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    • 专利摘要:
    Enzymatic derivative based on enzyme immobilized on laminar zeolites. The present invention relates to an enzymatic derivative comprising a hydrolytic enzyme, preferably naringinase, immobilized through covalent bonds on laminar zeolites, as well as its preparation method and its uses, preferably as a biocatalyst. The immobilization of naringinase by the described method is able to increase its thermal stability, its stability at different pHs, its catalytic activity and its affinity for the substrate, with respect to the free enzyme without immobilizing. Furthermore, the enzymatic derivative of the invention is capable of being reused in more than 20 consecutive cycles without diminishing its catalytic activity, showing a catalytic stability superior to that observed when using other different supports. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2711819A1
    申请号:ES201731285
    申请日:2017-11-03
    公开日:2019-05-07
    发明作者:Canós Avelino Corma；Chornet Sara Iborra；Carceller José Miguel Carceller；Marco Filice；Juliana Cristina Bassan；Galán Julián Paul Martínez；Rubens Monti
    申请人:Universidade Estadual Paulista Julio de Mesquita Filho UNESP；Consejo Superior de Investigaciones Cientificas CSIC；Universidad Politecnica de Valencia；
    IPC主号:
    专利说明:

    [0001]
    [0002] Enzymatic derivative based on enzyme immobilized on laminar zeolites
    [0003]
    [0004] The present invention relates to a hydrolytic enzyme, naringinase, both commercial and purified forms, immobilized through covalent bonds on lamellar zeolites and their characterization as a biocatalyst. Naringinase is distinguished by being a complex with two activities, an a-rhamnosidase and another pglucosidase capable of releasing rhamnose and glucose respectively from the substrate. The present invention gives the enzyme greater thermal stability, greater stability against pH, greater affinity for the substrate and increases the catalltic activity.
    [0005]
    [0006] BACKGROUND
    [0007]
    [0008] Examples of immobilized naringinase on mesoporous silicates such as MCM-41 are found in the literature where the enzyme is covalently immobilized by crosslinking with glutaraldehyde (Applied Surface Science, (2011), 257: 4096-4099). A similar enzyme called p-glucosidase was immobilized by electrostatic bonds on SBA-15 (J. Porous Mater, (2010), 17: 657-662). The use of different organic supports has also been described, such as: cellulose triacetate films (Journal of Food Science, (1998), 63: 61-65), taninaminohexylcellulose supports (Agric. Biol. Chem., (1978), 42 (10): 1847-1853) and wood chips (J. Chem. Tech. Biotech., (2005), 80: 1160-1165). Other strategies used in the immobilization of naringinase, have been the obtaining of a sol-gel formed by polyvinyl alcohol (PVA) (Appl. Biochem. Biotechnol., (2010), 160: 2129 2147; Food Chemistry, (2007), 104: 1177-1182), as! such as enzymatic immobilization known as entrapment of the enzyme, in sodium or calcium alginate beads (World Journal of Microbiology & Biotechnology, (1999), 15: 501-502; Indian Journal of Chemical Technology, (2003), 10: 701- 704; Enzyme Microb. Technol., (1996), 18: 281-285).
    [0009]
    [0010] Two important aspects that the supported enzymes must possess for industrial use in biotechnological applications are: that the immobilized enzyme must have a high affinity for the substrate and that in addition the enzyme derivative must be highly stable, maintaining its catallic activity during several reaction cycles . Despite the multiple works related to immobilization of naringinase on different supports the use of these laminar materials to covalently immobilize this enzyme had not been described until then and proved as a biocatalyst.
    [0011]
    [0012] The immobilization of naringinase on silicate materials such as MCM-41 has been carried out satisfactorily (Applied Surface Science, (2011), 257: 4096 4099) however in this work 65% of the initial activity of the biocatalyst is lost after 6 reuses. Another material used for the immobilization of a glucosidase, (J. Porous Mater, (2010), 17: 657-662) was the SBA-15 and in this work it is shown that the enzyme loses affinity for the substrate (Glc-pNP) .
    [0013]
    [0014] With respect to the catalytic stability of the immobilized naringinase-based enzymatic derivatives on different supports, a loss of activity after consecutive reuse has been observed on supports such as: alginate-PVA spheres (Appl. Biochem. Biotechnol. (2010), 160: 2129-2147, PVA-Cryogel (Food Chemistry, (2007), 104, 1177-1182), and with other supports such as adsorption or adsorption / cross-linking with glutaraldehyde, (European Food Research and Technology, (2006), 224, 55-60); (Enzyme and Microbial Technology, (2007), 40, 442 446).
    [0015]
    [0016] In view of the foregoing, there is a need in the state of the art to provide alternative enzymatic derivatives able to increase the yield of the processes, present an increase in their stability with respect to different temperatures and pHs, as well as to allow a simple and efficient recovery of the enzyme for future reactions, without the activity and speed of the same being diminished along the consecutive reaction cycles to which it can be subjected to said enzyme.
    [0017]
    [0018] DESCRIPTION OF THE INVENTION
    [0019]
    [0020] The present invention relates to an enzymatic derivative which can comprise at least one hydrolytic enzyme, preferably a naringinase (EC 3.2.1.40) covalently immobilized on lamellar zeolites, preferably where the zeolite is selected from ITQ2 or ITQ6, more preferably the Zeolite is functionalized with aldehyde groups, giving rise to GITQ2 or GITQ6. The hydrolytic enzyme is preferably selected from any of the following list commercial naringinase, purified naringinase, from the fungus Penicilliun decumbens, and combinations thereof, as well as its method of obtaining and using it.
    [0021]
    [0022] According to the preferred application, two enzymatic derivatives consisting of the commercial naringinase enzyme (Crude) or purified (Pure), covalently immobilized on zeolites of laminar structure (ZD), preferably ITQ2, and more preferably where said zeolite is functionalized, have been prepared. preferably with aldehyde groups, GITQ2. In the present invention it is demonstrated that, the naringinase enzyme supported on laminar zeolites, preferably laminar zeolites functionalized with aldehyde groups, more preferably the zeolite is GITQ2, shows advantages with respect to the free enzyme in terms of its stability, its catalytic activity and its affinity by the substrate.
    [0023]
    [0024] In the present invention, the use of zeolites with a high external surface, such as laminar zeolites, is proposed as a support for the covalent immobilization of naringinase, both of the commercial enzyme and of the purified enzyme. Laminar zeolites are crystalline materials with an external surface area greater than or equal to 350 m2g-1. The laminar zeolites used in the present invention are preferably formed of monolamines, 2.5 nm thick and bilamines, which have a high external and accessible surface area (> 600 m2g "1) .The nanolayers are structured by a hexagonal distribution of" Cups "present on both sides of the zeolite leaves These limes are delimited by rings of 12 members, which show an external opening of approximately (0.7 x 0.7) nm being connected with the cups on the opposite side, in the same sheet of zeolite, through a double hexagonal prism with 6-membered rings Additionally, sinusoidal channels delimited by 10-membered rings are present around the calyxes, along the internal part of each individual layer.To immobilize the enzyme, these supports have been modified by the anchoring of aldehyde groups, with which the enzymes can react giving rise to covalent bonds, thus forming a layer of stable enzyme also limiting the leaching of the enzyme in the reaction medium.
    [0025]
    [0026] According to the present invention, the purification of the enzyme naringinase consists of a single purification step using ion exchange chromatography by the technique of "batch." These conditions allow the complete separation and purification of the naringinase present in the commercial starting extract.
    [0027]
    [0028] The functionalization of the lamellar zeolite with aldehyde groups is carried out by treating the material, previously activated in vacuum at 200 ° C for 2 h, with 3-aminopropyltriethoxysilane in a zeolite / 3-aminopropyltriethoxysilane weight ratio of between 0.1-15 at the reflux of Anhydrous toluene for 1 to 72 h at 50-200 ° C. Subsequently, the solid is filtered and washed with toluene and hexane, and after drying at 25 ° C the material functionalized with amino groups (material N-ZD) is obtained. The functionalization with aldehyde groups is carried out by treatment under magnetic agitation of the N-ZD material with a NaH2P04 buffer at pH = 7-10 of a 200mM concentration containing glutaraldehyde in a percentage between 1-95% at room temperature for a period between 5-48 h. After this time, the material is filtered and washed with a NaH2PO4 buffer solution of 500mM concentration at pH = 7-10. The material is subsequently dried at a temperature of 25 ° C, thus obtaining! the laminar zeolite functionalized with aldehyde groups (G-ZD). In a preferred embodiment, the lamellar zeolite functionalized with aldehyde groups is GITQ2.
    [0029]
    [0030] For this invention it was observed that by supporting the enzyme naringinase on the laminar zeolite ITQ2 (GITQ2), the enzyme not only maintains its activity but also has better catallic characteristics compared to those reported for other supports. These improvements are: an affinity for the substrate superior to the free enzyme and superior to other enzymatic derivatives previously described in the bibliography (see Table 1) and also presents a catallotic stability superior to that observed with other enzymatic derivatives based on naringinase immobilized on other supports .
    [0031]
    [0032] As discussed above, the enzyme derivative has been shown to have a relative activity higher than that of the free enzyme. For example, at pH between 4 and 6, the relative activity is 5-17% higher than that of the free enzyme. The maximum relative activity of the enzyme according to the present invention corresponds to an incubation pH between 4 and 5. In addition, the temperature can also affect the relative activity. According to a preferred embodiment, the enzymatic derivative has a relative activity higher than 30-70% at 50 ° C and higher than 70-80% at 90 ° C with its maximum relative activity in a range of temperatures preferably between 50 ° C and 80 ° C. ° C.
    [0033] Furthermore, as described in the present invention, the enzyme derivative has also been shown to have a greater catalytic stability than the free enzyme. Thus, the enzymatic derivative possesses a catalytic stability between 80 ° C and 100 ° C superior to the crude and pure free enzyme. Specifically, it can have a catalytic stability at 80 ° C between 7 and 9% higher than the pure and crude free enzyme respectively and 100% higher than the crude and pure free enzyme at a temperature of 100 ° C. According to the present invention, the enzyme derivative can have a Michaelis-Menten constant (Km) between 0.1 and 1mM. In addition, the enzyme derivative may have high affinity for the specific substrate, with a lower KM for the free enzyme (crude and pure).
    [0034]
    [0035] It is important to note that the enzymatic derivative, according to the present invention, can be reused. Specifically, it can be used in more than 20 cycles without reducing its catalytic activity. After each reaction cycle, the enzyme derivative is washed with the buffer solution and reused in a new reaction.
    [0036]
    [0037] Thus, a first aspect of the invention relates to an enzymatic derivative characterized in that it comprises at least one enzyme naringinase (EC 3.2.1.40) immobilized covalently on a support of zeolites of laminar structure.
    [0038]
    [0039] In a preferred embodiment, the enzymatic derivative is characterized in that naringinase is derived from the fungus Penicilium decumbens.
    [0040]
    [0041] In another preferred embodiment, naringinase is selected from the list consisting of commercial naringinase, purified naringinase or combinations thereof.
    [0042]
    [0043] In another preferred embodiment, the enzymatic derivative of the invention is characterized in that the layered structure zeolite is selected from ITQ2 or ITQ6.
    [0044]
    [0045] In another preferred embodiment, the enzyme derivative of the invention is characterized in that the naringinase is in a ratio by weight enzyme: support of between 0.2 to 300 mg of enzyme per gram of support, more preferably between 0.2 to 70 mg of enzyme per gram of support.
    [0046]
    [0047] In another preferred embodiment, the enzymatic derivative of the invention is characterized in that it has a KM between 0.1 and 1mM.
    [0048] Another aspect of the present invention relates to the method of obtaining the enzymatic derivative described above and which may comprise, at least, the following steps:
    [0049] a) Contact the support of laminar zeolite with a compound derived from an aminoalkyltriethoxysilane,
    [0050] b) Filter, wash and dry the support obtained in step a),
    [0051] c) Functionalize the support of stage b) with aldehldo groups, d) Filter, wash and dry the functionalized support obtained in step c), e) Dissolve the enzyme naringinase (EC 3.2.1.40) in a buffer solution, f) Contact the support of step d) with the enzyme from step e), g) Filter, wash and dry the enzyme derivative obtained in step f).
    [0052]
    [0053] In a preferred embodiment, the method of obtaining the derivative of the invention is characterized in that the weight ratio of the laminar zeolite: 3-aminopropyltriethoxysilane of step a) ranges from 0.1 to 15.
    [0054]
    [0055] In another preferred embodiment, the process for obtaining the derivative of the invention is characterized in that the aminoalkyltriethoxysilane derivative of step a) is preferably 3-aminopropyltriethoxysilane.
    [0056]
    [0057] In another preferred embodiment, the process for obtaining the derivative of the invention is characterized in that step a) is carried out for a time between 1 to 72 h, preferably 24 h and at a temperature between 50 ° C to 200 ° C , preferably 120 ° C.
    [0058]
    [0059] In another preferred embodiment, the process for obtaining the derivative of the invention is characterized in that it is carried out in the presence of toluene and hexane. In another preferred embodiment, the drying of step b) is carried out at a temperature of 25 ° C.
    [0060]
    [0061] In another preferred embodiment, the method of obtaining the derivative of the invention is characterized in that the functionalization of step c) is carried out by treating the support with glutaraldehyde at a preferred percentage of between 1 95%, in the presence of a buffer solution. , preferably NaH2P04, at a concentration preferably between 25 to 200 mM and a preferred pH of between 3 to 11, more preferably between 7 to 10, at room temperature, by magnetic agitation and for a preferred time of between 5-48h.
    [0062] In another preferred embodiment, the process for obtaining the derivative of the invention is characterized in that the washing of step d) is carried out in a buffer solution preferably of NaH 2 PO 4, at a concentration preferably between 25 to 500 mM and at a pH preferred from 7 to 10.
    [0063]
    [0064] In another preferred embodiment, the method of obtaining the derivative of the invention is characterized in that the naringinase enzyme of step e) is dissolved in a buffer solution at a concentration of between 1 to 200 mM and at a pH of between 3 to 11.
    [0065]
    [0066] In another preferred embodiment, the process for obtaining the derivative of the invention is characterized in that step f) is carried out under stirring for a time of between 0.1 to 24 hours and at a temperature of between 4 ° C to 80 ° C, preferably between 25 ° C to 50 ° C.
    [0067]
    [0068] In another preferred embodiment, the method of obtaining the derivative of the invention is characterized in that the enzyme: support ratio is between 0.2 and 300 mg of enzyme per gram of support, preferably between 0.2 to 70 mg of enzyme per gram of support.
    [0069]
    [0070] Enzymatic derivatives of naringinase (crude and purified) immobilized on G-ZD, preferably on ITQ2 (GITQ2), were obtained by contacting the corresponding enzyme with the support in an enzyme / support ratio between 0.2 and 300 mg of enzyme per gram of support, in a buffer solution with a concentration between 1 and 200 mM. The mixture was subjected to gentle agitation at a pH between 3 and 11, preferably between 7 and 10. The incubation of the enzyme with the support is carried out for a time between 0.1 and 24 h at a temperature between 4 and 80 oC, preferably between 25-50oC. The materials obtained were characterized and the optimum reaction, pH, Ta and thermal stability conditions were evaluated. Kinetic parameters were also studied to determine the affinity of the enzymatic derivatives for the substrate.
    [0071]
    [0072] By "substrate" is meant any substance that contains a precursor of a glycoslide nature of a sugar component such as rhamnose and glucose.
    [0073]
    [0074] From the KM values obtained, it was deduced that the affinity for the substrate of the synthesized materials is greater than that of the enzymes (crude and pure) in free form.
    [0075] The apparent Km of free naringinase and immobilized on different supports or materials has been calculated by several authors in order to assess the affinity for the substrate. In order to compare the increase in the affinity of naringinase (raw and pure) immobilized on G-ZD, where the ZD is preferably ITQ2 (GITQ2), with other supports, the quotient between the Km of the free enzyme and the Km of the immobilized enzyme (CKm). The value of this quotient shows that the affinity for the substrate is greater for the enzymatic derivatives of the present invention, than the affinity described in the case of the immobilization of naringinase on other materials (see Table 1).
    [0076]
    [0077] Table 1. Quotients of the KM
    [0078]
    [0079]
    [0080]
    [0081] CKm = Quotient between the Km of the free enzyme and the Km of the immobilized enzyme
    [0082]
    [0083] Another aspect of the invention relates to the hydrolysis process of glycosides in the presence of the enzymatic derivative of the invention, characterized in that it comprises the following steps:
    [0084] a) Dissolve at least one glycoside in a buffer solution at pH between 4 to 7 and at a temperature between 50 to 70 ° C for at least 10 minutes, preferably under agitation.
    [0085] b) Cool the dissolution of stage a),
    [0086] c) Contacting the enzymatic derivative of the invention with the dissolution of step b),
    [0087] d) Heat the mixture of section c) to a temperature comprised between 50 to 100 ° C, for a time comprised between 5 to 60 minutes,
    [0088] e) Separate by centrifugation the solid of step d) and wash it in a buffer solution,
    [0089] f) Recover the supernatant of step d) and analyze its composition.
    [0090]
    [0091] In a preferred embodiment of the hydrolysis process, this is characterized in that glycosides are selected from the list comprising flavonoids, ramnollpidos and glycopeptides. In another more preferred embodiment, the flavonoids are selected from the list comprising naringin, hesperidin and / or combinations thereof. In another still more preferred embodiment, the flavonoid is naringin.
    [0092]
    [0093] In another preferred embodiment of the hydrolysis process, it is characterized in that the glycoside: enzyme weight ratio, considering the amount of enzyme on the support, ranges from 2 to 10, preferably from 2 to 5.
    [0094]
    [0095] In another preferred embodiment of the hydrolysis process, this is characterized in that the temperature of step d) ranges from 50 to 80 ° C.
    [0096]
    [0097] In another preferred embodiment of the hydrolysis process, this is characterized in that the time of step d) ranges from 5 to 30 minutes.
    [0098]
    [0099] In another preferred embodiment of the hydrolysis process, this is characterized in that the solid obtained in step e) comprises the enzymatic derivative of the invention, which can be reused again.
    [0100]
    [0101] In another preferred embodiment of the hydrolysis process, this is characterized in that the supernatant of the invention comprises the hydrolyzed compounds, preferably flavonoids, free sugars and intermediate glycosides.
    [0102] In another preferred embodiment of the hydrolysis process of glycosides, this is characterized in that when the derivative of the invention comprising raw naringinase is used, the molar composition of flavonoids, free sugars and intermediate glycosides, is comprised between 10 and 25% molar of flavonoids, 25-60% of free sugars and 10-25% of intermediate glycosides.
    [0103]
    [0104] In another preferred embodiment of the glycoside hydrolysis process, this is characterized in that when the derivative of the invention comprising pure naringinase is used, the molar composition of flavonoids, free sugars and intermediate glycosides is between 3 and 10 mol% of flavonoids, 30-60% of free sugars and 25-40% of intermediate glycosides.
    [0105]
    [0106] Another aspect of the invention relates to the use of the enzymatic derivative described herein as a biocatalyst.
    [0107]
    [0108] The term "biocatalyst", as used herein, refers to any biological entity, preferably an enzyme, capable of catalyzing the conversion of a substrate into a product, in this case the biotransformation of glycosides into sugars.
    [0109]
    [0110] The term "immobilized biocatalyst" refers to enzymes, which are in such a state that they can be reused. It is related to the physical confinement of an enzyme so that its catalltic activity is retained and can be reused. In a preferred embodiment of the present invention, the immobilized biocatalyst is an enzyme.
    [0111]
    [0112] The present invention also relates to the use of the enzymatic derivative described in the present invention for the selective hydrolysis of substrates, preferably glycosides, more preferably glycosides which are selected from the list consisting of flavonoid glycosides, glycosides glycoside and glycopeptides, more preferably flavonoid glycosides .
    [0113]
    [0114] According to a preferred embodiment, the substrate can be a flavonoid glycoside selected preferably from naringin, hesperidin, rutin and combinations thereof, and more preferably is naringin.
    [0115] In another preferred embodiment, the use of the derivative of the invention for the hydrolysis of glycosides is carried out at a pH of between 4 to 7, preferably between 4 to 5. In another preferred embodiment, the hydrolysis is carried out at a temperature between 50 ° C to 100 ° C, preferably between 50 ° C to 80 ° C.
    [0116]
    [0117] According to a particular embodiment, when the enzyme derivative comes from the purified naringinase, the a-rhamnosidase activity increases, obtaining rhamnose and prunin preferably in the hydrolysis of naringin.
    [0118]
    [0119] According to another particular embodiment, when the enzymatic derivative comes from the naringinase of the commercial Penicilliun decumbens fungus (without purifying), the activity of pglucosidase and a-rhamnosidase are similar (50/50) obtained after the hydrolysis of naringinase, preferably rhamnose, glucose and naringenin.
    [0120]
    [0121] Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be apparent in part from the description and in part from the practice of the invention.
    [0122]
    [0123] BRIEF DESCRIPTION OF THE FIGURES
    [0124]
    [0125] Figure 1 Molecula p-Nitrophenyl-alpha-L-Ramnopyranoside (Rha-pNP).
    [0126] Figure 2. Molecula p-Nitrophenyl-pD-glucoside (Glc-pNP).
    [0127] Figure 3. SDS-PAGE. 1-Molecular mass pattern; 2-Commercial Naringinase; 3-Pure Naringinase.
    [0128] Figure 4. Process of immobilization of the commercial enzyme on the GITQ2 support.
    [0129] Figure 5. Graph of the immobilization of the pure enzyme on the GITQ2 support.
    [0130] Figure 6. Reaction of hydrolysis of p-Nitrophenyl-pD-glucoside (Glc-pNP).
    [0131] Figure 7. Reaction of hydrolysis of p-Nitrophenyl-alpha-L-Ramnopyranoside (Rha-pNP).
    [0132] Figure 8. Lineweaver-Burk plot to determine the KM of crude naringinase in free and immobilized form.
    [0133] Figure 9. Lineweaver-Burk plot to determine the KM of pure naringinase in free and immobilized form.
    [0134] Figure 10. Catallotic stability of the commercial enzymatic derivative in the hydrolysis of naringin.
    [0135] Figure 11. Catallotic stability of the purified enzymatic derivative in the hydrolysis of naringin.
    [0136]
    [0137] The present invention is illustrated by the following examples which are not intended to be limiting:
    [0138]
    [0139] EXAMPLES
    [0140]
    [0141] Example 1. Synthesis of the laminar zeolite ITQ2
    [0142]
    [0143] Synthesis of the zeolite MWW (MCM-22) pure sllice.
    [0144]
    [0145] 113.34 g of an aqueous solution of N, N, N-trimethyl-1-adamantammonium hydroxide (7.5% by weight) are mixed with 26.9 g of water and 5.12 g of hexamethyleneimine (Sigma-Aldrich). Subsequently, 0.97 g of sodium chloride and 10 g of silica (Aerosil 200, Degussa) are added to the mixture, keeping the resulting gel in agitation for 30 minutes. Finally, the gel is transferred to a Teflon sheath autoclave, and heated to a temperature of 150 ° C for 9 days in dynamic (60 rpm). The solid obtained after the hydrothermal crystallization is filtered and washed with abundant water, and finally dried at 100 ° C.
    [0146]
    [0147] Preparation of the ITQ2 zeolite pure sllice
    [0148]
    [0149] The synthesis of the ITQ2 material was carried out in the following manner: 5 g of the pure zeolite MWW synthesized above were dispersed in 20 g of water. Then, 100 g of an aqueous solution of hexadecyltrimethylammonium hydroxide (25% by weight, 50% exchange Br / OH), and 30 g of another aqueous tetrapropylammonium solution (40% by weight, 30% exchange Br / are added). OH). The resulting mixture (pH 12.5) is heated to 55 ° C and stirred vigorously for 16 h to facilitate swelling between zeolite sheets. At this point, the suspension is treated in an ultrasonic bath (50 W, 50 Hz) for 1 h to disperse the zeolite sheets. By addition of HCl (6M), the pH is decreased to about 3, to facilitate the flocculation of the delaminated solid, which is recovered by centrifugation. Once washed with distilled water and then dried at 60 ° C for 12 h, the solid is treated at 540 ° C, first for 3 h in atmosphere of N2, and then for 6 h in air.
    [0150]
    [0151] Preparation of pure silica ITQ6 zeolite
    [0152]
    [0153] The synthesis of the ITQ6 material was carried out in the following way: 10 g of silica (Aerosil 200, Degussa), 2.3 g of alumina (boehmite, Catapal B), 9.2 g of NH4F (Aldrich, 98% purity), 3.1 g of HF, 49.8% concentration), 26 g of 4-amino-2,2,6,6-tetramethylpiperidine (Fluka, 98% purity) and 27.9 g of MiliQ deionized water in an autoclave at 175 ° C for 5 days. The resulting product, after being filtered, washed 3 times with water and dried at 60 ° C, was suspended in an aqueous solution of cetyltrimethylammonium bromide (CTA Br-) and tetrapropylammonium hydroxide (TPA OH-) and refluxed. for 16 h at 95 ° C. The delamination was carried out by placing the suspension in an ultrasonic bath (50 W, 40 kHz) for 1 h, maintaining a pH of 12.5 and 50 ° C. Finally, the solid phase was thoroughly washed with water, dried at 100 ° C and calcined at 580 ° C for 7 hours producing ITQ6.
    [0154]
    [0155] Example 2. Functionalization of ITQ2 or ITQ6 zeolites with aidehldo groups.
    [0156]
    [0157] First, zeolite (500mg), either ITQ2 or ITQ6, is activated at 200 ° C in vacuum for 2h. At room temperature, 50 ml of anhydrous toluene and 240 pL of (3-aminopropyl) triethoxysilane are added to the solid and the mixture is refluxed for 24 h at 120 ° C. After this time, the solid is filtered in vacuo and washed with toluene and n-hexane, obtaining the material functionalized with amino groups (N-ITQ). For functionalization with aldehyde groups, zeolite N-ITQ (0.5 g) is placed in contact with 20mL of a NaH2P04 buffer solution at pH = 7 200mM, with 10% glutaraldehyde and maintained under magnetic agitation for 24 h. After this time the solid is filtered and washed with a buffer solution of NaH2PO4 (25mM) at pH = 7. Subsequently, the material is dried at a temperature of 25 ° C, obtaining the material GITQ, preferably GITQ2 or GITQ6, functionalized with aldehyde groups.
    [0158]
    [0159] Example 3. Purification of naringinase and electrophoresis of the commercial enzyme and the purified fraction.
    [0160]
    [0161] The commercial naringinase (Raw) was purified obtaining the naringinase fraction (Pure). Commercial naringinase (Raw) presents a-rhamnosidase activity and p glucosidase, when purified, the a-rhamnosidase activity is increased and the pglucosidase activity is considerably reduced, thus increasing the selectivity of the biocatalyst.
    [0162]
    [0163] Purification method:
    [0164]
    [0165] 500 mg of commercial naringinase (Raw) was dialysed in 50 mL of milliQ water for 24 hours at a temperature of 5 ° C. Subsequently it was incorporated into an ion exchange resin (DEAE-Sephacel Pharmacia, Sweden) equilibrated with phosphate buffer (NaH2PO4 / Na2HPO4) 5mM, pH 6.8. The mixture was left under agitation with rollers in a closed bottle for one hour. The proteins bound to this gel were eluted with a salt gradient. A solution (50 mL) of 0.1 M NaCl selectively desorbed the retained (Pure) protein, obtaining pure naringinase. The results of the purification are shown in Table 2. This table shows that at the beginning of the purification the naringinase contains the two activities (a-rhamnosidase and pglucosidase) in about 50/50 and that after the purification shows a considerable amount of the activity of p-glucosidase.
    [0166]
    [0167] To measure the enzymatic activity of crude and pure naringinase, synthetic substrates were used: p-Nitrophenyl-alpha-L-Rhynopyranoside (Rha-pNP) and p-Nitrophenyl-p-D-glucopyranoside (Glc-pNP). In Table 2 it can be observed how the rhamnosidase activity per mg of enzyme has increased considerably after purification.
    [0168]
    [0169] Table 2. Glucosidase and rhamnosidase activity of naringinase according to the degree of purification
    [0170]
    [0171]
    [0172] The pure and crude enzymes were subjected to an electrophoresis technique on polyacrylamide gel under denaturing conditions, in the presence of sodium dodecyl sulphate (SDS-PAGE). As can be seen in Figure 3, the pure enzyme solution with rhamnosidase activity showed a single electrophoretic band (SDS-PAGE) with a molecular weight of approximately 66 KD (SDS-PAGE) (Figure 3).
    [0173]
    [0174] Example 4. Immobilization of commercial naringinase (Raw) and purified naringinase (Pure).
    [0175]
    [0176] The immobilization of the enzyme naringinase, both crude and pure, on the GITQ2 support is carried out by contacting 100 mg of support with 3 mg of enzyme, crude or pure, in 3 ml of phosphate buffer at pH 7, subjected to gentle agitation during 24h
    [0177]
    [0178] To determine the degree of immobilization with the contact time, different supernatants were taken from the supernatant that were analyzed according to the Bradford method of protein determination. To do this, each sample of 0.1mL of sample is added with 1mL of Bradford solution, the sample is shaken and after 10 min, the optical density is determined by spectrophotometry at an A = 595nm. Figures 4 and 5 show the disappearance of the enzyme from the supernatant for crude and pure naringinase respectively, showing in both cases that the immobilization of the enzyme is total after 24 hours of contact time.
    [0179]
    [0180] Example 5. Determination of enzymatic activities.
    [0181]
    [0182] (Relative activity Units / mg Protein)
    [0183] 1 Enzyme activity unit corresponds to the release of 1 pmol of pNP per minute.
    [0184]
    [0185] The activities of pure and crude naringinase were determined by incubating the enzyme (0.05 mL of free enzyme solution or enzyme derivative (1 mg protein / mL) with 0.05 mL (5 mM) of the specific substrate Rha-pNP or Glc-pNP as the case may be. , in 0.7 ml of 50 mM citrate buffer (pH 4.5) at 50 ° C for 5 minutes The release of pnitrophenol is estimated by adding 0.8 ml of 1 M sodium carbonate to the mixture and then determining the optical density at 405 nm.
    [0186] Example 6. Determination of the catalytic activity as a function of the incubation temperature.
    [0187]
    [0188] (Relative activity Units / mg Protein)
    [0189] 1 Enzyme activity unit corresponds to the release of 1 pmol of pNP per minute.
    [0190]
    [0191] The α-rhamnosidase activity of the enzymes (crude and pure) in free and immobilized form was determined after the incubation of the reaction medium (Example 5) at different temperatures ranging from 30 ° C-100 ° C. An enzymatic unit of activity corresponds to the release of 1 pmol of pNP per minute. The relative activity as a function of the incubation temperature is shown in Table 3. As observed, in general the activities of the immobilized enzymes were less affected by the different incubation temperatures, which shows that the immobilization has an important effect on the stabilization of the three-dimensional structure of naringinase.
    [0192]
    [0193] Table 3. Optimum temperature naringinase free (Raw and Pure) and immobilized.
    [0194]
    [0195]
    [0196]
    [0197]
    [0198] Example 7. Determination of the catalytic activity as a function of the incubation pH.
    [0199]
    [0200] The enzymes (crude and pure) in free and immobilized form were incubated in the presence of the substrate Rha-pNP, in a universal buffer (Mcllvaine) of a variable pH of 3.0 to 8.0. An enzymatic unit of activity corresponds to the release of 1 pmol of pNP per minute. The measures of the activities were carried out for the different values of Incubation pH under the conditions described at the beginning of this section (Example 5). The activity as a function of pH is shown in Table 4.
    [0201]
    [0202] Table 4. Optimal pH Free and immobilized Naringinase
    [0203]
    [0204]
    [0205]
    [0206]
    [0207] Example 8. Stability of the catalytic activity as a function of the incubation temperature.
    [0208]
    [0209] The free enzymes (pure and crude) and immobilized (pure and crude) were incubated separately for a period of one hour, in buffer (50 mM sodium citrate pH 4.5) at temperatures between 30-100 ° C, after which the Rha-pNP substrate and is subjected to an activity test under the conditions of Example 5. As can be seen in Table 5, at higher temperature the activity of the enzymes immobilized on GITQ2 is considerably higher than that of the free enzymes.
    [0210]
    [0211] Table 5. Free and immobilized naringinase thermal stability.
    [0212]
    [0213]
    [0214] Example 9: Calculation of Michaelis constants (KM)
    [0215]
    [0216] The enzymes (crude and pure) in free and immobilized form were incubated with the specific substrate Rha-pNP, keeping the reaction time constant, and varying substrate concentrations. The KM constants were determined by the Lineweaver-Burk method.
    [0217]
    [0218] As can be seen, both crude and pure naringinase immobilized on GITQ2 have KM values lower than those corresponding to the free enzyme, which shows the increase in the affinity for the substrate when immobilizing the enzyme.
    [0219]
    [0220] Example 10: Stability against leachate in salt gradient.
    [0221]
    [0222] In order to determine if the immobilization of the enzyme was through covalent bonds, the enzyme derivative was subjected to a salt gradient. For this, three immobilizations of the crude enzyme were performed on the GITQ2, at three different pHs (4.5, 7 and 10) and after checking that in the cases of pH 7 and 10 the immobilization of the enzyme was 100-95%, Two salt gradients were made at 0.5M and at 1M. As seen in Table 6, at the immobilization pH of 7 the amount of protein desorbed from the material is 0, this indicates that at these pH the bond between the enzyme and the support is through covalent bonds.
    [0223]
    [0224] Table 6. Protein Desorption vs. NaCI Gradient
    [0225]
    [0226]
    [0227]
    [0228] * Expressed in pgEnzyme / mL
    [0229] Example 11. Hydrolysis of naringin
    [0230]
    [0231] Hydrolysis of naringin was performed by incubating the enzymatic derivative with 8.6 mM of naringin in 3 mL of citrate buffer pH = 4.550 mM at 50 ° C and 750 revolutions per minute for 30 minutes.
    [0232]
    [0233] The sugars were determined by HPLC (Waters 1525 Binary HPLC Pump) using a detector (Refractive index waters 2410) with a column (ICE-COREGEL 87H3), in isocratic mode of acidified water H2SO4 (4mM) and a flow 0.6 ml / min at 75 ° C. The sample of the reaction crude is diluted with the fructose standard (previous calibration) and filtered with 0.22 pm Nylon filters. The flavonoids naringin, prunin and naringenin are determined by HPLC. For this, the reaction crude was diluted in ethanol, and filtered with 0.22 pm Nylon filters and analyzed by HPLC (Shimadzu LC-20 ADXR Liquid Chromatograph) using a Diode Array Detector SPD-M20A detector (A = 280 nm). The column used was a Mediterranean SEA 185pm 25 x 0.46 with a flow of 0.8mL / min. A water / acetonitrile gradient was used according to Table 7. Calibrated previously using naringin, prunin and naringenin.
    [0234]
    [0235] Table 7. HPLC solvent gradient
    [0236]
    [0237]
    [0238]
    [0239]
    [0240] Table 8. Results of naringin hydrolysis using immobilized crude naringinase on GITQ2
    [0241]
    [0242]
    [0243]
    [0244]
    [0245]
    [0246] Table 9. Results of the hydrolysis of naringin using pure naringinase immobilized on GITQ2
    [0247]
    [0248]
    [0249]
    [0250]
    [0251] As observed in Table 8 after 30 min of reaction, the enzymatic derivative of naringinase-Raw results in a practically quantitative conversion, the main products being naringin, rhamnose and glucose. In the case of the enzymatic derivative of Naringinasa-Pura, a practically quantitative conversion is also obtained, in which case the main products are prunin and rhamnose.
    [0252]
    [0253] Example 12. Study of catalyst reusability
    [0254] Once the first use is finished, the enzymatic derivative is centrifuged at 6000 revolutions per minute for 5 minutes, then it is washed 5 times with the 50mM citrate buffer pH = 4.5 and the reuse is performed as described in Example 11. After of 10 cycles the production capacity of the GITQ2-Raw 10.25 mg naringenin / mg Enzyme-Raw. While after 10 cycles the production capacity of the GITQ2-Pura is 28.14 mg prunin / mg Enzyme-Pure.
    权利要求:
    Claims (40)
    [1]
    Enzymatic derivative characterized in that it comprises at least one enzyme naringinase (EC 3.2.1.40) immobilized covalently on a support of zeolites of laminar structure.
    [2]
    2. Enzymatic derivative according to claim 1 wherein the naringinase comes from the fungus Penicilium decumbens.
    [3]
    3. Enzymatic derivative according to any of claims 1 to 2 wherein the naringinase is selected from the list consisting of commercial naringinase, purified naringinase or combinations thereof.
    [4]
    4. Enzymatic derivative according to any of claims 1 to 3 wherein the layered structure zeolite is selected from ITQ2 or ITQ6.
    [5]
    5. Enzymatic derivative according to any of claims 1 to 4 wherein the naringinase is in an enzyme weight ratio: support between 0.2 to 300 mg of enzyme per gram of support.
    [6]
    6. Enzymatic derivative according to claim 4 wherein the naringinase is in a ratio by weight enzyme: support between 0.2 to 70 mg of enzyme per gram of support.
    [7]
    7. Enzymatic derivative according to any of claims 1 to 6, characterized in that it has a KM between 0.1 and 1mM.
    [8]
    8. Process for obtaining an enzymatic derivative according to claims 1 to 7, characterized in that it comprises, at least, the following steps:
    a) Contact the support of laminar zeolite with a compound derived from an aminoalkyltriethoxysilane,
    b) Filter, wash and dry the support obtained in step a),
    c) Functionalize the support of stage b) with aldehldo groups,
    d) Filter, wash and dry the functionalized support obtained in step c), e) Dissolve the enzyme naringinase (EC 3.2.1.40) in a buffer solution, f) Contact the support of step d) with the enzyme from the stage e), and g) Filter, wash and dry the enzymatic derivative obtained in step f).
    [9]
    9. The method of obtaining according to claim 8 wherein the weight ratio of the laminar zeolite: 3-aminopropyltriethoxysilane of step a) ranges from 0.1 to 15.
    [10]
    The obtaining procedure according to any of claims 8 to 9 wherein the aminoalkyltriethoxysilane derivative of step a) is 3-aminopropyltriethoxysilane.
    [11]
    The method of obtaining according to any of claims 8 to 10 wherein step a) is carried out for a time between 1 to 72 h and at a temperature of between 50 ° C to 200 ° C.
    [12]
    The obtaining procedure according to any of claims 8 to 11, wherein stage a) is carried out for a period of 24 hours and at a temperature of 120 ° C.
    [13]
    The obtaining process according to any of claims 8 to 12, wherein the washing of step b) is carried out in the presence of toluene and hexane.
    [14]
    The method of obtaining according to any of claims 8 to 13 wherein the drying of step b) is carried out at a temperature of 25 ° C.
    [15]
    The method of obtaining according to any of claims 8 to 14 wherein the functionalization of step c) is carried out by treating the support with glutaraldehyde under magnetic stirring in the presence of NaH2P04 buffer and at a pH of between 7 to 10 for a time of between 5-48h.
    [16]
    16. The obtaining procedure according to claim 15 wherein the NaH2P04 buffer is at a concentration of between 25 to 200 mM and the glutaraldehyde in a percentage of between 1 to 95%.
    [17]
    17. The method of obtaining according to any of claims 15 to 16 wherein the concentration of glutaraldehyde is 10%.
    [18]
    18. The process of obtaining according to any of claims 8 to 17 wherein the washing of step d) is carried out in a buffer solution of NaH2PO4 at a concentration of between 25 to 500 mM and at a pH of between 7 to 10.
    [19]
    The obtaining process according to any of claims 8 to 18, wherein the drying of step d) is carried out at a temperature of 25 ° C.
    [20]
    The method of obtaining according to any of claims 8 to 19 wherein the naringinase enzyme of step e) is dissolved in a buffer solution at a concentration of between 1 to 200 mM and at a pH of between 3 to 11.
    [21]
    21. Obtaining procedure according to claim 20 wherein the pH is from 7 to 10.
    [22]
    22. Method of obtaining according to any of claims 8 to 21 wherein stage f) is carried out under agitation for a time between 0.1 to 24 hours and at a temperature between 4 ° C to 80 ° C.
    [23]
    23. Obtaining procedure according to claim 22 wherein the temperature ranges from 25 ° C to 30 ° C.
    [24]
    24. Method according to any of claims 8 to 23 wherein the enzyme: support ratio is between 0.2 to 300 mg of enzyme per gram of support.
    [25]
    25. Process according to claim 24 wherein the enzyme: support ratio is between 0.2 to 70 mg of enzyme per gram of support.
    [26]
    26. Procedure for hydrolysis of glycosides in the presence of the enzymatic derivative according to any of claims 1 to 7, comprising the following steps:
    a) Dissolve at least one glycoside in a buffer solution at pH between 4 and 7 and at a temperature between 50 and 70 ° C for 10 minutes, b) Cool the solution of step a)
    c) Contacting the enzyme derivative according to any of claims 1 to 7 with the dissolution of step b),
    d) Heat the mixture of section c) to a temperature comprised between 50 to 100 ° C, for a time comprised between 5 to 60 minutes, e) Separate by centrifugation the solid of step d) and wash it in a buffer solution,
    f) Recover the supernatant of step d) and analyze its composition.
    [27]
    27. Hydrolysis process according to claim 26 wherein the glycosides are selected from the list comprising flavonoids, ramnollpidos and glycopeptides.
    [28]
    28. Hydrolysis process according to claim 27 wherein the flavonoids are selected from the list comprising naringin, hesperidin and / or combinations thereof.
    [29]
    29. Hydrolysis process according to claim 28 wherein the flavonoid is naringin.
    [30]
    30. Hydrolysis process according to any of claims 26 to 29 wherein the glycoside: enzyme weight ratio ranges from 2 to 10.
    [31]
    31. Hydrolysis process according to claim 30 wherein the glycoside: enzyme weight ratio ranges from 2 to 5.
    [32]
    32. Hydrolysis process according to any of claims 26 to 31 wherein the temperature of step d) ranges from 50 to 80 ° C.
    [33]
    33. The hydrolysis process according to any of claims 26 to 32, wherein the time of step d) ranges from 5 to 30 minutes.
    [34]
    34. Use of the enzymatic derivative according to any of claims 1 to 7 in the hydrolysis of glycosides.
    [35]
    35. Use according to claim 34 wherein the glycosides are selected from the list comprising flavonoids, ramnollpidos and glycopeptides.
    [36]
    36. Use according to claim 35 wherein the flavonoids are selected from the list comprising naringin, hesperidin and / or combinations thereof.
    [37]
    37. Use according to claim 36 wherein the flavonoid is naringin.
    [38]
    38. Use according to any of claims 34 to 37 wherein the hydrolysis is carried out at a pH of between 4 to 6.
    [39]
    39. Use according to any of claims 34 to 38 wherein the hydrolysis is carried out at a temperature between 50 ° C to 100 ° C.
    [40]
    40. Use of the enzymatic derivative according to any of claims 1 to 7 as a biocatalyst.
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    同族专利:
    公开号 | 公开日
    ES2711819B2|2020-03-19|
    WO2019092294A1|2019-05-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4971812A|1989-08-31|1990-11-20|National Science Council|Immobilized penicillium sp. naringnase and its use in removing naringin and limonin from fruit juice|
    CN105707640A|2016-03-07|2016-06-29|三峡大学|Citrus juice debitterizing method|
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