.
    c ') addition of sodium borohydride to its final concentration of 0.12M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [15]
    15. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.6 mg per ml of buffer solution,
    b) addition of CU2SO45H2O to the solution obtained in the previous step at 20-25 ° C, in which the CU2SO45H2O is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in step b) during 16 h.
    c ') addition of sodium borohydride to its final concentration of 0.12M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [16]
    16. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in the that the Cu2S04-5H20 is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in step b) for 16 h.
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [17]
    17. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in which Cu2S04-5H20 is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in the step b) for 16 h.
    c ') addition of H202 to its final concentration of 0.1 M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [18]
    18. A process according to claim 4 wherein the process comprises the following steps:
    a) addition, stirring the lipase B of the Antarctic Candida, to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in which Cu2S04-5H20 is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in the step b) for 16 h.
    c ') addition of NaOH to a final concentration of 0.5 M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [19]
    19. A process according to claim 4 wherein the process comprises the following steps:
    a) addition, stirring the lipase B of the Antarctic Candida, to a 0.1 M sodium phosphate buffer solution, in which the protein is added in a proportion of 0.3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in which is added Cu2S04-5H20 in a proportion of 10 mg per ml of buffer solution,
    c) incubation of the solution obtained in step b) for 16 h.
    c ') addition of sodium borohydride to its final concentration of 0.012 M,
    d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [20]
    20. A process according to claim 4 wherein the process comprises the following steps:
    a) Stirring addition of Antarctic Candida lipase B to a 0.1 M sodium phosphate buffer solution (pH 7), in which the protein is added in a proportion of 3 mg per ml of buffer solution,
    b) addition of Cu2S04-5H20 to the solution obtained in the previous step at 20-25 ° C, in which Cu2S04-5H20 is added in a proportion of 10mg per ml of buffer solution, c) incubation of the solution obtained in the stage b) for 3 days.
    c ') the optional addition of sodium borohydride to a final concentration of O, 12M, d) Collect, wash with water and dry by lyophilization the hybrid material obtained in the previous step.
    [21]
    21. Use of the material described in any of claims 1-3 as a catalyst.
    [22]
    22. Use according to claim 21 to degrade Bisphenol-A.
    [23]
    23. Use according to claim 21 for the reduction of 4-nitrophenol to 4-aminophenol.
    [24]
    24. Use according to claim 21 for the degradation of H202.
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    WO2020239673A1|2020-12-03|
    ES2795844B2|2021-07-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CN109529945B|2018-12-03|2019-11-08|清华大学|A kind of macromolecule-enzyme-metal composite nano catalyst and its controllable synthesis method|
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    PCT/EP2020/064407| WO2020239673A1|2019-05-24|2020-05-25|Hybrid material comprising a protein matrix and copper nanoparticles therein, process for preparing the same and use thereof|
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