阅读说明：本技术 一种二维类石墨烯碳GMCs固定化酶及其制备方法和应用 (Two-dimensional graphene-like carbon GMCs immobilized enzyme and preparation method and application thereof ) 是由 时杰 郑磊 后华清 上官慧娟 邓乾春 张珊 于 2021-09-09 设计创作，主要内容包括：本发明提供了一种二维类石墨烯碳GMCs固定化酶及其制备方法和应用,其制备方法包括：将碳源和氮源按质量比0.02-0.2：1研磨混匀后,在惰性气体保护下依次经550-650℃碳化和750-850℃保温45-100min石墨化,收集后研磨得到二维类石墨烯碳GMCs；将二维类石墨烯碳GMCs加入到PBS中,超声处理后加入酶溶液,经常温振荡、离心、洗涤和真空冷冻干燥后即得。本发明所制备的二维类石墨烯碳GMCs具有较大的比表面积可以为酶的负载提供位点,增加酶的负载率,减少酶的堆积,且有利于酶催化过程中的质量转移；制备的二维类石墨烯碳GMCs固载的酶用于催化叶黄素与琥珀酸酐酯化具有较高的酯化率。(The invention provides a two-dimensional graphene-like carbon GMCs immobilized enzyme, a preparation method and an application thereof, wherein the preparation method comprises the following steps: mixing a carbon source and a nitrogen source according to a mass ratio of 0.02-0.2: 1 grinding and uniformly mixing, sequentially carbonizing at 650 ℃ under 550 and 850 ℃ under the protection of inert gas, graphitizing at 850 ℃ for 45-100min under 750 and grinding after collection to obtain two-dimensional graphene-like carbon GMCs; adding the two-dimensional graphene-like carbon GMCs into PBS, adding an enzyme solution after ultrasonic treatment, and carrying out normal temperature oscillation, centrifugation, washing and vacuum freeze drying to obtain the graphene-like carbon GMCs. The two-dimensional graphene-like carbon GMCs prepared by the method have larger specific surface area, can provide sites for loading of enzymes, increase the loading rate of the enzymes, reduce the accumulation of the enzymes, and are beneficial to mass transfer in the enzyme catalysis process; the prepared two-dimensional graphene-like carbon GMCs immobilized enzyme has higher esterification rate when used for catalyzing esterification of lutein and succinic anhydride.)

1. A preparation method of two-dimensional graphene-like carbon GMCs immobilized enzyme is characterized in that,

the method comprises the following steps:

s1, mixing a carbon source and a nitrogen source according to the mass ratio of 0.02-0.2: 1, under the protection of inert gas, sequentially carbonizing at 550-650 ℃ and preserving heat at 750-850 ℃ for 45-100min for graphitization, collecting a product, and grinding to obtain two-dimensional graphene-like carbon GMCs;

and S2, adding the two-dimensional graphene-like carbon GMCs into PBS, carrying out ultrasonic treatment, adding an enzyme solution, oscillating at normal temperature, and sequentially carrying out centrifugation, washing and vacuum freeze drying to obtain the two-dimensional graphene-like carbon GMCs immobilized enzyme.

2. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1, characterized in that,

in the step S1, in the step S,

the temperature rise rate of carbonization is less than 3 ℃/min, preferably 2-2.5 ℃/min;

and/or the temperature rise rate of the graphitization is less than 2 ℃/min, preferably 1.2-1.75 ℃/min.

3. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1 or 2, characterized in that,

in the step S1, in the step S,

the carbonization temperature is 580-625 ℃, and preferably 600 ℃;

and/or the graphitization temperature and the heat preservation time are 780-845 ℃ and 50-75min, preferably 800 ℃ and 60min respectively.

4. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1, characterized in that,

in the step S1, in the step S,

the carbon source is one or more of glucose, sucrose, chitosan and cellulose, and is preferably glucose;

and/or the nitrogen source is one or more of dicyandiamide, melamine, cyanamide, urea and guanidine hydrochloride, and is preferably dicyandiamide.

5. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 4, wherein,

in step S1, the ratio of the amounts of the carbon source and the nitrogen source added is 0.025: 1.

6. the preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1, characterized in that,

in step S2, the amount of PBS added is 20 to 50mL, preferably 30mL, for each 100mg of the two-dimensional graphene-like carbon GMCs.

7. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1, characterized in that,

in step S2, the addition amount of the enzyme solution is 500g, preferably 300g, for every 100mg of the two-dimensional graphene-like carbon GMCs, wherein the mass of the enzyme contained in the enzyme solution is controlled to be 100-;

further preferably, the concentration of the enzyme solution is 5-20g/mL, preferably 15 g/mL.

8. The preparation method of the two-dimensional graphene-like carbon GMCs immobilized enzyme according to claim 1 or 7, characterized in that,

in the step S2, in the step S,

the rotating speed and the time of the oscillation are respectively 130-180rpm and 8-15 h;

and/or the centrifugation and washing times are the same and are 2-5 times.

9. The two-dimensional graphene-like carbon GMCs immobilized enzyme prepared by the preparation method of any one of claims 1-8.

10. Use of the preparation method of any one of claims 1 to 8 or the two-dimensional graphene-like carbon GMCs immobilized enzyme of claim 9 in enzymatic synthesis of lutein ester.

Technical Field

The invention relates to the technical field of enzyme immobilization, and particularly relates to a two-dimensional graphene-like carbon GMCs immobilized enzyme, and a preparation method and application thereof.

Background

The research of the catalyst is always concerned, and the catalyst has the effects of accelerating the reaction rate, improving the yield, shortening the time and reducing the cost in both basic research and industrial production; however, the conventional catalysts including acid, alkali and transition metal catalysts have the problems of high reaction temperature, difficulty in dissolving in organic solvents, easy corrosion of reaction vessels, difficulty in separation, troublesome post-treatment, low catalytic efficiency, serious pollution and the like.

Biocatalysts have attracted considerable research interest as a new catalyst. Lipase is an important biocatalyst, has the advantages of easy reaction at normal temperature and pressure, high reaction rate, specific catalytic action and the like compared with non-biocatalysts, is widely applied to industrial production of food processing, medicines, textiles, biosensors, biodiesel and the like, and is closely related to the development of life science. The lipase can catalyze hydrolysis and synthesis of ester bonds, has high regioselectivity and enantioselectivity, can catalyze many types of reactions, but is catalyzed only at an oil-water interface, and has a remarkable catalytic effect on hydrolysis of a substrate only when the substrate is in a micro-particle, small polymerization dispersion state or emulsified particles; in addition, lipase is susceptible to factors such as reaction system temperature, pH, solvent, reaction substrate concentration, etc., so that the activity of the lipase is reduced or even inactivated. Meanwhile, because the lipase is difficult to recover, the reuse rate of the lipase is low, and the use cost is greatly increased.

The lutein is one of six carotenoids existing in human serum, has the functions of resisting oxidation, enhancing the immune system of a human body, delaying age-related macular degeneration and the like, and is an important dietary supplement for human; however, lutein is very susceptible to decomposition under light or heat conditions due to its rich unsaturated bonds. The hydroxyl groups at two ends of the lutein are esterified with different fatty acids or anhydrides to form lutein diester, so that the stability and the bioavailability of the lutein diester are effectively improved, and different functions and activities of the lutein can be endowed. The lutein diester is prepared by catalyzing esterification with the immobilized lipase, so that the structure of the lutein can be prevented from being damaged.

The existing synthesis method of lutein ester (natural extraction method, chemical synthesis method and the like) has the problems of complex and complicated process flow, low extraction efficiency, solvent pollution, environmental pollution and the like; the method for preparing the lutein ester by the biological enzyme synthesis method has the advantages of high efficiency, strong selectivity, strong specificity, mild catalytic medium, environmental friendliness and the like, and is a promising method. However, free lipase is extremely sensitive to reaction conditions, is easy to inactivate, has high cost, and is difficult to realize application in large-scale industrial production. Currently, the commercial immobilized enzyme Novo 435 is mostly used, and solvents with strong toxicity and pollution, such as toluene, methyl tert-butyl ether (MTBE) or methyl isobutyl ketone (MIBK), are used as lutein esterification media, so that the application of the lutein ester in the field of food is not preferable.

Therefore, there is an urgent need to develop a green and safe immobilized lipase and a solvent to realize efficient enzymatic synthesis of lutein esters.

Disclosure of Invention

The invention aims to provide a two-dimensional graphene-like carbon GMCs immobilized enzyme, and a preparation method and application thereof.

The invention adopts the following technical scheme:

a preparation method of a two-dimensional graphene-like carbon GMCs immobilized enzyme comprises the following steps:

s1, mixing a carbon source and a nitrogen source according to the mass ratio of 0.02-0.2: 1, under the protection of inert gas, sequentially carbonizing at 550-650 ℃ and preserving heat at 750-850 ℃ for 45-100min for graphitization, collecting a product, and grinding to obtain two-dimensional graphene-like carbon GMCs;

and S2, adding the two-dimensional graphene-like carbon GMCs into PBS, carrying out ultrasonic treatment, adding an enzyme solution, oscillating at normal temperature, and sequentially carrying out centrifugation, washing and vacuum freeze drying to obtain the two-dimensional graphene-like carbon GMCs immobilized enzyme.

Further, in the above technical solution, in step S1, the temperature increase rate in the carbonization process is less than 3 ℃/min, preferably 2-2.5 ℃/min.

Further, in the above technical solution, in step S1, the temperature increase rate in the graphitization process is less than 2 ℃/min, preferably 1.2-1.75 ℃/min.

Still further, in the above technical solution, in step S1, the carbonization temperature of the carbonization is 580-625 ℃, preferably 600 ℃.

Still further, in the above technical solution, in step S1, the graphitization temperature and the heat preservation time are 780-.

Specifically, in the above technical solution, in step S1, the carbon source is one or more of glucose, sucrose, chitosan and cellulose, and is preferably glucose.

Specifically, in the above technical solution, in step S1, the nitrogen source is one or more of dicyandiamide, melamine, cyanamide, urea and guanidine hydrochloride, and is preferably dicyandiamide.

Still further, in the above technical solution, in step S1, the ratio of the amounts of the carbon source and the nitrogen source added is 0.025: 1.

further, in the above technical solution, in step S2, the amount of PBS added is 20 to 50mL, preferably 30mL, per 100mg of the two-dimensional graphene-like carbon GMCs.

Further, in the above technical solution, in step S2, the amount of the enzyme solution added is controlled to control the mass of the enzyme contained in the enzyme solution to be 500g, preferably 300g, for each 100mg of the two-dimensional graphene-like carbon GMCs.

Specifically, in the above technical solution, in step S2, the concentration of the enzyme solution is 5-20g/mL, preferably 15 g/mL.

Still further, in the above technical solution, in step S2, the rotation speed and time of the oscillation are 130-180rpm and 8-15h, respectively.

Still further, in the above technical solution, in step S2, the centrifugation and washing are performed the same number of times, and both the number of times is 2 to 5.

The invention also provides the two-dimensional graphene-like carbon GMCs immobilized enzyme prepared by the preparation method.

The invention also provides an application of the preparation method or the two-dimensional graphene-like carbon GMCs immobilized enzyme in enzymatic synthesis of lutein ester.

Compared with the prior art, the invention has the beneficial effects that:

(1) the two-dimensional graphene-like carbon GMCs prepared by the invention has larger specific surface area and can provide sites for enzyme loading, so that on one hand, the loading rate of the enzyme is increased, on the other hand, the accumulation of the enzyme is reduced, in addition, the larger specific surface area is favorable for mass transfer in the enzyme catalysis reaction process, and the catalysis efficiency is improved;

(2) compared with free enzyme, the two-dimensional graphene-like carbon GMCs immobilized enzyme prepared by the invention can endow the enzyme with reusability, thereby reducing the cost, improving the thermal stability and pH adaptability and widening the application range of the enzyme;

(3) the raw materials selected in the process of preparing the two-dimensional graphene-like carbon GMCs have wide sources, low price, low use cost, simple preparation process and simple operation, and have good development prospect in practical application;

(4) the two-dimensional graphene-like carbon GMCs prepared by the method can be used for immobilizing various enzymes with different types, and has good universality, and the prepared two-dimensional graphene-like carbon GMCs immobilized enzyme is used for catalyzing esterification of lutein and succinic anhydride, and has higher esterification rate compared with free enzyme.

Detailed Description

The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.

The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.

All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.

In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

In the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.

In the examples of the present invention, the raw materials used were all conventional commercially available products.

Example 1

The embodiment of the invention provides a preparation method of a two-dimensional graphene-like carbon GMCs immobilized enzyme, which specifically comprises the following steps:

s1, respectively taking glucose and dicyanodiamine as a carbon source and a nitrogen source, and mixing the components in a mass ratio of 0.025: 1, grinding and uniformly mixing a carbon source and a nitrogen source by using an agate mortar, transferring the mixture into an alumina crucible, transferring the mixture into a tube furnace, heating the mixture to 600 ℃ from normal temperature at a heating rate of 2.5 ℃/min under the protection of nitrogen, carbonizing the mixture, heating the mixture to 800 ℃ at a heating rate of 1.667 ℃/min, preserving the heat for 60min for graphitization, naturally cooling the mixture to room temperature to obtain a blocky fluffy black product, grinding the product to obtain two-dimensional graphene-like carbon GMCs, weighing and storing the product in a dry sample bottle;

s2, weighing 0.1g of the two-dimensional grapheme-carbon GMCs prepared in the step S1, adding the two-dimensional grapheme-carbon GMCs into 30mL of PBS, carrying out ultrasonic treatment, adding 20mL of CALB solution with the concentration of 15g/mL, oscillating at the rotation speed of 150rpm for 12h at normal temperature, and finally carrying out centrifugation, washing and vacuum freeze drying to obtain the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme.

Analyzing and detecting the microstructure and the pore structure of the two-dimensional graphene-like carbon GMCs prepared in the step S1, so that the prepared two-dimensional graphene-like carbon GMCs powder is fluffy and has a porous structure formed by stacking; according to the BET test result, the specific surface area is as high as 372.2m 2/g.

In addition, melamine and urea are respectively selected as nitrogen sources, glucose is selected as a carbon source, the processes are selected and the same process parameters are adopted to prepare two-dimensional graphene-like carbon GMCs and two-dimensional graphene-like carbon GMCs immobilized CALB enzyme; the specific surface areas of the two-dimensional graphene-like carbon GMCs prepared by selecting melamine and urea as nitrogen sources in a BET test are 303.5m2/g and 212.6m2/g respectively.

Example 2

The embodiment of the invention provides a preparation method of a two-dimensional graphene-like carbon GMCs immobilized enzyme, which specifically comprises the following steps:

s1, respectively taking chitosan and melamine as a carbon source and a nitrogen source, wherein the mass ratio of the chitosan to the melamine is 0.05: 1, grinding and uniformly mixing a carbon source and a nitrogen source by using an agate mortar, transferring the mixture into an alumina crucible, transferring the mixture into a tube furnace, heating the mixture to 785 ℃ from normal temperature at a heating rate of 2.5 ℃/min under the protection of nitrogen, carbonizing the mixture, heating the mixture to 800 ℃ at a heating rate of 1.6 ℃/min, preserving the temperature for 60min for graphitization, then naturally cooling the mixture to room temperature to obtain a blocky fluffy black product, grinding the product to obtain two-dimensional graphene-like carbon GMCs, and weighing and storing the product in a dry sample bottle;

s2, weighing 0.1g of the two-dimensional grapheme-carbon GMCs prepared in the step S1, adding the two-dimensional grapheme-carbon GMCs into 35mL of PBS, carrying out ultrasonic treatment, adding 15mL of CALB solution with the concentration of 20g/mL, oscillating at the rotation speed of 175rpm for 10h at normal temperature, and finally carrying out centrifugation, washing and vacuum freeze drying to obtain the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme.

The two-dimensional grapheme-like carbon GMCs prepared by respectively using chitosan and melamine as a carbon source and a nitrogen source in the XPS and EDS detection step S1 and adopting the process parameters show that the N content is 24%, and the surface of the two-dimensional grapheme-like carbon GMCs contains more multi-electron groups containing N, O.

In addition, dinitrile diamine and urea are respectively selected as nitrogen sources, sucrose is selected as a carbon source, the two-dimensional graphene-like carbon GMCs are prepared by the process and the same process parameters, and the XPS and the EDS are also adopted for detecting the two-dimensional graphene-like carbon GMCs, so that the results show that the N content is respectively 18% and 13%. The reason is mainly analyzed as follows: both chitosan and melamine molecules contain N atoms, one melamine monomer contains 6N atoms, and the N atoms are not lost in the calcining process, so that the surface of the prepared two-dimensional carbon material has higher multi-electron groups; the groups can fix the enzyme through hydrogen bonds or electrostatic adsorption, thereby improving the cycle stability and the temperature tolerance of the fixed enzyme.

Example 3

The embodiment of the invention provides a preparation method of a two-dimensional graphene-like carbon GMCs immobilized enzyme, which specifically comprises the following steps:

s1, respectively taking glucose and dicyanodiamine as a carbon source and a nitrogen source, and mixing the components in a mass ratio of 0.025: 1, grinding and uniformly mixing a carbon source and a nitrogen source by using an agate mortar, transferring the mixture into an alumina crucible, transferring the mixture into a tube furnace, heating the mixture to 600 ℃ from normal temperature at a heating rate of 2.5 ℃/min under the protection of nitrogen, carbonizing the mixture, heating the mixture to 700 ℃, 750 ℃, 800 ℃ and 850 ℃ at a heating rate of 1.667 ℃/min respectively, preserving the heat for 60min for graphitization, and then naturally cooling the mixture to room temperature to obtain a blocky fluffy black product, grinding the blocky fluffy black product to obtain two-dimensional graphene-like carbon-based Cs, weighing and storing the two-dimensional graphene-like carbon-based Cs in a dry sample bottle;

s2, weighing 0.1g of two-dimensional grapheme-carbon GMCs obtained by graphitization at different temperatures in the step S1, adding the two-dimensional grapheme-carbon GMCs into 30mL of PBS, performing ultrasonic treatment, adding 20mL of CALB solution with the concentration of 15g/mL, oscillating at the rotation speed of 150rpm for 12h at normal temperature, and finally performing centrifugation, washing and vacuum freeze drying to obtain the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme.

The results of comparative analysis of different graphitization temperatures on two-dimensional graphene-like carbon GMCs immobilized CALB enzyme show that:

graphitizing at a higher temperature, and enabling oxygen-containing groups on the surface of the graphene carbon to fall off, so that the graphitization degree of the two-dimensional graphene-like carbon GMCs surface is higher; due to the reduction of the oxygen-containing groups, the hydrophobicity of the surface of the material is improved; for enzyme immobilization, because the enzyme has a cover structure and is immobilized on the surface of a hydrophobic material, the cover structure of the enzyme is favorable for opening to a certain extent, so that the internal active center is exposed, and the catalytic activity of the enzyme is favorably improved; therefore, the calcination temperature can be increased to a certain extent, and the hydrophobicity of the surface of the material is increased, so that the enzyme catalytic activity is improved, but the oxygen-containing groups on the surface are greatly reduced by continuously increasing the calcination temperature, and the load rate of the enzyme is reduced by reducing the surface charged groups, and the interaction such as hydrogen bonds between the material and the enzyme is reduced, so that the cycling stability and the temperature tolerance of the enzyme are not influenced.

Result verification

Respectively detecting the catalytic efficiency, the circulation stability and the like of the two-dimensional grapheme-carbon GMCs immobilized enzymes prepared in the embodiments 1 to 3, and taking CALB which does not adopt the two-dimensional grapheme-carbon GMCs as a load as a blank control group; the sample information of each test group is specifically as follows: test group 1 is the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme prepared by heat preservation graphitization at 800 ℃ by taking glucose and dicyandiamide as a carbon source and a nitrogen source in example 1, test group 2 is the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme prepared by heat preservation graphitization at 800 ℃ by taking chitosan and melamine as a carbon source and a nitrogen source in example 2, and test group 3 is the two-dimensional grapheme-carbon GMCs immobilized CALB enzyme prepared by heat preservation graphitization at 750 ℃ by taking glucose and dicyandiamide as a carbon source and a nitrogen source in example 3.

The specific test results are shown in table 1 below.

TABLE 1 results of Performance test of samples

Analyzing the results of table 1, it can be seen that: CLAB is fixed on two-dimensional graphene-like carbon GMCs with large specific surface area and fluffy and porous structures, so that the catalytic activity and the cycling stability of the immobilized enzyme on lutein esters can be improved, and the obtained immobilized enzyme has high stability in a wide temperature range.

The reason for this is that, on the one hand, the prepared multilayer two-dimensional graphene-like carbon GMCs have a higher specific surface area, and the enzyme immobilization is catalyzed to facilitate the mass transfer in the catalysis process, reduce the accumulation of catalytic products and facilitate the catalysis, and in addition, the fluffy porous structure can improve the loading rate of the enzyme, thereby improving the amount of the enzyme which is a catalytic active substance, improving the contact between the enzyme and a reaction substrate in the catalysis process, further improving the catalytic esterification rate, and meanwhile, the surface of the carrier material contains functional groups such as amino groups and the like, and can immobilize the enzyme through the actions of hydrogen bonds, electrostatic adsorption and the like, thereby ensuring that the enzyme on the surface of the immobilized enzyme is not easy to separate in the recycling process, thereby improving the cycling stability of the immobilized enzyme, and further improving the temperature stability because the enzyme is immobilized on the surface of the carrier through the actions of hydrogen bonds, electrostatic adsorption and the like, the structure of the enzyme is not easy to change, and the enzyme structure is stable and the activity is maintained along with the temperature rise to a certain degree.

The two-dimensional graphene-like carbon GMCs prepared by the embodiment of the invention has larger specific surface area and can provide sites for enzyme loading, so that the loading rate of the enzyme is increased, the enzyme accumulation is reduced, the larger specific surface area is favorable, and the mass transfer in the enzyme catalysis reaction process is realized, thereby improving the catalysis efficiency; compared with free enzyme, the two-dimensional graphene-like carbon GMCs immobilized enzyme can endow the enzyme with reusability, so that the cost is reduced, the thermal stability and the pH adaptability are improved, and the application range of the enzyme is wider; the two-dimensional graphene-like carbon GMCs prepared by the embodiment of the invention can be used for immobilizing various enzymes with different types, and has good universality.

It should be noted that the above embodiments are only for further illustration and description of the technical solutions of the present invention, and are not further limitations of the technical solutions of the present invention, and the modifications made by those skilled in the art without outstanding substantive features and significant advances are all within the protection scope of the present invention.