CN112342210A

CN112342210A - Cyclodextrin graft porous rubber bead and application thereof in steroid conversion

Info

Publication number: CN112342210A
Application number: CN202011064702.8A
Authority: CN
Inventors: 申雁冰; 王敏; 郭俊超; 谢培培; 杜俊; 夏梦雷; 骆健美
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-02-09
Anticipated expiration: 2040-09-30
Also published as: CN112342210B

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to a cyclodextrin graft porous gel bead, a preparation method and an application thereof in biotransformation. The porous rubber bead is prepared by uniformly mixing sodium alginate, cyclodextrin-carboxymethyl cellulose graft and graphene oxide in water according to a certain proportion, and then dropping the mixture into a metal ion solution to form balls. The glue beads have higher mechanical strength, are beneficial to repeated use, can efficiently recycle cyclodextrin, reduce the loss of the cyclodextrin and facilitate separation in the circulation process; the method has the advantages that the pore diameter is small and regular, the number of pores is large, the porosity is higher, the specific surface area is large, the mass transfer resistance is small, the method is not only beneficial to the rapid contact of cyclodextrin and a substrate, the conversion rate is improved, and the reaction period is shortened; and plays a certain role in promoting the enzyme activity in the cells, and shows good cell compatibility.

Description

Cyclodextrin graft porous rubber bead and application thereof in steroid conversion

The technical field is as follows:

the invention belongs to the technical field of biological catalysis, and particularly relates to a cyclodextrin graft porous gel bead, a preparation method and an application thereof in biotransformation.

Background art:

steroids, second only to antibiotics, are the second main group of drugs that have the function of regulating the metabolism and physiological actions of various substances in the body of a living body. Compared with the traditional chemical synthesis method, the microbial conversion method can form a plurality of steroid drug active intermediates, and the product purity is high. The cyclodextrin as solubilizer can increase the solubility of the hydrophobic steroid, and the special cavity structure can encapsulate the steroid substrate, thereby improving the bioavailability and the yield of the steroid compound. However, the high price of cyclodextrin restricts its large-scale application in the field of biocatalysis. Cyclodextrin is immobilized on a polymer carrier through a chemical grafting technology, the cyclodextrin can be prepared into a water-insoluble polymer material from a water-soluble powder material, the defect that the cyclodextrin is difficult to recover is overcome, the cyclodextrin can be recycled, the industrial use cost is reduced, but the grafting rate is low, and part of materials are harmful to cells, so that finding a harmless grafting carrier and adopting a proper method to improve the cyclodextrin immobilization amount are important problems to be solved urgently.

The carboxymethyl cellulose is a cellulose-based derivative with an ether structure, which is an anionic cellulose ether and is generated by alkalization and etherification reactions of natural cellulose serving as an initial raw material, and is easy to carry out graft polymerization in an aqueous solution, so that the carboxymethyl cellulose is widely concerned. It can be used as stabilizer, dispersant, adhesive, emulsifier, thickener, suspending agent and sizing agent, and is widely used in food, medicine, daily chemical, petroleum, paper making, textile, building and other fields.

Sodium Alginate (SA) is a linear natural polymer formed by irregularly connecting two structural units of (1,4) -beta-D-mannuronic acid (M) and (1,4) -alpha-L-guluronic acid (G), has the advantages of good biocompatibility, safety, no toxicity, low price and the like, and the guluronic acid in the structure can be crosslinked into divalent or trivalent metal ions (such as Ca)²⁺、Ba²⁺) The gel with strength and elasticity is formed, so that the sodium alginate microspheres are prepared, but the formed sodium alginate hydrogel has poor mechanical strength, poor stability and difficulty in recycling, and is not beneficial to balling when the concentration is too high.

Graphene (GO) is a new carbonaceous material with a single-layer two-dimensional structure formed by tightly stacking carbon atoms, graphene oxide is an important derivative of graphene, has a good nano-sheet structure and a large specific surface area, and has a large number of hydroxyl groups, epoxy groups and carbonyl groups at the edges and carboxyl groups on the surface, so that the graphene oxide has good hydrophilicity and is easy to form chemical bonds or hydrogen bonds with polymers, and the graphene oxide has great potential in the aspect of improving various performances of polymer composite materials.

The applicant finds that if carboxymethyl cellulose easy to graft polymerize is grafted with cyclodextrin to form powdery cyclodextrin-carboxymethyl cellulose graft, the graft is embedded and fixed into gel spheres by compounding graphene oxide with sodium alginate. Not only can improve the mechanical strength of the gel ball and be beneficial to the repeated use of the gel ball, but also can efficiently recycle the cyclodextrin, reduce the loss of the cyclodextrin and be convenient for separation in the circulating process.

The invention content is as follows:

in order to achieve the purpose, the invention provides a cyclodextrin graft porous rubber bead which is prepared by uniformly mixing sodium alginate, a cyclodextrin-carboxymethyl cellulose graft and graphene oxide according to a certain proportion and then dropping the mixture into a metal ion solution to form a ball.

Specifically, the preparation method of the cyclodextrin graft porous gel bead (hereinafter referred to as β -CD-CMC-GO porous gel bead) is as follows:

(1) graphite is placed in a sulfuric acid and phosphoric acid system, potassium permanganate is added, and graphene oxide is prepared under the ultrasonic-assisted condition;

further, the ultrasonic power is 200-;

(2) adding a small amount of graphene oxide powder into a beaker filled with a proper amount of water, performing ultrasonic dispersion, adding a certain amount of cyclodextrin-carboxymethyl cellulose graft and sodium alginate into the graphene oxide aqueous solution uniformly dispersed by ultrasonic dispersion, stirring for a period of time at a certain reaction temperature, and standing for defoaming;

further, graphene oxide accounts for 0.1% -1% (m: v) of water;

further, the sodium alginate: cyclodextrin-carboxymethylcellulose graft: the weight ratio of the graphene oxide is 1: 1-10: 0.1 to 1;

further, the reaction temperature is 10-60 ℃, and the stirring time is 0.5-3 h; the stirring speed is 50-300 rpm;

further, the preparation method of the cyclodextrin-carboxymethyl cellulose graft is as follows: weighing cyclodextrin and carboxymethyl cellulose according to the mass ratio of 1:0.5-5, adding into 2-15% of sodium hydroxide solution, and stirring for dissolving; adding epoxy chloropropane according to the volume ratio of the epoxy chloropropane to the sodium hydroxide solution of 0.2-1:5, stirring for 1-2h at 20-30 ℃ until the mixture becomes gel-like, stopping stirring, and raising the temperature to 50-70 ℃ for 2-6 h; alternately washing the product with a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate is free of epichlorohydrin and cyclodextrin; drying at 30-80 deg.C to constant weight, and grinding into powder;

further, the mass sum of the cyclodextrin and the carboxymethyl cellulose in the sodium hydroxide solution has the concentration of 0.12-0.3 g/ml;

(3) dripping the mixed solution obtained in the step (2) into a solution containing metal cations by using an injector, fixing for a period of time, and washing by using a reaction solution to obtain the cyclodextrin graft porous gel beads;

further, the metal cation is selected from Ba²⁺，Ca²⁺，Co²⁺，Ni²⁺，Zn²⁺，Mn²⁺，Fe²⁺One or any combination of several of them; the concentration of the metal cations is 0.1-0.5 mol/L;

further, the fixed time is 1-6 h;

further, the reaction solution is Tris-HCl buffer, normal saline or deionized water.

The invention also provides the application of the beta-CD-CMC-GO porous gel bead in the biological catalysis process of steroid drugs;

the application specifically comprises the steps of adding the colloidal beads serving as a dissolution promoter into a reaction system according to the mass of the colloidal beads and the mass of the reaction system of 1:3-5(m: v) in the biological catalysis process of the steroid drugs, filtering after the reaction is finished, collecting a reaction product from filtrate, washing the filtrate, namely the cyclodextrin graft porous colloidal beads, for 1-10 times by using a reaction solution, and reusing the filtrate in the biological catalysis reaction of the steroid drugs, thereby realizing the recycling of a cyclodextrin medium.

The porous rubber beads can be circulated for about 20 times;

the reaction solution is Tris-HCl buffer solution, normal saline or deionized water and the like, and the pH value is 7.0-7.6;

further, the cyclodextrin is beta-cyclodextrin, HP-beta-cyclodextrin, RM-beta-cyclodextrin or SBE-beta-cyclodextrin;

the porous rubber beads can be fixed again to prolong the recycling times, and the fixing method comprises the following specific steps: and (3) putting the porous rubber beads with the reduced mechanical properties into the metal cation solution for fixing for 2 hours again, washing the reaction solution, collecting the rubber beads, and storing in a refrigerator at 4 ℃ for later use.

Has the advantages that:

(1) according to the invention, the cyclodextrin-carboxymethyl cellulose graft, the graphene oxide and the sodium alginate are compounded for the first time to prepare the rubber beads with small and regular pore diameter and large number of pores, the porosity is higher, the specific surface area is large, the mass transfer resistance is small, the rapid contact between the cyclodextrin and a substrate is facilitated, the conversion rate is improved, the reaction period is shortened, and the problem of cyclic utilization of the cyclodextrin is solved through solid loading;

compared with the prior art, the graphene oxide can form hydrogen bond action with CMC and sodium alginate, the formed pore diameter is small and regular, the porosity of the rubber beads is improved, the swelling of the rubber beads is reduced, and the recycling frequency of the rubber beads is improved; after the graphene oxide is added, the hardness of the rubber beads is increased, the rotary shearing force of the shake flask can be resisted, the circulation is facilitated, and the circulation frequency is increased.

(2) According to the invention, the purpose of utilizing the sodium alginate gel beads modified by graphene oxide to immobilize the cyclodextrin-carboxymethyl cellulose graft is realized for the first time, the cyclic application of a cyclodextrin medium is realized, the steroid catalysis efficiency is improved, the production cost is reduced, the environmental pollution is reduced, and the application value and the popularization prospect are good.

(3) The cyclodextrin graft porous gel bead provided by the invention plays a certain role in promoting the enzyme activity (dehydrogenase) in cells and shows good cell compatibility. The dehydrogenase activity or 11 alpha dehydrogenase activity of the resting cells of the Arthrobacter simplex and the Aspergillus ochraceus treated by the cyclodextrin graft porous colloidal beads beta-CD-CMC-GO is increased to different degrees, and the enzyme activity can be increased by about 20 percent compared with that of a blank control group.

(4) The cyclodextrin recycling process method is simple and convenient, and cost saving is convenient to realize.

Description of the drawings:

FIG. 1 swelling degree of different kinds of rubber beads;

FIG. 2 hardness of different kinds of rubber beads;

FIG. 3 scanning electron micrographs of different types of rubber beads

The surface structure of the beta-CD-CMC-GO rubber beads, (b) the surface structure of the beta-CD-CMC-GO rubber beads, (c) the internal structure of the beta-CD-CMC rubber beads, (d) the internal structure of the beta-CD-CMC-GO rubber beads;

FIG. 4 effect of different kinds of glue beads on the dehydrogenase activity of mold;

FIG. 5 effect of different types of glue beads on bacterial dehydrogenase enzyme activity;

the specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The technical solution of the present invention will be further described with reference to the following specific examples.

EXAMPLE 1 rubber bead Performance measurement

(1) Preparation of rubber beads

The preparation method of SA (sodium alginate) gel beads comprises the steps of accurately weighing 2g of sodium alginate, slowly adding the sodium alginate into 100mL of deionized water, forming a uniform solution under the condition of stirring, and then adding 0.25mol/L CaCl by using an injector₂Fixing the solution for 2h, and finally washing with deionized water to collect SA glue beads.

The preparation of the beta-CD and CMC grafts was as follows: weighing 5g of beta-cyclodextrin and carboxymethyl cellulose according to the mass ratio of 1:1, adding the beta-cyclodextrin and carboxymethyl cellulose into 50mL of 7% sodium hydroxide solution, and stirring and dissolving at 30 ℃; adding epoxy chloropropane according to the volume ratio of the epoxy chloropropane to the sodium hydroxide solution of 1:6.25, stirring for 1h at 30 ℃ until the mixture becomes gel-like, stopping stirring, and raising the temperature to 60 ℃ for 6 h; alternately washing the product with a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate is free of epichlorohydrin and cyclodextrin; drying to constant weight at 55 deg.C, grinding into powder (grafting rate of 60 + -2.5 μmol/g).

The preparation method of the beta-CD-CMC colloidal beads comprises the steps of accurately weighing 2g of sodium alginate and 10g of beta-CD-CMC grafts, slowly adding the sodium alginate and the beta-CD-CMC grafts into 100mL of deionized water, stirring the mixture at 25 ℃ and 100rpm for 1 hour to form a uniform solution, and then injecting 0.25mol/L CaCl by using an injector₂Fixing the solution for 2h, and finally washing and collecting the beta-CD-CMC glue beads by deionized water.

The preparation method of the beta-CD-CMC-GO rubber bead comprises the steps of accurately weighing 0.2g of graphene oxide, adding the graphene oxide into 100mL of deionized water, carrying out ultrasonic treatment for 30min, accurately weighing 2g of sodium alginate and 10g of beta-CD-CMC graft, slowly adding the sodium alginate and the beta-CD-CMC graft into the solution, and stirring at 25 ℃ and 100rpm for 1h to form the beta-CD-CMC-GO rubber beadHomogenizing the solution, standing for defoaming, and then adding 0.25mol/L CaCl by using a syringe₂Fixing the solution for 2 hours, and finally washing and collecting the beta-CD-CMC-GO rubber beads by deionized water.

(2) Degree of swelling

The Swelling Ratio (SR) of the cyclodextrin graft gel beads was measured by immersing the dried cyclodextrin graft gel beads (W1) in Tris-HCl buffer (pH 7.2). Until the swelling reached equilibrium, it was then separated by filtration and the surface water was taken off by adsorption on filter paper and the weight was recorded as (W2). Data are recorded and the SR is calculated using the formula.

Wherein W1 is the dry weight of the cyclodextrin graft gel beads and W2 is the weight of the swollen cyclodextrin graft gel beads in Tris-HCl buffer.

The results are shown in fig. 1, where fig. 1 shows swelling degree data of different kinds of gel beads, in which SAbeads are pure sodium alginate gel beads, β -CD-CMC is cyclodextrin graft gel beads without graphene oxide, and β -CD-CMC-GO is cyclodextrin graft porous gel beads with graphene oxide. The test result shows that the swelling degree of the SAbeads is 91% +/-1, the swelling degree of the beta-CD-CMC is 85% +/-1.2, and the swelling degree of the beta-CD-CMC-GO is 62% +/-1.3.

(3) Hardness of

The hardness of the cyclodextrin grafts was determined by texture analysis. The detailed test conditions comprise that the speed before the test is 3mm/s, the test speed is 1mm/s, the speed after the test is 1mm/s, the compression degree is 50 percent, the trigger force is 5g, 4 test beads are selected each time, and each group of test beads are tested in parallel for 3 times.

As a result, as shown in fig. 2, the hardness of the beads was significantly increased and the degree of swelling was decreased after the addition of GO, which were in inverse proportion to each other. The addition of GO plays a role in reducing the swelling degree of the rubber beads and increasing the mechanical strength of the rubber beads, thereby increasing the recycling frequency of the rubber beads.

And (3) testing results: the hardness of the SAbeads is 676 +/-10, the hardness of the beta-CD-CMC is 683 +/-10, and the hardness of the beta-CD-CMC-GO is 781 +/-12.

(4) Scanning electron microscope

It is seen from fig. 3 that after GO is added, the surface and the inside of the product both have a rough porous structure, and probably due to the hydrogen bonding effect existing before graphene oxide, sodium alginate and sodium carboxymethyl cellulose, the formed colloidal beads have small and regular pore diameters, a large number of pores, high porosity and large specific surface area. The structure can reduce mass transfer resistance, is beneficial to the in and out of the substrate, further promotes the contact of cyclodextrin and cells in the rubber beads with the substrate, and accelerates the dissolution of the substrate, thereby accelerating the initial conversion rate and shortening the reaction period. And powerful conditions are provided for fixing cells in the later period, and the opportunity that the cells in the rubber beads escape in the circulation process is reduced, so that a better circulation transformation effect is achieved.

(5) Influence on the Activity of the enzyme

A. Effect on the Activity of mold (Aspergillus ochraceus) dehydrogenase (11. alpha. dehydrogenase)

1g of resting cells of aspergillus ochraceus (OD is 0.5 after ten-fold dilution) were weighed accurately, 20mL of Tris-HCl buffer solution was added, 5g of the cyclodextrin graft porous gel beads prepared in step (1), namely beta-CD-CMC-GO (experimental group), and a control group, namely beta-CD-CMC (4.86g, the amount of cyclodextrin in the gel beads is controlled to be the same as that in the experimental group)), gel beads, 5g of pure SA gel beads and a blank group without any gel beads were added.

The experimental, control and blank groups were placed on a shaker at 28 ℃ and 200rpm for two hours. And then centrifuging to obtain resting cells, adding 1mL of PBS and 2mL of 0.5% TTC solution into the resting cells, carrying out water bath at 37 ℃ for 30min, adding 0.5mL of formaldehyde to stop the reaction, centrifuging for 5min at 5000r/min, washing precipitates by using the PBS, adding 4mL of 80% acetone into the precipitates after oscillating, carrying out ultrasonic extraction, measuring the light absorption value of the supernatant at 485nm after centrifuging, and reflecting the activity of 11 alpha dehydrogenase according to the size of the light absorption value, wherein the higher the light absorption value is, the higher the activity of the dehydrogenase of the cells is.

As shown in FIG. 4, the absorbance values of the experimental β -CD-CMC-GO gel beads are 0.356. + -. 0.015, the absorbance values of the β -CD-CMC control are 0.302. + -. 0.013, the absorbance values of the pure SA control are 0.301. + -. 0.012, and the absorbance values of the blank without any gel beads are 0.301. + -. 0.011. The results show that the activity of Aspergillus ochraceus 11 alpha dehydrogenase is improved by about 17.9% under the action of the beta-CD-CMC-GO colloidal beads.

In addition, researches show that the activity of the dehydrogenase is reduced by the single beta-CD (influence of a high cyclodextrin structure on the characteristics of simple arthrobacter [ D ]. Tianjin science and technology university, 2017.), and the beta-CD-CMC-GO colloidal beads provided by the invention not only solve the problem of cytotoxicity caused by the beta-CD, but also improve the enzyme activity.

B. Effect on the Activity of bacterial (Arthrobacter simplex) dehydrogenase (C1, 2-position dehydrogenase)

A resting cell suspension with a thallus concentration (twenty times OD ═ 0.5 by dilution) was prepared, 20mL of resting cell suspension was accurately weighed and 5g of β -CD-CMC-GO porous beads (experimental group) were added, and the control group was 4.86g of β -CD-CMC beads (amount of cyclodextrin in the beads was controlled to be the same as that in the experimental group), 5g of pure SA beads, and a blank group without any beads. And (3) placing the experimental group and the blank control group in a shaking table with the temperature of 34 ℃ and the rpm of 180 for two hours, then taking 1mL of cell suspension, adding 1mL of PBS and 2mL of 0.5% TTC solution, carrying out water bath at the temperature of 37 ℃ for 30min, adding 0.5mL of formaldehyde to stop the reaction, centrifuging at the speed of 5000r/min for 5min, adding 4mL of 80% acetone into the precipitate after washing the precipitate by the PBS, oscillating, carrying out ultrasonic extraction, measuring the light absorption value of the supernatant at the position of 485nm after centrifuging, and reflecting the activity of dehydrogenase according to the size of the light absorption value, wherein the higher the light absorption value indicates that the activity of the dehydrogenase of the.

The results are shown in FIG. 5, which shows that the absorbance values of the experimental group of beta-CD-CMC-GO are 0.410 + -0.012, the control group of beta-CD-CMC beads is 0.343 + -0.013, the control group of pure SA is 0.341 + -0.012, and the blank group without any beads is 0.341 + -0.011. The cell dehydrogenase activity is improved by about 19.8 percent under the action of beta-CD-CMC-GO.

Example 2 preparation of porous gel beads of beta-Cyclodextrin grafts and their use in the dehydrogenation of cortisone acetate C1, 2-position

1. Preparation of beta-cyclodextrin graft porous gel beads (beta-CD-CMC-GO):

preparing a carboxymethyl cellulose-beta-cyclodextrin graft, dissolving 5g of carboxymethyl cellulose in 50mL of 7% NaOH solution, adding 5g of beta-cyclodextrin, adding 8mL of epoxy chloropropane after the beta-cyclodextrin and the carboxymethyl cellulose are completely dissolved, continuing stirring for 2h at 20 ℃ until the mixture becomes gel, stopping stirring, and placing the gel at 60 ℃ for 6 h. And after the reaction is finished, alternately washing the product by using a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate does not contain epichlorohydrin and beta-cyclodextrin. Then the mixture is put into an oven to be dried and ground into powder to obtain the beta-cyclodextrin-carboxymethyl cellulose graft.

Accurately weighing 0.2g of graphene oxide, adding the graphene oxide into 100mL of deionized water, carrying out ultrasonic treatment for 30min, accurately weighing 2g of sodium alginate and 5g of beta-CD-CMC graft, slowly adding the sodium alginate and the beta-CD-CMC graft into the solution, stirring at 25 ℃ and 100rpm for 1h to form a uniform solution, standing for defoaming, and adding 0.25mol/L CaCl by using an injector under magnetic stirring₂Fixing in the solution for 2h to obtain porous gel beads with diameter of 3-5mm, washing the gel beads with Tris-HCl buffer solution, suspending in the above buffer solution, and storing in a refrigerator at 4 deg.C.

2. Biotransformation

(1) Preparation for biotransformation

The microbial strain adopts Arthrobacter simplex (TCCC 11037), which can realize the dehydrogenation conversion of C1, 2-position of steroid compound and convert Cortisone Acetate (CA) into prednisone acetate;

slant medium (g/L): 10 portions of glucose, 10 portions of yeast extract, 20 portions of agar and 7.2 portions of pHs;

seed (fermentation) medium (g/L): 10 parts of glucose, 10 parts of corn steep liquor, 5 parts of peptone, 2.5 parts of monopotassium phosphate and 7.2 parts of pHs;

preparation of resting cells of Arthrobacter simplex:

TCCC 11037 is cultured for 18 hours under the conditions of 32 ℃, 160r/min and 30/250mL of seed culture medium, then is inoculated into a 500mL shaking flask filled with 120mL of fermentation culture medium according to the inoculation amount of 5 percent, is cultured for 20 hours under the conditions of 32 ℃ and 110r/min, cells obtained by fermentation culture are centrifuged at 5000r/min, are washed three times by Tris-HCl buffer solution with the pH value of 7.2, and are resuspended in Tris-HCl buffer solution with the pH value of 7.2 to obtain the resting cell bacterial suspension of the Arthrobacter simplex;

(2) biotransformation system

Weighing 0.06g of CA into a 100mL triangular flask, adding 15mL of Tris-HCl (pH 7.2) and 5mL of Arthrobacter simplex resting cell bacterial suspension (OD value is 2), adding 5g of the carboxymethyl cellulose-beta-cyclodextrin graft porous colloidal beads prepared in the previous step into an experimental group (the grafted beta-cyclodextrin is 0.1062g in precise amount), adding equivalent amount of cyclodextrin grafted beta-CD-CMC colloidal beads and equivalent amount of pure SA colloidal beads into a control group, converting at 34 ℃ at 180r/min for 8h, and testing the substrate conversion rate by an HPLC method;

a cyclic utilization process of cyclodextrin;

using the porous gel beads for the biocatalytic reaction of the CA, collecting the porous gel beads after the reaction is finished, washing the porous gel beads for 3 times by using Tris-HCl (pH 7.2), wherein the using amount is 50mL per gram of the gel beads, reusing the porous gel beads for biocatalysis of cortisone acetate after washing, keeping the using amount unchanged, and measuring the substrate conversion rate after each circulation by using an HPLC method;

after 10 times of circulation, the mechanical strength of the porous rubber beads is reduced, and the porous rubber beads need to be fixed again, and the steps are as follows:

throwing the beta-CD-CMC-GO porous rubber beads with reduced mechanical properties into CaCl₂Fixing the solution for 2h again, washing the reaction solution with Tris-HCl (pH 7.2) and collecting the gel beads for 3 times, and storing in a refrigerator at 4 ℃ for later use;

the results show that: the conversion rate of the pure SA glue bead control group is 81%, the primary conversion rate of the beta-CD-CMC glue beads is 93%, the mechanical strength of the beta-CD-CMC glue beads and the pure SA glue beads is poor in the recycling process, the beta-CD-CMC glue beads and the pure SA glue beads are fixed again after 5 times of circulation, the mechanical strength of the beta-CD-CMC glue beads and the pure SA glue beads cannot resist the shearing force of a shaking table, and two kinds of glue beads are broken after ten times of circulation and cannot be recycled. The primary conversion rate of the beta-CD-CMC-GO porous rubber beads is 96%, the beta-CD-CMC-GO porous rubber beads can be used for 10 times after being fixed after being circulated for 10 times, and the circulation times can reach 20 times. The initial conversion rate of the beta-CD-CMC-GO porous gel beads is that of the control group of pure SA gel beads (0.8 multiplied by 10)^-2g/L min^-1) 2.8 times of the amount of the cyclodextrin grafted on the control group, and is beta-CD-CMC (1.62 multiplied by 10) glue beads with the same amount of cyclodextrin grafted on the control group^-2g/L min^-1) 1.38 times of that of the crude product, which was recycled 20 times, and the final conversion of CA was 91%, as shown in tables 1-3.

TABLE 1 beta-CD-CMC-GO porous gel beads

TABLE 2 beta-CD-CMC beads

Number of cycles	1	2	3	4	5	6	7	8	9	10
											Conversion rate	93％	92％	92％	91％	91％	91％	91％	90％	90％	89％

TABLE 3 pure SA glue beads

Number of cycles	1	2	3	4	5	6	7	8	9	10
											Conversion rate	81％	81％	81％	80％	81％	80％	80％	80％	80％	81％

Example 3 preparation of HP-beta-Cyclodextrin graft porous gel beads and their use in the dehydrogenation of cortisone acetate C1, 2-position

The procedure was as in example 1 except for the following.

1. Preparation of HP-beta-cyclodextrin graft porous gel beads (HP-beta-CD-CMC-GO porous gel beads):

preparing a carboxymethyl cellulose-HP-beta-cyclodextrin graft, dissolving 5g of carboxymethyl cellulose in 50mL of 7% NaOH solution, adding 7g of HP-beta-cyclodextrin, adding 8mL of epoxy chloropropane after the HP-beta-cyclodextrin and the carboxymethyl cellulose are completely dissolved, continuing stirring at 25 ℃ for 1.5h until the mixture becomes gel, stopping stirring, and placing the gel at 70 ℃ for 5 h. And after the reaction is finished, alternately washing the product by using a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate does not contain epichlorohydrin and HP-beta-cyclodextrin. Then the mixture is put into an oven to be dried and ground into powder to obtain the HP-beta-cyclodextrin-carboxymethyl cellulose graft.

Accurately weighing 0.2g of graphene oxide, adding the graphene oxide into 100mL of deionized water, carrying out ultrasonic treatment for 30min, accurately weighing 2g of sodium alginate and 6g of HP-beta-CD-CMC graft, slowly adding the sodium alginate and the HP-beta-CD-CMC graft into the solution, stirring at 25 ℃ and 200rpm for 2h to form a uniform solution, and adding 0.25mol/L CaCl by using an injector under magnetic stirring₂Fixing in the solution for 2h to obtain porous gel beads with diameter of 3-5mm, washing the gel beads with Tris-HCl buffer solution, suspending in the above buffer solution, and storing in a refrigerator at 4 deg.C.

2. Biotransformation

Weighing 0.06g of CA into a 100mL triangular flask, adding 15mL of Tris-HCl (pH 7.2) and 5mL of Arthrobacter simplex resting cell bacterial suspension (OD value is 2) into an experimental group, adding 6g of the carboxymethyl cellulose-HP-beta-cyclodextrin graft porous colloidal beads (the grafted HP-beta-cyclodextrin amount is 0.052g) prepared in the step, adding a control group of HP-beta-CD-CMC colloidal beads and pure SA colloidal beads grafted with the same amount of HP-cyclodextrin into the experimental group, converting the mixture at 34 ℃ for 8h at 180r/min, and testing the substrate conversion rate by an HPLC method;

a cyclic utilization process of cyclodextrin;

charging the porous rubber beads with reduced mechanical properties into CaCl₂Fixing the solution for 2h again, washing the reaction solution, collecting the gel beads for 3 times, and storing in a refrigerator at 4 ℃ for later use;

the results show that: the conversion rate of the pure SA glue bead control group is 81%, the primary conversion rate of the HP-beta-CD-CMC glue beads is 94%, the mechanical strength of the HP-beta-CD-CMC glue beads and the pure SA glue beads is poor in the recycling process, the HP-beta-CD-CMC glue beads and the pure SA glue beads are fixed again after 5 times of circulation, the mechanical strength of the HP-beta-CD-CMC glue beads and the pure SA glue beads cannot resist the shearing force of a shaking table, and two kinds of glue beads are broken after ten times of circulation and cannot be recycled. The initial conversion rate of the HP-beta-CD-CMC-GO porous rubber beads is 95%, the HP-beta-CD-CMC-GO porous rubber beads can be fixed after being circulated for 10 times, the HP-beta-CD-CMC-GO porous rubber beads can still be continuously used for 10 times, and the circulation times can reach 20 times. The initial conversion rate of HP-beta-CD-CMC-GO porous gel beads was control pure SA gel beads (0.8X 10)^-2g/L min^-1) 2.6 times of the reference group, and is HP-beta-CD-CMC (1.37 multiplied by 10) glue beads (grafted with cyclodextrin with the same quantity as the reference group)^- ²g/L min^-1) 1.52 times of that of the crude product, which was recycled 20 times, and the final conversion of CA was 91%, as shown in tables 4 to 6.

TABLE 4 HP-beta-CD-CMC-GO porous gel beads

TABLE 5 HP-beta-CD-CMC beads

Number of cycles	1	2	3	4	5	6	7	8	9	10
											Conversion rate	94％	92％	93％	92％	91％	91％	91％	91％	90％	89％

TABLE 6 pure SA glue beads

EXAMPLE 4 preparation of RM-. beta. -Cyclodextrin graft porous gel beads and their use in the dehydrogenation of cortisone acetate C1, 2-position the procedure is as in example 1 except as follows.

1. Preparation of RM-beta-cyclodextrin graft (RM-beta-CMC-GO) porous gel beads:

preparing a carboxymethyl cellulose-RM-beta-cyclodextrin graft, dissolving 5g of carboxymethyl cellulose in 50mL of 7% NaOH solution, adding 9g of RM-beta-cyclodextrin, adding 8mL of epoxy chloropropane after the RM-beta-cyclodextrin and the carboxymethyl cellulose are completely dissolved, continuing stirring for 2h at 25 ℃ until the mixture becomes gel, stopping stirring, and placing the gel at 50 ℃ for 4 h. And after the reaction is finished, alternately washing the product by using a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate does not contain epichlorohydrin and RM-beta-cyclodextrin. Then the mixture is put into an oven to be dried at the temperature of 80 ℃ and ground into powder to obtain the RM-beta-cyclodextrin-carboxymethyl cellulose graft.

Accurately weighing 0.2g of graphene oxide, adding the graphene oxide into 100mL of deionized water, carrying out ultrasonic treatment for 30min, accurately weighing 2g of sodium alginate and 6.5g of RM-beta-CD-CMC graft, slowly adding the sodium alginate and the RM-beta-CD-CMC graft into the solution, stirring at 25 ℃ and 150rpm for 1h to form a uniform solution, standing for defoaming, adding 0.25mol/L CaCl by using an injector under magnetic stirring₂Fixing in the solution for 2h to obtain porous gel beads with diameter of 3-5mm, washing the gel beads with Tris-HCl buffer solution, suspending in the above buffer solution, and storing in a refrigerator at 4 deg.C.

2. Biotransformation

Weighing 0.06g of CA into a 100mL triangular flask, adding 15mL of Tris-HCl (pH 7.2) and 5mL of Arthrobacter simplex resting cell bacterial suspension (OD value is 2) into an experimental group, adding 6.5g of RM-beta-CMC-GO porous gel beads prepared in the step (the grafted RM-beta-cyclodextrin amount is 0.073g), adding a control group of RM-beta-CD-CMC gel beads grafted with the same amount of RM-beta-CD and pure SA gel beads into the experimental group, converting the RM-beta-CD-CMC gel beads grafted with the same amount of RM-beta-CD for 8h at 34 ℃ at 180r/min, and testing the substrate conversion rate by an HPLC method;

a cyclic utilization process of cyclodextrin;

the results show that: the conversion rate of the pure SA glue bead control group is 83%, the primary conversion rate of the RM-beta-CD-CMC glue beads is 92%, the mechanical strength of the RM-beta-CD-CMC glue beads and the pure SA glue beads is poor in the recycling process, the RM-beta-CD-CMC glue beads and the pure SA glue beads are fixed again after 5 times of circulation, the mechanical strength of the RM-beta-CD-CMC glue beads and the pure SA glue beads cannot resist the shearing force of a shaking table, and the two glue beads are broken after ten times of circulation and cannot be recycled. The initial conversion rate of the RM-beta-CD-CMC-GO porous rubber beads is 95%, the RM-beta-CD-CMC-GO porous rubber beads can be used for 10 times after being fixed after being circulated for 10 times, and the circulation time can reach 20 times. The initial conversion rate of RM-beta-CD-CMC-GO porous gel beads is that of control group pure SA gel beads (0.8X 10)^-2g/L min^-1) 2.9 times of the amount of the cyclodextrin grafted by the control group, and is RM-beta-CD-CMC (1.69 multiplied by 10) glue beads with the same amount of cyclodextrin grafted by the control group^- ²g/L min^-1) 1.38 times of that of the crude product, which was recycled 20 times, the final conversion of CA was 91%, as shown in tables 7 to 9.

TABLE 7 RM-beta-CMC-GO porous gel beads

TABLE 8 RM-beta-CD-CMC beads

Number of cycles	1	2	3	4	5	6	7	8	9	10
											Conversion rate	92％	92％	92％	91％	91％	91％	91％	90％	91％	89％

TABLE 9 pure SA glue beads

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims

1. A porous glue bead of cyclodextrin graft is characterized in that the porous glue bead is prepared by uniformly mixing sodium alginate, cyclodextrin-carboxymethyl cellulose graft and graphene oxide in water according to a certain proportion and then dropping the mixture into a metal ion solution to form balls.

2. The porous gel bead of cyclodextrin graft of claim 1, wherein the ratio of sodium alginate: cyclodextrin-carboxymethylcellulose graft: the weight ratio of the graphene oxide is 1: 1-10: 0.1-1.

3. The porous cyclodextrin graft bead of claim 2, wherein graphene oxide comprises 0.1% -1% of water.

4. A process for the preparation of the porous gel beads of cyclodextrin grafts according to any of the claims 1 to 3, characterized by the following steps:

(1) adding graphene oxide powder into a beaker filled with water, performing ultrasonic dispersion, adding cyclodextrin-carboxymethyl cellulose graft and sodium alginate into a graphene oxide aqueous solution uniformly subjected to ultrasonic dispersion, stirring for reaction, and standing for defoaming;

(2) and (3) dripping the mixed solution obtained in the step (1) into a solution containing metal cations by using an injector, fixing, and washing by using a reaction solution to obtain the cyclodextrin graft porous gel bead.

5. The preparation method according to claim 4, wherein the reaction temperature in the step (1) is 10-60 ℃, and the stirring time is 0.5-3 h; the stirring speed is 50-300 rpm.

6. The method of claim 4, wherein the cyclodextrin-carboxymethylcellulose graft is prepared by the following method: weighing cyclodextrin and carboxymethyl cellulose according to the mass ratio of 1:0.5-5, adding into 2-15% of sodium hydroxide solution, and stirring for dissolving; adding epoxy chloropropane according to the volume ratio of the epoxy chloropropane to the sodium hydroxide solution of 0.2-1:5, stirring until the mixture becomes gel, stopping stirring, raising the temperature to 50-70 ℃, and keeping the temperature for 2-6 h; alternately washing the product with a large amount of distilled water and ethanol until the eluate is neutral, and until the eluate is free of epichlorohydrin and cyclodextrin; drying to constant weight, and grinding into powder.

7. The process according to claim 6, wherein the sum of the masses of cyclodextrin and carboxymethylcellulose is present in the sodium hydroxide solution in a concentration of 0.12 to 0.3 g/ml.

8. The method according to claim 4, wherein the metal cation is Ba²⁺，Ca²⁺，Co²⁺，Ni²⁺，Zn²⁺，Mn²⁺，Fe²⁺One or any combination of several of them; the concentration of the metal cations is 0.1-0.5 mol/L; the fixed time is 1-6 h.

9. Use of the cyclodextrin graft porous gel beads of any one of claims 1-3 in a steroid drug biocatalytic process.

10. The application of claim 9, wherein the gel beads are used as a cosolvent, the gel beads and the reaction system are added into the reaction system according to the mass ratio of the gel beads to the reaction system of 1:3-5, after the reaction is finished, the gel beads are filtered, the filtrate is used for collecting a reaction product, and the filtrate, namely the cyclodextrin graft porous gel beads, is washed by the reaction solution for 1-10 times and then is reused for the steroid drug biocatalysis reaction, so that the cyclic utilization of the cyclodextrin medium is realized.

11. The use of claim 10, wherein said porous beads are re-immobilized for extended cycles, said immobilization being as follows: and (3) putting the porous rubber beads with the reduced mechanical properties into the metal cation solution for fixing for 2 hours again, and washing and collecting the rubber beads by using the reaction solution.

12. The porous cyclodextrin graft polymer bead of claim 1, wherein the cyclodextrin is β -cyclodextrin, HP- β -cyclodextrin, RM- β -cyclodextrin or SBE- β -cyclodextrin.