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    • 专利摘要:
    Recyclable biological catalyst obtained from the black mass of discarded batteries for the synthesis of alkyl esters of volatile fatty acids. The present invention refers to a recyclable biological catalyst characterized in that it comprises binary oxide nanoparticles, with a spinel-like structure, silanized and comprising reactive amino groups on their surface and a glycoprotein with oxidized glycidic chains where the glycoprotein is covalently immobilized on the binary oxide through secondary amines and the procedure for obtaining said catalyst. Furthermore, the present invention relates to the use of said catalyst in the synthesis of alkyl esters of volatile fatty acids and to the process of synthesis of alkyl esters of volatile fatty acids. Therefore, the present invention is of interest in the urban solid waste management industry and, in turn, in the biocatalyst industry. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2785774A1
    申请号:ES201930303
    申请日:2019-04-03
    公开日:2020-10-07
    发明作者:Orzanco Alicia Prieto；Gutierrez Maria Molina；Gomez Felix Antonio Lopez；Diez Irene Garcia；Romo Lorena Alcaraz；Hernandez Maria Jesus Martinez
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0002] Recyclable biological catalyst obtained from black mass of discarded batteries for the synthesis of alkyl esters of volatile fatty acids
    [0004] The present invention refers to a recyclable biological catalyst characterized in that it comprises binary oxide nanoparticles of the formula, with a spinel-like structure, silanized and comprising reactive amino groups on their surface and a glycoprotein with oxidized glycid chains where the glycoprotein is covalently immobilized on binary oxide through secondary amines and the process for obtaining said catalyst. Furthermore, the present invention relates to the use of said catalyst in the synthesis of alkyl esters of volatile fatty acids and to the process of synthesis of alkyl esters of volatile fatty acids.
    [0006] Therefore, the present invention is of interest in the urban solid waste management industry and, in turn, in the biocatalyst industry.
    [0008] BACKGROUND OF THE INVENTION
    [0010] Short chain volatile fatty acid esters contribute to the natural flavor and aroma of fruits and vegetables and are widely used as additives in the pharmaceutical, cosmetic and food industries. Although they can be extracted from natural sources, the concentrations are very low. For this reason, for industrial purposes they are usually produced by chemical synthesis at high temperatures using non-selective catalysts. Under these conditions, unwanted side products are generated and esters cannot be labeled as natural products. Today, consumers' preference for natural foods and the pursuit of green and sustainable chemical processes have fueled interest in bio-based chemicals. The synthesis of esters by biocatalysis provides a solution to the above problems, since the reactions are specific, selective, clean and take place under mild conditions. Therefore, enzymatically produced esters comply with European and American regulations for natural compounds, with the expected annual growth rate of their global market being around 6.4% by 2016 2021. The lipases from the fungi Candida rugosa, Candida antarctica, Rhizopus oryzae, Thermomyces lanuginosus or Rhizomucor miehei are among the most biocatalytic frequently used for the synthesis of ester flavors and aromas such as butyl valerate.
    [0012] Among the main advantages of biological catalysts over chemical ones, we can mention their selectivity, their specificity, and their "green" character, since they are efficient under mild reaction conditions and do not generate toxic by-products. However, the cost of enzymes is high. Immobilization is one of the main tools to reduce the impact of biocatalysts on the final costs of bioprocesses. This strategy allows the enzyme to be separated from the rest of the components of the reaction mixture, using it in successive catalytic cycles without significant loss of activity and also facilitating the separation of the products. All this translates into less expensive, environmentally clean and simpler processes, which justifies the growing number of patents that protect applications related to the use of immobilized biocatalysts.
    [0014] The materials used as supports for immobilization can be organic, inorganic or hybrid. In general, inorganic ones are preferred because of their greater resistance, especially microbiological and mechanical, and because they can be chemically modified allowing the protein to be fixed by different procedures.
    [0016] Therefore, it is necessary to continue developing new materials for this purpose.
    [0018] BRIEF DESCRIPTION OF THE INVENTION
    [0020] The present invention proposes the use of materials derived from battery recycling as low-cost, sustainable inorganic support for the immobilization of glycoproteins with oxidized carbohydrate chains. The use of energy accumulators is essential for our daily activity, but, from an environmental perspective, it is essential both to properly manage these post-consumer products and to recover and reuse their valuable components, in this case, zinc and manganese that are part of the anode. and cathode of batteries and accumulators.
    [0022] In the present invention, discarded batteries of the alkaline and Zn / C type are used to synthesize a binary mixed oxide of Zn / Mn in nanometric size, with Zn 0 stoichiometry. 25 Mn 2 . 75 O 4 and tetragonal symmetry with space group I41 / amd corresponding to a spinel-like structure. Said binary oxide is used as a support for the covalent immobilization of an enzymatic crude with lipase activity in the present invention, specifically of a glycoprotein with oxidized carbohydrate chains; the glycoprotein is covalently immobilized on the binary oxide through secondary amines. The biological catalyst formed is recyclable because it maintains its catalytic activity throughout several reaction cycles carried out under the same conditions.
    [0024] In order to immobilize the glycoprotein with oxidized carbohydrate chains to binary oxide obtained from discarded batteries, this oxide must be silanized and have reactive amino groups on its surface; Said amino groups are those that react, in the presence of a reducing agent, with the aldehydes of the oxidized glycoprotein chains to form the covalent bond between the binary oxide and the glycoprotein in the form of secondary amines. The process for forming the recyclable biological catalyst of the present invention therefore comprises a stage of obtaining the binary oxide from discarded batteries, a stage of silanization and incorporation of amino groups on the surface of the binary oxide by means of silanation and a stage of immobilization covalent with the help of a reducing agent.
    [0026] The present invention further relates to the use of said recyclable biological catalyst in the synthesis of alkyl esters of volatile fatty acids comprising the transesterification and / or esterification of a volatile fatty acid with an alcohol in the presence of the catalyst of the invention.
    [0028] DETAILED DESCRIPTION OF THE INVENTION
    [0030] In a first aspect, the present invention refers to a recyclable biological catalyst (hereinafter "the catalyst of the present invention") characterized in that it comprises
    [0031] • a binary oxide with the formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15, with a spinel-like structure that is in the form of nano-sized particles, where said particles are silanized and comprise reactive amino groups on its surface, and
    [0032] • a glycoprotein with oxidized glycid chains,
    [0033] where the glycoprotein is covalently immobilized on the binary oxide through secondary amines.
    [0035] In a preferred embodiment of the recyclable biological catalyst of the present invention, said catalyst comprises:
    [0037] • a binary oxide of the formula Zn 0 , 25 Mn 2 , 75 O 4 , with a spinel-like structure that is in the form of nano-sized particles, where said particles are silanized and comprise reactive amino groups on their surface, and
    [0038] • a glycoprotein with oxidized glycid chains,
    [0040] where the glycoprotein is covalently immobilized on the binary oxide through secondary amines.
    [0042] The binary oxide of formula ZnxMnyO 4 , where x = 0.25-0.85 and y = 2.75-2.15, of the recyclable biological catalyst of the present invention has a spinel-like structure and is in the form of nano-sized particles. In a preferred embodiment of the catalyst of the present invention, the binary oxide is in the form of particles of size between 35 nm and 50 nm.
    [0044] In the recyclable biological catalyst of the present invention, the binary oxide of formula ZnxMnyO 4 , where x = 0.25-0.85 and y = 2.75-2.15, acts as a support for the glycoprotein with oxidized glycid chains. In order to support the glycoprotein, the binary oxide must be silanized and have reactive amino groups on its surface. By the term "reactants" is understood in the present invention as those amino groups capable of reacting with the aldehyde groups of the oxidized glycoprotein chains in the presence of a reducing agent. Preferably, the concentration of reactive amino groups on the surface of the The support is between 5 pmol / gparticle and 20 pmol / gparticle The glycoprotein is covalently immobilized on the binary oxide through the secondary amines formed.
    [0046] For the purposes of the present invention, covalent immobilization between the binary oxide comprising reactive amino groups on its surface and the glycoprotein occurs through the formation of secondary amines; in the presence of a reducing agent the Aldehydes of the oxidized glycoprotein chains react with the reactive amino groups found on the surface of the binary oxide of formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15, to form an imine (Schiff's base) which is subsequently stabilized by reductive amination to form a secondary amine. The immobilization provides a covalent attachment to the support at multiple points that results in improved catalyst efficiency. It should be noted that immobilization maintains the structural integrity of the enzyme, whose protein sequence does not intervene in the interaction with the binary oxide of the formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15 .
    [0048] The glycoproteins to which the present invention refers are glycoproteins from the group of carboxylic ester hydrolases (EC 3.1.1), preferably with triacylglycerol lipase activity (EC 3.1.1.3), generally known as lipases. Within the group of enzymes generally called lipases, there is a group known as "strict lipases" which are enzymes that only act on glycerides, and another group known as "versatile lipases" which, in addition to acting on glycerides, also act against esters other than glycerides. Similarly, some carboxyl esterases (EC 3.1.1.1) and sterol esterases (EC3.1.1.13) have broad substrate specificity and also exhibit lipase activity thanks to their activity against glycerides. In the particular case of sterol esterases with lipase activity, it has recently been proposed that they be called "versatile lipases" since some of them even have more activity on glycerides than on sterol esters.
    [0050] In a preferred embodiment, the glycoprotein is preferably any enzyme with lipase activity, more preferably it is a lipase, both strict and versatile, and / or a sterol esterase.
    [0052] Glycoproteins are proteins that naturally contain one or more carbohydrate chains that are covalently linked to certain asparagine, serine, and threonine during protein biosynthesis. In the case of its binding to asparagine, the amino group of the side chain is associated with the carbohydrate through a W-glycosidic bond, while with serine and threonine an O-glycosidic bond is established by condensing the hydroxyl of its side chains with another of the monosaccharide. The carbohydrate portion of the glycoproteins is highly variable, ranging from monosaccharides to long and complex chains of different composition, structure, and degree of branch.
    [0054] In a more preferred embodiment the glycoprotein is a versatile lipase naturally synthesized by the dimorphic filamentous fungus Ophiostoma piceae. Said native sterol esterase / lipase or versatile lipase is a glycoprotein of molecular mass 66 kDa that comprises 8% glycosylation.
    [0056] In the present invention, the term "sterol esterase gene" refers to a nucleotide sequence encoding the sterol esterase / lipase or versatile lipase from O. piceae and is identified by SEQ. ID NO: 1.
    [0058] For the purposes of the present invention, the terms, glycoprotein with lipase activity, sterol esterase / lipase and versatile lipase, are used interchangeably throughout the present invention and refer to the group of proteins that present lipase activity, being able to be strict lipases and / or versatile lipases. The native form of the sterol esterase / lipase or versatile lipase of the present invention comprises the complete amino acid sequence of the protein encoded by the sterol esterase gene of O. piceae, including the signal peptide (SEQ. ID NO: 2). The mature form of said enzyme comprises the amino acid sequence encoded by the sterol esterase gene, not including the signal peptide and is identified with SEQ ID NO: 3. Hereinafter, the native enzyme will be referred to as the mature form, without signal peptide, for differentiate it from recombinant forms.
    [0060] In the present invention, the terms "heterologous expression" and "heterologous gene" refer to the introduction of a foreign (heterologous) gene into an organism in order to modify its genetic material and expression products. For the purposes of the present invention, said organism is a methylotrophic yeast, preferably Pichia pastons.
    [0062] In a particular embodiment, the mature amino acid sequence encoded by the sterol esterase gene is expressed in the yeast P. pastoris, using the pre-propeptide of Saccharomyces cerevisiae factor a as signal peptide, giving rise to the versatile sterol esterase / lipase or lipase. recombinant described in the present invention. This versatile recombinant lipase presents modifications in its amino acid sequence, which comprises between 6-8 new amino acids at the N-terminal end, added on the amino acid sequence of the mature native protein. In a more preferred embodiment, the recombinant versatile lipase derived from O. piceae and expressed in the yeast P. pastoris (OPEr), comprises the sequence SEQ ID NO: 4 or SEQ ID NO: 5.
    [0064] In another preferred embodiment, the versatile lipase of the present invention is selected from the list consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In a more preferred embodiment , the versatile lipase enzyme is selected from the list consisting of: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
    [0066] For the purposes of the present invention, in addition to the versatile lipases of SEQ ID NOs: 3, 4 and 5, any person skilled in the art can use any native or recombinant glycoprotein of any biological origin with known lipase activity, such as native lipases or recombinants produced by fungi other than O. piceae, for example: Candida sp., Candida rugosa, Melanocarpus albomyces, Candida antárctica ( Cal A, Cal B), Thermomyces lanuginosus, Rhizopus oryzae, Mucor miehei, Aspergillus oryzae, Trichoderma reesei, Aspergillus niger, Nectria haematococca, or Plicaturiopsis crispa .
    [0068] In a more preferred embodiment, the glycoprotein, preferably the versatile lipase, immobilized on binary oxide nanoparticles of the formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15, with spinel-like structure according to described in the present invention, it is preferably the versatile lipase comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
    [0070] The term "glycoprotein with oxidized glycid chains" refers to a glycoprotein, as described above, which, through an oxidation reaction, has generated aldehyde groups in its glycid chains.
    [0072] Examples of oxidizing agents capable of generating aldehyde groups on glycoprotein glycid chains are
    [0074] - enzymes, which are selected from the list consisting of oxidoreductases, aldose oxidases, alcohol dehydrogenases and oxidases,
    [0076] - and oxidizing chemical agents that are selected from among 2,2,6,6-tetramethyl-1piperidinyloxy radical (TEMPO), Dess-Martin periodinate, lead tetraacetate Pb- (O 2 CCH 3 ) 4 , sodium bismuthate (NaBiO 3 ), periodic acid, periodates as sodium periodate (NaIO 4 ) and potassium periodate (KIO 4 ).
    [0078] Another aspect of the invention refers to the process for obtaining the catalyst of the present invention, characterized in that it comprises the following steps:
    [0079] a) obtain a binary oxide with the formula ZnxMnyO 4 , where x = 0.25-0.85 and y = 2.75-2.15, and a spinel-like structure by means of:
    [0080] a1) Leaching in a constant acid medium of black mass at a concentration of 300 g / l until obtaining a Zn concentration of between 27.5 g / L and 46 g / L and a Mn concentration of between 110 g / L and 127 g / L, discarding the resulting solids by filtration, and
    [0081] a2) selective alkaline precipitation of the solution resulting from the filtration of step (a1) using from 2.4 L to 3 L of alkaline medium in concentrations greater than 6 M,
    [0082] b) silanize and incorporate amino groups on the surface of the binary oxide obtained in step (a),
    [0083] c) oxidize the glycoprotein glycid chains, and
    [0084] d) covalently immobilize the binary oxide comprising reactive amino groups on its surface obtained in step (b) and the glycoprotein with oxidized carbohydrate chains obtained in step (c) with the help of a reducing agent by d1) reaction between aldehydes of the oxidized carbohydrate chains of the glycoprotein obtained in step (c) and the reactive amino groups of the surface of the binary oxide obtained in step (b) in the presence of a reducing agent, and
    [0085] d2) reductive amination of the imines formed in step (d1) with a reducing agent
    [0087] The binary oxide of the formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15, and spinel-like structure is obtained from the black mass obtained from discarded batteries.
    [0089] In the present invention, "black mass" is understood as that inorganic solid obtained in the dismantling stages of batteries and that contains electrolytes, graphite and manganese and zinc oxides that constitute the anode and cathode of batteries. The main components of batteries are manganese dioxide (electrode positive), zinc (negative electrode), electrolyte (KOH or ZnCl 2 + NH 4 CI) and steel (battery cover). These wastes generate great concern in society due to the negative impact on public health and the environment that they produce, mainly due to their high content of heavy metals. The mechanical separation constitutes the starting point of the obtaining of black masses and aims to separate the electrodes, steel, paper, and plastics through cutting-shredding stages, magnetic separation, dimensional separation (screening), ECS separation (using eddy currents) and grinding the dust fraction.
    [0091] In the present invention the binary oxide of formula ZnxMnyO 4 , where x = 0.25 - 0.85 and y = 2.75 - 2.15, and spinel-like structure is obtained from (step (a1)) a leaching in constant acid medium of black mass at a concentration of 300 g / l until obtaining a Zn concentration of between 27.5 g / L and 46 g / L and a Mn concentration of between 110 g / L and 127 g / L. The resulting solids are discarded by filtration. Preferably, the acid medium used in this step (a1) is an acidic solution of 25 % v / v of water, 25 % v / v of H 2 O 2 and 50 % v / v of HCl of 35 % purity. A lower percentage of H 2 O 2 does not adequately dissolve Mn and a higher percentage increases the foams in the dissolution process and causes the reactor to overflow.
    [0093] This step of leaching in an acid medium is followed by a selective alkaline precipitation (stage (a2)) using between 2.4 L to 3 L of alkaline medium in concentrations greater than 6 M. Preferably the alkaline medium of stage (a2) it is a solution of NaOH or KOH, preferably NaOH.
    [0095] The next stage of the process of the present invention is stage (b) relating to the silanization and incorporation of amino groups on the surface of the binary oxide of formula Zn 0.25 Mn 2.75 O 4 and spinel type structure. The reagent for silanation and amino functionalization is preferably selected from the list consisting of (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane, 3 [2- (2- 25-aminoethylamino) ethylamino] propyltrimethoxysilane, 3- ( 2-aminoethylamino) -propyldimethoxymethylsilane, [3- (2-aminoethylamino) propyl] trimethoxysilane, 3-aminopropyldimethylmethoxysilane and 3-aminopropyl (diethoxy) methylsilane.
    [0097] The oxidation of the glycid chains of a glycoprotein with lipase activity of step (c) is preferably carried out in the presence of an oxidizing agent. For purposes of the present invention, by oxidizing agent is understood any compound known in the art capable of oxidizing another in contact with it. Preferably the oxidizing agent is selected from those capable of generating aldehyde groups in carbohydrate chains. In yet another more preferred embodiment, the oxidizing agent capable of generating aldehyde groups in carbohydrate chains is selected from the list consisting of: enzymes, which are selected from the list consisting of oxidoreductases, aldose oxidases, alcohol dehydrogenases and oxidases, and agents oxidizing chemicals. In yet another more preferred embodiment of the present invention the oxidizing chemical agents are selected from 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO), Dess-Martin periodinate, lead tetraacetate Pb- (O 2 CCH 3 ) 4 , sodium bismuthate (NaBiO 3 ), periodic acid or periodates. Periodates are particularly preferred and more preferably sodium (NaIO 4 ) and potassium (KIO 4 ) periodate.
    [0099] According to a preferred embodiment, the oxidizing chemical agent is applied in liquid form, generally dissolved in aqueous solvent, which facilitates and enhances its oxidative action. According to a preferred embodiment, the concentration of the oxidizing agent solution is between 5 mM to 50 mM, more preferably 10 mM.
    [0101] Once aldehyde groups have been generated in the glucidic chains, in step (d1), the aldehydes of the oxidized glycoprotein chains of the glycoprotein obtained in step (c) react with the reactive amino groups on the surface of the binary oxide obtained in step ( b) to form imines (Schiff's base) in the presence of the reducing agent. The reducing agent of step (d1) is preferably selected from the list consisting of sodium borohydride (NaBH 4 ), lithium hydride, lithium aluminum hydride, sodium cyanoborohydride, lithium cyanoborohydride, borans such as trimethyl amino borane (TMAB) or the a-picolin borane, sodium triacetoxyborohydride, or sodium borohydride modified with polyvalent metal salts or activated by acids.
    [0103] If said reducing agent is applied in the form of a solution, the reducing action is enhanced, therefore, in a preferred embodiment of the present invention, the reducing agent is a solution of trimethyl amino borane in a concentration between 100 mM and 300 mM. Preferably a solution with a concentration of 150 mM.
    [0105] Finally, the imines formed in step (d1) are converted into stable secondary amines (covalent bond) by reductive amination in one step (d 2 ); said stage (d 2 ) It is therefore a stabilization stage where covalent bonds are formed in the form of secondary amines between the glycoprotein and the binary oxide. Preferably, the reducing agent of step (d2) is selected from among sodium borohydride (NaBH 4 ), lithium hydride, lithium aluminum hydride, sodium cyanoborohydride, lithium cyanoborohydride, borans or the a-picolin borane, sodium triacetoxyborohydride , or sodium borohydride modified with polyvalent metal salts or activated by acids.
    [0107] Another aspect of the present invention refers to the use of the catalyst of the invention for the synthesis of alkyl esters of volatile fatty acids, preferably for the synthesis of butyl valerate.
    [0109] By the term "volatile fatty acid alkyl ester" is understood in the present invention as that alkyl ester derived from short chain fatty acids or volatile fatty acids that are a subgroup of fatty acids with carbon chains of less than six carbon atoms, such as acetic acid. propionic acid. isobutyric acid. butyric acid, isovaleric acid, valeric acid and caproic acid, among others. Their volatility is due to the short carbon chain they possess, in contrast to long-chain fatty acids , which are solid at room temperature The part of the ester that is an alkyl is a short chain alkyl, preferably of between 4 and 7 carbon atoms.
    [0111] Another aspect of the present invention refers to the process for the synthesis of alkyl esters of volatile fatty acids that comprises the transesterification and / or esterification of a volatile fatty acid with an alcohol in the presence of the catalyst of the invention. Preferably the alcohol is a C 1 -C 4 short chain alcohol, more preferably the alcohol is butanol, even more preferably it is 1-butanol in the presence of isooctane.
    [0113] In a preferred embodiment of the volatile fatty acid alkyl ester synthesis process, the volatile fatty acid is valeric acid.
    [0115] In another preferred embodiment of the volatile fatty acid alkyl ester synthesis process, the transesterification and / or esterification reaction is carried out at a temperature of between 20 ° C to 50 ° C.
    [0116] In another preferred embodiment of the volatile fatty acid alkyl ester synthesis process, the molar ratio of the volatile fatty acid to the alcohol is between 1: 1 to 1: 3.
    [0118] 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.
    [0120] BRIEF DESCRIPTION OF THE FIGURES
    [0122] FIG. 1 Scheme of the process carried out to obtain binary oxides
    [0124] FIG. 2 X-ray diffraction patterns of binary metal oxides.
    [0126] FIG. 3 Recyclability of the biocatalyst G1-CH-OPEr in butyl valerate synthesis reactions.
    [0128] FIG. 4 Thermal stability of free and immobilized OPEr on the G1 support through its oxidized carbohydrate chains.
    [0130] FIG. 5 Stability at different pH values of free and immobilized OPEr on the G1 support through its oxidized carbohydrate chains.
    [0132] EXAMPLES
    [0134] The invention will now be illustrated by means of tests carried out by the inventors, which show the effectiveness of the product of the invention.
    [0136] Synthesis and characterization of binary metal oxides G1, G4 and G5
    [0138] In the present invention, binary metal oxides are synthesized starting from a mass black from the mechanical recycling of spent alkaline and Zn / C cells and batteries.
    [0140] The binary metal oxide called G1 corresponds to the product object of the present invention, while the products G4 and G5 are presented as comparative data.
    [0142] The synthesis of binary metal oxides with different Zn / Mn ratios was carried out from a waste generated in the recycling of alkaline cells and batteries through stages of (1) acid leaching (with HCl) of Zn and Mn , followed by (2) the precipitation of the cations Zn2 + and Mn2 + in an alkaline medium. In this way, Mn-Zn oxides with different Mn / Zn molar ratios (1,4-11) of the ZnxMn 3 -xO 4 type (0.25 <x> 1.25) and with a spinel-type crystalline structure can be obtained. . Figure 1 summarizes the process carried out.
    [0144] Disused cells and batteries were crushed under an atmosphere of N 2 . The resulting product was subjected to a magnetic separation step to remove the steel and finally, the plastic and paper components were separated by sieves. The product resulting from these stages is made up of the electrodes (graphite) and the electrolytes of the cells and batteries (zinc, manganese and potassium salts). We call this product the initial black mass. The analysis of the chemical composition by X-ray fluorescence (XRF) of the initial black mass is presented in Table 1.
    [0146] Table 1. Chemical composition of the starting black mass (% by weight)
    [0151] The black mass is mainly composed of Mn (36.8 wt.%) And Zn (23.7 wt.%). Mineralogically, the main crystalline phases are zincite (ZnO), hetaerolite (ZnMn 2 O 4 ) and sylvite (KCl), with wt% being the percentage by weight.
    [0153] The leaching of the black mass was carried out using 1 liter of a solution formed by 250 mL of milliQ water, 500 mL of hydrochloric acid (HCl, 32% -35% richness) and 250 mL of hydrogen peroxide (H 2 O 2, 39% purity) for different black mass / leaching solution (S / L). These tests were carried out at room temperature for one hour using mechanical stirring, 500 rpm and after that time, they were filtered using a Millipore Holder filter at 7 bar pressure.
    [0155] The spinel of formula Zn 0, 25 Mn 2 O 4 75 (G1), is obtained when dissolve 300 g of black mass in 1L of acid solution; we refer to a solution that is made up of a mixture of 25% v / v of water, 25% v / v of H 2 O 2 (30% by weight solution of H 2 O 2 in water) and 50% v / v of 35% pure HCl (11.33M). The pH of this solution is between 3.5 and 3.7.
    [0157] Table 2 summarizes the results obtained after the analysis by Atomic absorption spectroscopy (AAS) (SpectraAA 200 spectrometer, Varian) of the different solutions obtained depending on the different operating conditions. The pH of these solutions is in all cases between 0 and 4 units.
    [0159] Table 2. Mn and Zn content and efficiency of the different leaching.
    [0164] Zn and Mn were selectively precipitated from the leached solutions of each of the LG1, LG4 and LG5 nanoparticles, using a 6M NaOH solution until reaching a pH value of ~ 12-14. Precipitations were carried out at room temperature, constantly using mechanical stirring at 500 rpm. Once the basic pH was reached, the solution was filtered and the resulting solid was dried in an oven at 80 ° C for 24 h.
    [0166] The LG1 solution is precipitated with 6M NaOH solution to obtain product G1. The 6M NaOH solution consumption is between 2.4 and 3.0 L for each liter of LG1. For the LG4 solution the consumption is between 2.0 and 2.4 L of 6M NaOH and for the LG5 solution, between 1.1 and 1.4 L of 6M NaOH.
    [0168] The resulting solids (G1, G4 and G5) were analyzed by X-ray diffraction to determine their crystalline nature and stoichiometry, using refinements of the structure by Rietveld analysis. Table 3 shows the chemical composition (weight percent) in terms of binary oxides of each of the resulting solids G1, G4, and G5.
    [0170] Table 3. Chemical composition in terms of binary oxides (% by weight) of each resulting solid G1, G4 and G5.
    [0175] The composition in crystalline phases was examined by XRD. Figure 2 shows the X-ray diffraction patterns for each of the solids G1, G4 and G5, and the reflections that can be indexed to a tetragonal symmetry with space group I41 / amd corresponding to a spinel-like structure, in agreement with JCPDS, Card No. 24-1133.
    [0177] The data obtained by XRD were analyzed by the Rietveld method. The corresponding diffraction profiles were refined considering the type structure spinel, where the Zn atoms were placed in the 4a crystallographic positions occupying the tetrahedral sites while the Mn atoms occupy the octahedral sites in the 4d positions. Table 4 shows the composition in crystalline phases of the synthesized samples obtained from the Rietveld refinements, observing the variation in the Mn / Zn ratios. The different mixed Mn / Zn oxides obtained in the synthesis have a high purity (95-96%). Regarding the network parameters for the ZnMn 2 O 4 phase, the values of the parameter a are slightly higher than those found in the literature, which are between 5,709 Á and 5,722 Á, while the values of the parameter b (9,240 Á) are similar to those obtained in the literature (they vary between 9,222 and 9,238 Á).
    [0179] Table 4. Stoichiometry of synthesized Zn / Mn oxides obtained by analysis of the XRD results by the Rietveld method.
    [0181]
    [0184] Synthesis of G1-CH, G4-CH and G5-CH: Immobilization of OPEr lipase on G1, G4 and G5 nanoparticles.
    [0186] Biocatalysts were synthesized from the three nanomaterials G1, G4 and G5, using them as supports for the immobilization of enzymes, specifically as support for an enzymatic crude rich in OPEr lipase (SEQ ID NOs: 4 and 5). Immobilization was carried out through the oxidized carbohydrate chains of said lipase by the procedure explained below.
    [0188]
    [0191] The nanoparticles of binary oxide of the formula Zn 0 , 25 Mn 2 , 75 O 4 , with structure type spinel were silanized and functionalized with 3-aminopropyl triethoxysilane (APTES, Sigma), a compound that incorporates reactive amino groups on the surface of the nanoparticle. Based on the method described by Chen et al. (International Journal of Molecular Sciences 2013, 14: 4613-4628), 1 g of the binary oxide nanoparticles of the formula Zn 0 , 25 Mn 2 , 75 O 4 , with spinel-like structure, is mixed with 9.7 mL of ethanol in an ultrasound bath for 20 min. 300 p, L of APTES are added, sonicated for 10 min. Stir in a mixer at 80 rpm and 28 ° C for 16 h. Once this time has elapsed, the nanoparticles are separated by centrifugation and the reaction liquid is removed, washing the functionalized particles with the amino groups on their surface with 50 mL of 50% ethanol in water six times. Finally, binary oxide nanoparticles of formula Zn 0, 25 Mn 2 O 4 75, with spinel structure type amino-functionalized dried in an oven aeration at 65 ° C. The density of accessible amino groups in the particles is assessed by the procedure described by del Campo et al. (Journal of Magnetism and Magnetic Materials 2005, 293: 33-40), showing that they present surface accessible amino groups in the range of 5-20 ^ mol / g support.
    [0193] Oxidation of the carbohydrate chains of OPEr lipase
    [0195] For OPEr lipase (SEQ ID NOs: 4 and 5) to bind covalently to the amino-functionalized supports on its surface, it is necessary to oxidize their carbohydrate chains. Briefly, NaIO 4 in 5 mM Tris-HCl pH 7 is added to a lipase solution with 20 mg / mL of total proteins, at a final concentration of 10 mM. The reaction is maintained for 3 h at 4 ° C, in the dark and without stirring, and is dialyzed against 20 mM Tris-HCl pH 7 to eliminate low molecular mass reagents and by-products. The oxidation of the glucidic chains of the lipase is carried out to generate aldehyde groups in the sugars of said enzyme, and later, during the immobilization process on the amino-functionalized supports, to covalently link these reactive groups to the amino groups of the nanoparticles.
    [0197] Immobilization of lipase OPEr.
    [0199] Briefly, one starts with 1 g of functionalized G1-CH, G4-CH and G5-CH nanoparticles to which 20 mM Tris-HCl pH 7 is added, sonicating for 10 min. The buffer by centrifugation at 5000 x g. In the same vial, 100 mM Tris-HCl buffer pH 8 is added, shaken and then the volume of the lipase preparation equivalent to an activity of 1.2 U of activity per mg of amino-functionalized support GX-CH ( corresponding to 20 micrograms of total protein per mg of support), and trimethyl amino borane (TMAB) at a final concentration of 150 mM. Shake and keep stirring at 80 rpm and 28 ° C for 16 h. Subsequently, the supernatant is removed, measuring its residual activity. The activity was determined using a model substrate, p-nitrophenyl butyrate (pNPB), whose hydrolysis allows a simple colorimetric monitoring of the activity, both in the supernatants of the immobilization reactions and in the immobilized biocatalyst.
    [0201] 30 mL of NaBH 4 (1 mg / mL) are added to the magnetic nanoparticles with the OPEr lipase (SEQ ID NO: 4 and 5) immobilized and it is allowed to react for 1 h. Finally, the OPEr preparations (SEQ ID NOs: 4 and 5) immobilized on the three supports are washed sequentially with 100 mM Tris-HCl pH 7 and 20 mM Tris-HCl pH 7, resuspending them in the latter solvent for storage at 4 ° C.
    [0203] The results of the immobilization reaction are shown in Table 5. In all three cases, the amount of residual activity detected in the supernatant after immobilization was practically nil, suggesting that all the activity contributed had been immobilized. However, the specific activity of the three biocatalysts produced corresponds to between 15% and 61% of the total activity offered (1.2 U / mg). This is probably due to the partial inactivation of the enzyme under the immobilization conditions, which also seems to depend on the composition of the nanoparticles.
    [0205] Table 5. Results of the immobilization reaction of OPEr on G1, G4 and G5 nanoparticles.
    [0207]
    [0208]
    [0211] Butyl valerate synthesis catalyzed by G1-CH-OPEr, G4-CH-OPEr and G5-CH-OPEr biocatalysts.
    [0213] The activity of the three biocatalysts G1-CH-OPEr, G4-CH-OPEr and G5-CH-OPEr was tested in a synthesis reaction of biotechnological interest, specifically in the production of butyl valerate, responsible for certain natural aromas, by means of direct esterification. The reaction mixture, which did not contain water, consisted of 11 total U of activity (measured against pNPB), 100 mM valeric acid and 200 mM of 1-butanol in isooctane, maintaining the reaction at 25 ° C with rotary stirring at 100 rpm for 8 h. The results are shown in Table 6.
    [0215] Table 6. Percentages of esterification of 1-butanol and valeric acid catalyzed by the three biocatalysts G1-CH-OPEr, G4-CH-OPEr and G5-CH-OPEr tested.
    [0220] All preparations were active, although with considerable differences. The biocatalyst G1-CH-OPEr was the one that showed the highest activity in this experiment, and was selected to evaluate the effect of the concentration of substrates on the synthesis yield of the ester as well as the recyclability in this reaction.
    [0222] In the specific case of the G5 support and despite the fact that the efficiency of the esterification of 1-butanol and valeric acid was 76.3% ± 4.9 under the conditions tested, the specific activity of the biocatalyst G5-CH-OPEr is only 0.2 U / mg (See Table 5). For this reason, the following experiments were performed with the G1-CH-OPEr and G4-CH-OPEr biocatalysts.
    [0224] G1-CH-OPEr and G4-CH-OPEr storage stability
    [0225] Two procedures were tested for the storage of the G1-CH-OPEr and G4-CH-OPEr biocatilizers. On the one hand, the biocatalysts were kept in buffer at 4 ° C for 1 month after which their activity was verified. On the other hand, a quantity of the biocatalyst was lyophilized and its activity was tested after resuspending them in buffer for 1 h. In Table 7 we can see that by keeping the catalysts in buffer at 4 ° C the activity is maintained for both types of immobilization and support. However, after lyophilization there is a great loss of activity.
    [0227] Table 7. Buffer stability at 4 ° C and after lyophilization
    [0232] Recyclability of G1-CH-OPEr and G4-CH-OPEr biocatalysts
    [0234] The recyclability of the biocatalysts G1-CH-OPEr and G4-CH-OPEr selected for the synthesis of butyl valerate was also tested, using a 100 mM acid concentration. The separation of the biocatalysts was carried out by centrifugation at 20,000 x g, washing the nanoparticles with Tris-HCl buffer and isooctane between cycles. This centrifugation speed is necessary for the biocatalyst to settle and be able to recover it for recycling. However, centrifugation causes a certain compaction of the biocatalyst, which can affect its activity. On the other hand, the biocatalyst G4-CH-OPEr sediments poorly even at this rate, so it is more difficult to recover.
    [0236] Despite everything, the results shown in Figure 3 allow us to verify the recyclability of both biocatalysts. These substrates are small in size and do not seem to present problems to diffuse despite the compaction of the biocatalyst, which is visible to the naked eye. With the G1-CH-OPEr biocatalyst, practically 100% esterification is achieved over 5 reaction cycles. The G4-CH-OPEr biocatalyst maintains 80-86% esterification in the first three cycles, but this value decays in subsequent cycles. For this reason, the G1-CH-OPEr biocatalyst was selected for subsequent trials.
    [0238] Effect of the concentration of the substrates on the activity of G1-CH-OPEr in the synthesis of butyl valerate
    [0240] The effect of the concentration of the substrates 1-butanol and valeric acid was tested, maintaining all the conditions of the initial experiment, including the molar ratio of the substrates (1: 2). The yield of catalyzed synthesis in each case is shown in Table 8.
    [0242] In view of the results, we can say that this biocatalyst is capable of synthesizing butyl valerate in the range of concentrations tested, although the efficiency of esterification decreases with increasing concentration of the substrates.
    [0244] Table 8. Percentages of esterification of different concentrations of 1-butanol and valeric acid (2: 1 molar ratio) catalyzed by G1-CH-OPEr.
    [0249] Stability of free OPEr lipase and G1-CH-OPEr biocatalyst at temperature and pH
    [0251] The thermostability of OPEr lipase (SEQ ID NOs: 4 and 5) was evaluated between 25 and 70 ° C, with the catalyst free (dissolved) or immobilized through its carbohydrate chains on the G1 support resuspended in Tris-HCl 20 buffer. mM at pH 7. Stability against different pH values was determined at 4 ° C in Britton & Robinson buffer adjusted to pH between 2 and 10. In both cases, 0.25 mg of catalyst was stirred, dissolved or resuspended in 500 pL of buffer, for 24 hours at 1200 rpm in a thermoblock at each of the temperatures and the pH analyzed. The residual activity was measured, taking the value obtained at 25 ° C and pH 7, respectively, as 100%.
    [0252] The results presented in Figure 4 show that the immobilization of the OPEr lipase with the support (G1-CH-OPEr) and the procedure presented here significantly improves the thermal stability of the glycoprotein, conserving 80% of activity after 24 h at 50 ° C and 45% at 60 ° C, while the free OPEr lipase (SEQ ID NO: 4 and 5) is little or not active under these conditions.
    [0254] Regarding the stability at pH, in Figure 5 it is observed that the activity of the biocatalyst G1-CH-OPEr remains at values close to 100% between pH 3 and 10, unlike the free enzyme (SEQ ID NOs: 4 and 5), whose activity decays below pH 6 and above pH 8.
    权利要求:
    Claims (21)
    [1]
    1. A recyclable biological catalyst characterized in that it comprises
    • a binary oxide with the formula ZnxMnyO 4 , where x = 0.25-0.85 and y = 2.75-2.15, with a spinel-like structure that is in the form of nano-sized particles, where said particles are silanized and comprise reactive amino groups on its surface, and
    • a glycoprotein with oxidized glycid chains,
    where the glycoprotein is covalently immobilized on the binary oxide through secondary amines.
    [2]
    2. The catalyst according to claim 1, wherein the binary oxide is in the form of particles of size between 35 nm and 50 nm.
    [3]
    3. The catalyst according to any of claims 1 or 2, wherein the concentration of reactive amino groups on the surface of the binary oxide is between 5 pmol / gparticle and 20 pmol / gparticle.
    [4]
    4. The catalyst according to any one of claims 1 to 3, wherein the glycoprotein is selected from a lipase and / or a sterol esterase with lipase activity.
    [5]
    5. The catalyst according to claim 4, wherein the versatile lipase is selected from any of the list consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
    [6]
    6. A process for obtaining the recyclable biological catalyst according to any of claims 1 to 5, characterized in that it comprises the following steps: a) obtaining a binary oxide of the formula ZnxMnyO 4 , where x = 0.25-0.85 and y = 2.75-2.15, and spinel-like structure by:
    a1) Leaching in a constant acid medium of black mass at a concentration of 300 g / l until obtaining a Zn concentration of between 27 g / L and 46 g / L and a Mn concentration of between 110 g / L and 127 g / L, discarding the resulting solids by filtration, and
    a2) selective alkaline precipitation of the solution resulting from the filtration of step (a1) using between 2.4 L to 3 L of alkaline medium in concentrations greater than 6 M,
    b) silanize and incorporate amino groups on the surface of the binary oxide obtained in step (a),
    c) oxidize the glycoprotein glycid chains, and
    d) covalently immobilizing the binary oxide comprising reactive amino groups on its surface, obtained in step (b) and the glycoprotein with oxidized carbohydrate chains obtained in step (c) with the help of a reducing agent through d1) reaction between the aldehydes of the oxidized glycoprotein chains of the glycoprotein obtained in step (c) and the reactive amino groups of the surface of the binary oxide obtained in step (b) in the presence of a reducing agent, and
    d2) reductive amination of the imines formed in step (d1) with a reducing agent
    [7]
    7. The process according to claim 6, wherein the acid medium of step (a1) is an acidic solution of 25% v / v of water, 25% v / v of H 2 O 2 and 50% v / v of HCl 35% purity.
    [8]
    8. The process according to any one of claims 6 or 7, wherein the alkaline medium of step (a2) is a solution of NaOH or KOH.
    [9]
    The process according to any one of claims 6 to 8, wherein the silanation and amino-functionalization reagent of step (b) is selected from the list consisting of (3-aminopropyl) triethoxysilane (APTES), (3- aminopropyl) trimethoxysilane, 3 [2- (2- (2- 25-aminoethylamino) ethylamino] propyltrimethoxysilane, 3- (2-aminoethylamino) -propyldimethoxymethylsilane, [3- (2-aminoethylamino) propyl] trimethoxysilane, 3-aminopropyldimethylmethoxysilane and 3-aminopropyl (diethoxysilane) methylsilane.
    [10]
    The process according to any one of claims 6 to 9, wherein the oxidizing agent of step (c) is selected from the list consisting of oxidoreductases, aldose oxidases, alcohol dehydrogenases and oxidases.
    [11]
    The process according to any one of claims 6 to 10, wherein the oxidizing agent of step (c) is selected from the list consisting of 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO), Dess-Martin periodinate, lead tetraacetate Pb- (O 2 CCH 3 ) 4 , sodium bismuthate (NaBiÜ 3 ), periodic acid and periodates.
    [12]
    12. The process according to any of claims 6 to 11, wherein the reducing agent of step (d1) is selected from the list consisting of: sodium borohydride (NaBH 4 ), lithium hydride, lithium aluminum hydride , sodium cyanoborohydride, lithium cyanoborohydride, trimethyl amino borane (TMAB), a-picolin borane, sodium triacetoxyborohydride, and sodium borohydride modified with polyvalent metal salts or activated by acids.
    [13]
    The method according to claim 12, wherein the reducing agent is a solution of trimethyl amino borane in a concentration between 100 mM and 300 mM.
    [14]
    The process according to any one of claims 6 to 13, wherein the reducing agent of step (d2) is selected from the list consisting of: sodium borohydride (NaBH 4 ), lithium hydride, lithium aluminum hydride , sodium cyanoborohydride, lithium cyanoborohydride, trimethyl amino borane (TMAB), a-picolin borane, sodium triacetoxyborohydride, and sodium borohydride modified with polyvalent metal salts or activated by acids.
    [15]
    15. Use of the catalyst according to any of claims 1 to 5 in the synthesis of alkyl esters of volatile fatty acids, preferably for the synthesis of butyl valerate.
    [16]
    16. Process for the synthesis of alkyl esters of volatile fatty acids comprising the transesterification and / or esterification of a volatile fatty acid with an alcohol in the presence of a catalyst according to any of claims 1 to 5.
    [17]
    17. Process according to claim 16, wherein the alcohol is a C1-C4 short chain alcohol.
    [18]
    18. Process according to any of claims 16 or 17, wherein the alcohol is 1-butanol in the presence of isooctane.
    [19]
    19. Process according to any one of claims 16 to 18, wherein the volatile fatty acid is valeric acid.
    [20]
    20. Process according to any one of claims 16 to 19, wherein the transesterification and / or esterification reaction is carried out at a temperature of between 20 ° C to 50 ° C.
    [21]
    21. Process according to any one of claims 16 to 20 wherein the molar ratio of the volatile fatty acid and the alcohol is between 1: 1 to 1: 3.
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    引用文献:
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    EP0274798A2|1986-12-19|1988-07-20|Unilever N.V.|Process for the preparation of esters|
    US20120065419A1|2009-03-02|2012-03-15|Arkema France|Method for producing ricinoleic acid ester by selective enzymatic transesterification|
    WO2010151085A2|2009-06-25|2010-12-29|Industry-Academic Cooperation Foundation, Yonsei University|Zinc-containing magnetic nanoparticle-based magnetic separation systems and magnetic sensors|
    CN105241938A|2015-09-14|2016-01-13|盐城工学院|Construction method and detection method of potassium ion nucleic acid aptamer photoelectrochemical sensor based on diluted magnetic semiconductor|
    WO2019002658A2|2017-06-28|2019-01-03|Consejo Superior De Investigaciones Científicas |Synthesis of biodiesel catalysed by an enzymatic crude immobilised on magnetic particles|
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