阅读说明：本技术 一种用酶固定化技术合成多酚类化合物的制备方法 (Preparation method for synthesizing polyphenol compound by enzyme immobilization technology ) 是由 杨缜 韦春媚 于 2019-10-21 设计创作，主要内容包括：本发明提供一种用酶固定化技术合成多酚类化合物的制备方法,所述制备方法包括：将金属离子或金属簇与有机配体和酪氨酸酶混合,对酪氨酸酶进行固定化,得到酪氨酸酶-金属有机骨架复合物；用所述酪氨酸酶-金属有机骨架复合物做为催化剂,以单苯酚类化合物作为反应物,合成所述多酚类化合物。本发明通过金属离子或金属簇与有机配体在酪氨酸酶的诱导下配位形成金属有机骨架,同时金属有机骨架形成酪氨酸酶的保护壳,实现对酪氨酸酶的固定化,得到的固定化酪氨酸酶具有较高的催化性能。固定化酪氨酸酶催化单苯酚类化合物合成邻位羟基化多酚类化合物,并添加还原剂以抑制多酚类化合物被进一步氧化,从而提高多酚类化合物产率。(The invention provides a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology, which comprises the following steps: mixing metal ions or metal clusters with an organic ligand and tyrosinase, and immobilizing the tyrosinase to obtain a tyrosinase-metal organic framework compound; and synthesizing the polyphenol compound by using the tyrosinase-metal organic framework compound as a catalyst and using a monophenol compound as a reactant. According to the invention, the metal ions or metal clusters and the organic ligands are coordinated under the induction of tyrosinase to form a metal organic framework, and meanwhile, the metal organic framework forms a protective shell of the tyrosinase, so that the tyrosinase is immobilized, and the obtained immobilized tyrosinase has high catalytic performance. The immobilized tyrosinase catalyzes the monophenol compound to synthesize the ortho-hydroxylated polyphenol compound, and a reducing agent is added to inhibit the polyphenol compound from being further oxidized, so that the yield of the polyphenol compound is improved.)

1. A preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology is characterized by comprising the following steps:

mixing metal ions or metal clusters, organic ligands and tyrosinase solution, and fixing tyrosinase on a metal organic framework generated by combining the metal ions or the metal clusters and the organic ligands to obtain a tyrosinase-metal organic framework compound;

and adding the tyrosinase-metal organic framework compound into a monophenol compound solution, adding a reducing agent, and reacting under a preset reaction condition to synthesize the polyphenol compound.

2. The method of claim 1, wherein the metal ions comprise Cu²⁺、Fe³⁺、Zn²⁺、Mn²⁺、Co²⁺、Cr³⁺、Ni²⁺、Ag⁺And Ca²⁺One or more of (a).

3. The method of claim 1, wherein the organic ligand is an azo heterocyclic organic ligand and/or a carboxyl group-containing organic compound.

4. The method of claim 3, wherein the azo-heterocyclic organic ligand is one or more of 2-methylimidazole, benzimidazole, N-acetylimidazole, 2, 4-dimethylimidazole, 2-undecylimidazole, 1-tritylimidazole, 2-mercapto-1-methylimidazole, 2' -bipyridine, 4' -dimethoxy-2, 2' -bipyridine, 2, 4-diaminopyridine, 1, 2, 4-triazole, and 5-aminotetrazole.

5. The method of claim 1, wherein the monophenol compound is an organic compound having a phenolic hydroxyl group.

6. The method of claim 1, wherein the reducing agent is L-ascorbic acid, citric acid, thiourea, NaBH₄And LiBH₄One or more of (a).

7. The method of claim 1, wherein the metal ions or metal clusters, the organic ligand and the tyrosinase are present in an amount of 50-80% by volume of the solution.

8. The method of claim 1, wherein the concentration of the metal ions or metal clusters in the solution of metal ions or metal clusters, organic ligands and tyrosinase is 0.1-80 mM.

9. The method of claim 1, wherein the concentration of the organic ligand in the mixture of the metal ions or metal clusters, the organic ligand and the tyrosinase solution is 0.1-640 mM.

10. The method of claim 1, wherein the predetermined reaction conditions are a reaction temperature of 10 ℃ to 60 ℃ and a reaction pH of 5.0 to 8.0.

Technical Field

The invention relates to the technical field of enzyme immobilization and enzyme catalysis, in particular to a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology.

Background

The polyphenol compound is a substance with two or more hydroxyl groups on a benzene ring, the unique polyhydroxy structure of the polyphenol compound enables the property of the polyphenol compound to be better than that of a monophenol compound, and the polyphenol compound has important significance on human health due to the effects of oxidation resistance, antibiosis, cancer resistance, tumor resistance, blood sugar and blood fat reduction, immunity enhancement and the like, and has great application and research values in the fields of biomedicine and health care.

At present, the polyphenol compounds are synthesized by a chemical method and a biological method, and compared with the chemical method, the biological method for synthesizing the polyphenol compounds by enzyme catalysis has the advantages of high specificity, high efficiency, environmental protection, easy recovery and the like, so the biological method is widely concerned in the synthesis technology of the polyphenol compounds.

Tyrosinase, also known as polyphenol oxidase, is a copper-containing oxidoreductase that catalyzes the oxidation of monophenols to ortho-diphenols and further oxidizes diphenols to ortho-benzoquinones. Free tyrosinase has the defects of poor stability, intolerance to extreme conditions, difficulty in recovery and the like, so that the tyrosinase is immobilized by utilizing an immobilization technology, and the immobilized tyrosinase can be used for catalytically synthesizing polyphenol compounds by using immobilized tyrosinase, and the immobilized tyrosinase has the advantages of easiness in reactor operation, convenience in product separation, improvement of enzyme reutilization and the like. However, the prior immobilization technology has the defects of high cost of used reagents and carriers, low immobilization efficiency, poor stability, more complex equipment for continuous operation and the like, so that the yield of the polyphenol compound synthesized by the traditional immobilization technology is not high. Therefore, it is still a goal of the field of enzyme immobilization to further develop a simpler and more applicable immobilization method and a support material having more excellent performance.

Accordingly, the prior art is yet to be improved and developed.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology, wherein the preparation process of the immobilization technology is simple, tyrosinase is immobilized by using the immobilization technology, and the problem that the yield of the polyphenol compounds synthesized by using the tyrosinase is low in the prior art is solved.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology comprises the following steps:

mixing metal ions or metal clusters, organic ligands and tyrosinase solution, and fixing tyrosinase on a metal organic framework generated by combining the metal ions or the metal clusters and the organic ligands to obtain a tyrosinase-metal organic framework compound;

and adding the tyrosinase-metal organic framework compound into a monophenol compound solution, adding a reducing agent, and reacting under a preset reaction condition to synthesize the polyphenol compound.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the metal ions comprise Cu²⁺、Fe³⁺、Zn²⁺、Mn²⁺、Co²⁺、Cr³⁺、Ni²⁺、Ag⁺And Ca²⁺One or more of (a).

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the organic ligand is an azo heterocyclic organic ligand and/or a carboxyl-containing organic compound.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology comprises the step of synthesizing the azo heterocyclic organic ligand by using one or more of 2-methylimidazole, benzimidazole, N-acetyl imidazole, 2, 4-dimethylimidazole, 2-undecylimidazole, 1-trityl imidazole, 2-mercapto-1-methylimidazole, 2' -bipyridyl, 4' -dimethoxy-2, 2' -bipyridyl, 2, 4-diaminopyridine, 1, 2, 4-triazole and 5-aminotetrazole.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the tyrosinase is derived from animals, plants and microorganisms.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the monophenol compound is an organic compound containing one phenolic hydroxyl group.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology comprises the step of using L-ascorbic acid, citric acid, thiourea and NaBH as a reducing agent₄And LiBH₄One or more of (a).

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology comprises the following steps of preparing a solution containing metal ions or metal clusters, an organic ligand and tyrosinase, wherein the tyrosinase accounts for 50-80% in volume fraction.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology comprises the step of synthesizing the polyphenol compound by using an enzyme immobilization technology, wherein the concentration of metal ions or metal clusters in a solution of the metal ions or metal clusters, an organic ligand and tyrosinase is 0.1-80 mM.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology comprises the following steps of adding metal ions or metal clusters, an organic ligand and tyrosinase solution, wherein the concentration of the organic ligand in the mixed solution is 0.1-640 mM.

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the preset reaction condition is that the reaction temperature is 10-60 ℃, and the reaction pH value is 5.0-8.0.

Zn is preferred in the invention²⁺And 2-methylimidazole, Zn under the induction of an enzyme molecule²⁺Forming zeolite imidazole ester framework-8 (ZIF-8) crystal by coordination with imidazolyl, growing into a protective shell of enzyme, and fixing tyrosinase to form tyrosinase @ zeolite imidazole ester framework-8 (TYR @ ZIF-8).

Has the advantages that: the invention provides a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technology. The tyrosinase-metal organic framework compound is synthesized by a one-pot method, so that metal ions or metal clusters and organic ligands are complexed under the induction of tyrosinase to form a metal organic framework, and the metal organic framework can further grow to form a protective shell of the tyrosinase in the reaction process, so that the immobilization of the tyrosinase is realized, and the immobilized tyrosinase has high catalytic performance. The metal organic framework compound with the immobilized tyrosinase catalyzes the monophenol compounds, and a reducing agent is added into a reaction system to inhibit the generation of quinone substances, so that the yield of the polyphenol compounds can be improved.

Drawings

FIG. 1 is a flow chart of a preparation method for synthesizing polyphenol compounds by using an enzyme immobilization technique according to an embodiment of the present invention.

FIG. 2 is a graphical representation of the effect of various conditions on the activity of tyrosinase @ zeolitic imidazolate framework-8 (TYR @ ZIF-8) as provided in example 1 of the present invention. Wherein FIG. 2A is a graph of the effect of 2-methylimidazole concentration on the activity of the resulting TYR @ ZIF-8; FIG. 2B is a graph of the effect of zinc acetate concentration on the activity of the resulting TYR @ ZIF-8; FIG. 2C is a graph showing the effect of reaction time on the activity of the resulting TYR @ ZIF-8.

Fig. 3 is a schematic view of an HPLC detection spectrum of piceatannol provided in embodiment 2 of the present invention.

FIG. 4 is a schematic diagram showing the effect of various factors on the catalytic synthesis of piceatannol by TYR @ ZIF-8 according to example 3 of the present invention. Wherein FIG. 4A is a graph showing the effect of L-ascorbic acid concentration on piceatannol yield; FIG. 4B is a graph showing the effect of enzymatic reaction temperature on piceatannol yield; FIG. 4C is a graph showing the effect of pH on piceatannol yield by enzymatic reaction; FIG. 4D is a graph showing the effect of TYR @ ZIF-8 as the amount of catalyst on piceatannol yield.

FIG. 5 is a schematic diagram of the response surface method for optimizing piceatannol yield according to example 4 of the present invention. FIG. 5A is a three-dimensional graph optimized by a response surface method; FIG. 5B is a graph showing the relationship between piceatannol yield and reaction time after optimization by the response surface method.

FIG. 6 is a schematic diagram of an HPLC detection profile of L-dopa provided by example 5 of the invention.

FIG. 7 is a graph showing the relationship between the yield of L-dopa produced and the reaction time in example 6 of the present invention.

Fig. 8 is a schematic diagram of an HPLC detection profile of 3' -hydroxytyrosol provided in embodiment 7 of the present invention.

FIG. 9 is a schematic diagram showing the relationship between the yield of 3' -hydroxytyrosol and the reaction time for preparing pterostilbene in example 8 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Referring to fig. 1, fig. 1 is a flow chart of a preferred embodiment of a method for synthesizing polyphenols by enzyme immobilization technology according to the present invention, which comprises the following steps:

s10, mixing the metal ions or the metal clusters, the organic ligands and the tyrosinase solution, and fixing the tyrosinase on a metal organic framework generated by the combination of the metal ions or the metal clusters and the organic ligands to obtain a tyrosinase-metal organic framework compound;

s20, adding the tyrosinase-metal organic framework compound into a monophenol compound solution, adding a reducing agent, reacting under a preset reaction condition, and synthesizing the polyphenol compound.

The synthetic method provided by the embodiment can ensure that tyrosinase has better fixing effect and catalytic performance, is simple to operate, can effectively improve the yield of polyphenol compounds, and has the following principle for realizing the effects:

the preparation method comprises the steps of mixing metal ions or metal clusters with organic ligands and tyrosinase, and fixing the tyrosinase by a one-pot method, wherein the tyrosinase induces the metal ions or the metal clusters and the organic ligands to form a hybrid porous metal organic framework through coordination, so as to form a protective shell of the tyrosinase, and the tyrosinase is fixed in the metal organic framework with a porous structure, so that the tyrosinase-metal organic framework compound is obtained. The tyrosinase-metal organic framework compound has good catalytic performance, under certain conditions, monophenol compounds can be oxidized into ortho-hydroxylated polyphenol compounds (such as o-diphenol), and the polyphenol compounds can be oxidized into quinone compounds (such as o-quinone).

The preparation method for synthesizing the polyphenol compound by using the enzyme immobilization technology is characterized in that the metal ions comprise Cu²⁺、Fe³⁺、Zn²⁺、Mn²⁺、Co²⁺、Cr³⁺、Ni²⁺、Ag⁺And Ca²⁺One or more of (a).

In some embodiments, the anion in the metal salt is Cl^-、NO₃ ^-、SO₄ ^2-、CH₃COO^-、PO₄ ³⁺But is not limited thereto.

In the embodiment, a complexation effect is generated between the metal ions and the organic ligand, so that the formed metal organic framework has good thermal stability, and the environmental tolerance of the tyrosinase-metal organic framework compound is further improved.

In some embodiments, the organic ligand is an azo heterocyclic organic ligand and/or a carboxyl-containing organic compound.

In some embodiments, the azoheterocyclic organic ligand is one or more of 2-methylimidazole, benzimidazole, N-acetylimidazole, 2, 4-dimethylimidazole, 2-undecylimidazole, 1-tritylimidazole, 2-mercapto-1-methylimidazole, 2' -bipyridine, 4' -dimethoxy-2, 2' -bipyridine, 2, 4-diaminopyridine, 1, 2, 4-triazole, 5-aminotetrazole.

In this example, a metal ion Zn is used²⁺Reacting with organic ligand 2-methylimidazole to construct zeolite imidazolate framework-8 (ZIF-8). ZIF-8 is one of MOF family, and the material has good advantages in pore volume, pore channel structure, stability, biocompatibility and the like. In the above Zn²⁺And 2-methylimidazole reaction system, and simultaneously adding tyrosinase to carry out one-pot synthesis to prepare tyrosinase @ zeolite imidazole ester skeleton-8 (TYR @ ZIF-8).

Specifically, the Zn²⁺The concentration in the mixed solution was 0.1 to 80 mM. The concentration of the 2-methylimidazole in the mixed solution is 0.1-640 mM. The volume fraction of the tyrosinase in the mixed solution is 50-80%. And stirring the mixed solution at 4 ℃ for 1-12 hours, centrifuging, collecting the precipitate, washing the precipitate for several times by using phosphate buffer solution or deionized water, and performing vacuum drying at room temperature overnight to obtain TYR @ ZIF-8.

Specifically, the tyrosinase is a tyrosinase crude enzyme liquid, and the preparation method of the tyrosinase crude enzyme liquid comprises the following steps: mashing white Agaricus bisporus and phosphate buffer (such as 50mM, pH6.0) at a mass (g) volume (mL) ratio of 1:2 in a homogenizer, filtering, centrifuging, and collecting supernatant to obtain tyrosinase crude enzyme solution.

In the embodiment, the crude tyrosinase liquid obtained by the preparation method has the volume fraction of 50-80% of the total reaction system volume in the enzyme immobilization reaction, so that the enzyme immobilization efficiency is better in the enzyme immobilization reaction process, and the enzyme activity of the immobilized enzyme is high in the dosage range.

In some embodiments, the monophenolic compound is an organic compound containing one phenolic hydroxyl group. The organic compound containing one phenolic hydroxyl group is one or more of L-tyrosine, 4-hydroxyphenylacetic acid, 4-hydroxyibuprofen phenol ester, p-phenoxy phenol, 3-chlorophenol, p-methylphenol, 2, 4-dichlorophenol, resveratrol and pterostilbene, but is not limited thereto.

In some embodiments, the reducing agent is L-ascorbic acid, citric acid, thiourea, NaBH₄And LiBH₄And the like, but is not limited thereto.

In the embodiment, the monophenol compounds are preferably L-tyrosine, resveratrol and pterostilbene, and the reducing agent is preferably L-ascorbic acid. Oxidizing the monophenol compounds into ortho-hydroxylated polyphenol compounds by using TYR @ ZIF-8 as a catalyst, wherein L-tyrosine, resveratrol and pterostilbene are catalyzed to form L-dopa, piceatannol and 3' -hydroxy pterostilbene in sequence.

In some embodiments, in the enzyme-catalyzed reaction of step S20, the reaction temperature is 10 ℃ to 60 ℃ and the reaction pH is 5.0 to 8.0.

In some embodiments, in the reaction system of step S20, the monophenol compound accounts for 15% to 40% (wt.%) of the total mass of the solute, the reducing agent accounts for 25% to 60% (wt.%) of the total mass of the solute, and the tyrosinase-metal-organic framework complex (TYR @ ZIF-8) is used in an amount of 2.5 to 10 mg/mL.

In this example, TYR @ ZIF-8 was used as a catalyst to oxidize monophenolic compounds to ortho-hydroxylated polyphenols, tyrosinase, a copper-containing oxidoreductase, in O₂With the participation of molecules, after the monophenol compounds are oxidized into the polyphenol compounds, the polyphenol compounds are further catalyzed to obtain the quinone compounds, which has adverse effect on the yield of the polyphenol compounds. The reducing agent is added into the catalytic reaction system, so that the generation of quinone compounds can be effectively inhibited, and the yield of polyphenol compounds is further improved.

The invention provides a polyphenol compound, which is prepared by the method.

The present invention will be described in detail below with reference to specific examples.