CN107022542B - Sodium alginate grafted derivative cyclodextrin immobilized cell and application thereof - Google Patents

Sodium alginate grafted derivative cyclodextrin immobilized cell and application thereof Download PDF

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CN107022542B
CN107022542B CN201710452656.0A CN201710452656A CN107022542B CN 107022542 B CN107022542 B CN 107022542B CN 201710452656 A CN201710452656 A CN 201710452656A CN 107022542 B CN107022542 B CN 107022542B
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sodium alginate
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申雁冰
王敏
于子棋
商志华
夏梦雷
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Tianjin University of Science and Technology
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Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to a sodium alginate graft derivative cyclodextrin immobilized cell and application thereof. The sodium alginate grafted and derived natural cyclodextrin immobilized cell constructed by the invention realizes simultaneous cyclic utilization of a cyclodextrin medium and the cell for the first time, achieves the repeated use of a dissolution promoting medium and a catalytic cell while improving the solubility of a steroid substrate, has the conversion rate of the steroid substrate still above 90% after 8 times of circulation, and has good application value and popularization prospect.

Description

Sodium alginate grafted derivative cyclodextrin immobilized cell and application thereof
The technical field is as follows:
the invention belongs to the technical field of biological catalysis, and particularly relates to a sodium alginate grafted derivative cyclodextrin immobilized cell in a microbial catalytic reaction and application thereof.
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. The industrial production of steroid drugs mainly modifies the structure of natural steroid compounds, and 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 the wide application of cyclodextrin in the field of biocatalysis, and the expansion of the application of cyclodextrin in biotransformation reaction by adopting a proper method is an important problem to be solved urgently.
The cyclodextrin is fixedly carried on a macromolecular carrier such as chitosan, sodium alginate and the like by a grafting technology, β -cyclodextrin can be prepared into a water-insoluble macromolecular material from a water-soluble powder material, the defect that the cyclodextrin is difficult to recover is overcome, the cyclodextrin can be recycled,the cost of industrial use is reduced. The grafted cyclodextrin still keeps a unique cavity structure and other excellent properties, and simultaneously has the characteristics of good mechanical property, stability and the like of a high-molecular carrier, so that the application space of the cyclodextrin is greatly widened, and the application value of the cyclodextrin is improved. Sodium alginate is a natural polyanionic polysaccharide macromolecular compound, is composed of G, M units, has viscosity on the surface after being wetted, and can make the solution have viscosity. It is safe, non-toxic, low in cost, good in biodegradability and biocompatibility, possesses the properties required by medicinal auxiliary material, and meets Ca2+、Zn2+When cations are used, chemical reaction can occur to form gel spheres, and the gel spheres can be used as slow-release and controlled-release materials of medicines and used for fixing substances with unstable properties to improve the stability and can also be used for fixing microorganisms, cells, enzymes and the like to realize cyclic utilization. At present, the application of sodium alginate mainly relates to the fields of pesticides, foods, biomedicines and the like, and is gradually extended to the aspect of treatment of heavy metal pollution.
Chinese patent CN 105754984-A discloses a sodium alginate composite immobilized microbial agent, a preparation method and application thereof, and discloses a sodium alginate composite immobilized microbial agent and a preparation method thereof, solving the problem that the prior art has no immobilization mode suitable for pseudomonas for quinclorac degradation. The preparation method comprises the steps of mixing a carrier culture medium with pseudomonas and sodium alginate, and then dropping the mixture into 1-4% calcium chloride for standing for 2-4 hours to prepare granules; the mass concentration of the sodium alginate is 2-6%; the carrier culture medium comprises an adsorption carrier and an inorganic salt liquid culture medium; the weight ratio of the adsorption carrier to the sodium alginate is 1-3: 1, and the adsorption carrier is composed of corncobs, bamboo charcoal and oil cake in a weight ratio of 1: 1-2: 1. The invention effectively improves the degradation efficiency of the thallus; the degradation rate of the immobilized bacteria agent is far higher than the sum of the physical adsorption efficiency of the blank pellets and the biodegradation efficiency of the free strains, and the effect of mutual promotion can be effectively achieved.
Sodium alginate is used as a natural polymer carrier, the molecule contains two hydrophilic groups of-COOH and-OH, the hydrophilicity is strong, epichlorohydrin can be used as a cross-linking agent to be grafted with cyclodextrin, and the formed cyclodextrin-sodium alginate graft can immobilize cells and realize the co-circulation of cyclodextrin and cells. At present, the technology of taking sodium alginate as a carrier and taking a natural polymer material grafted cyclodextrin as a dissolution promoter to be applied to the biotransformation of insoluble compounds and carrying out co-cyclic utilization on cyclodextrin and cells is not visible.
The invention content is as follows:
in order to solve the technical problems, the invention provides a cyclodextrin medium for steroid biocatalysis reaction and a method for catalyzing cell recycling, which specifically comprises the following steps: in the steroid biocatalysis reaction, derivative cyclodextrin-sodium alginate immobilized cell colloidal beads are used as a catalyst, after the reaction is finished, filtration is carried out, filtrate is used for collecting reaction products, and the filtrate, namely the derivative cyclodextrin-sodium alginate immobilized cell colloidal beads, is washed for 1-8 times by using a reaction solution and then is reused for the steroid biocatalysis reaction, so that the cyclic utilization of a cyclodextrin medium and cells is realized;
the reaction solution is Tris-HCl buffer solution, normal saline or pure water and the like, and the pH value is 7.0-7.6;
the dosage of the reaction solution for washing the cell gel beads is 10-100m L per gram of the cell gel beads;
the preparation method of the derivative cyclodextrin-sodium alginate immobilized cell gel bead comprises the following steps:
(1) sodium alginate grafted cyclodextrin
Weighing sodium alginate and derivative cyclodextrin according to the mol ratio of 1:0.2-1:1.5, adding into a triangular flask, adding 45-55 times (mass volume ratio) of distilled water, and stirring in water bath at 30-70 deg.C to dissolve completely;
adding epoxy chloropropane according to the volume ratio of the epoxy chloropropane to the distilled water of 0.05-1:50, simultaneously dropwise adding 0.5 mol/L NaOH solution according to the volume ratio of the epoxy chloropropane to the NaOH solution of 0.05-1:10, dropwise adding for 10min, and then reacting for 1.5-6.5 h;
(2) preparation of immobilized cell gel beads
Cooling the reaction liquid in the step (1), adding the thalli with the final concentration of 1-30 g/L for restingThe cells are stirred evenly and added with CaCl of 0.1-0.5 mol/L by a syringe under magnetic stirring2In solution, the gel beads are placed in CaCl2Continuously soaking in the solution for 1-6h, filtering, and washing with the reaction solution for 1-8 times to obtain derivative cyclodextrin-sodium alginate immobilized cell gel beads;
the reaction solution is Tris-HCl buffer solution, normal saline or pure water and the like, and the pH value is 7.0-7.6;
the derivative cyclodextrin is hydroxypropyl- β -cyclodextrin, methyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin, carboxymethyl- β -cyclodextrin, hydroxyethyl- β -cyclodextrin, sulfo- β -cyclodextrin, hydroxypropyl-gamma-cyclodextrin or methyl-gamma-cyclodextrin;
the derivative cyclodextrin-sodium alginate immobilized cell gel beads can be recycled for more than 8 times under the condition of not supplementing cyclodextrin and microbial cells;
the derivatized cyclodextrin-sodium alginate immobilized cell gel beads can prolong the recycling times through activation, and the activation method is as follows:
(1) adding 10g of the derivative cyclodextrin-sodium alginate immobilized cell gel beads with reduced catalytic efficiency into a fermentation medium of 30m L for activation, and carrying out shake culture at 160r/min and 32 ℃ for 20h in a shaking table;
the fermentation medium is used for fermenting and culturing the thalli;
(2) filtering the fermentation liquid to obtain rubber beads after the culture is finished, washing the rubber beads with the reaction solution, and placing the rubber beads into CaCl2Fixing the solution for 2h again, washing the reaction solution and collecting the rubber beads for 1-8 times, and storing in a refrigerator at 4 ℃ for later use;
the activated derived cyclodextrin-sodium alginate immobilized cell gel beads can be recycled for 3-5 times.
Has the advantages that:
(1) the invention realizes the cyclic utilization of cyclodextrin medium and cells for the first time, the conversion rate of steroid substrate is still more than 90% after 8 times of circulation, the purpose of reducing production cost while improving the efficiency of steroid catalytic reaction is achieved, and the invention has good application value and popularization prospect.
(2) The invention can improve the initial conversion rate of the steroid substrate biocatalysis and the final conversion rate.
(3) The cyclodextrin recycling process method is simple, convenient and fast, convenient to implement and cost-saving.
The specific implementation mode is as follows:
the following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: derivative cyclodextrin-sodium alginate immobilized cell gel bead and application thereof in cortisone acetate C1,2Application in site dehydrogenation reaction
The microorganism strain adopts Arthrobacter simplex (TCCC 11037), which can realize C of steroid compound1，2Transforming position dehydrogenation to transform Cortisone Acetate (CA) into prednisone acetate;
slant culture medium (g/L) glucose 10, yeast extract 10, agar 20, pH 7.2;
a seed culture medium (g/L) including glucose 10, corn steep liquor 10, peptone 5, dipotassium hydrogen phosphate 2.5, and pH 7.2;
fermentation medium (g/L) glucose 10, corn steep liquor 15, peptone 5, dipotassium hydrogen phosphate 2.5, pH 7.2;
preparation of resting cells of Arthrobacter simplex:
TCCC 11037 is cultured for 18 hours under the conditions of 32 ℃, 160r/min and 30/250m L seed culture medium loading, then is inoculated into a 250m L shaking flask containing 30m L fermentation culture medium according to the inoculation amount of 5 percent, is cultured for 20 hours under the conditions of 32 ℃ and 160r/min, cells obtained by fermentation culture are centrifuged at 5000r/min and washed for 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;
preparing a derivative cyclodextrin grafted sodium alginate immobilized cell:
accurately weighing 0.99g (5.0mmol) of sodium alginate, adding hydroxypropyl- β -cyclodextrin with the molar ratio of 1:0.2 into a triangular flask with the diameter of 100m L, adding about 50m L distilled water, electrically stirring in a water bath at the temperature of 30 ℃ to completely dissolve the sodium alginate, adding 0.6m L of epoxy chloropropane, simultaneously dropwise adding 10m L0.5 mol/L of NaOH solution, dropwise adding the NaOH solution for about 10min, and after about 3.5hStopping the reaction, after the reaction liquid is cooled, adding the thalline resting cell suspension to ensure that the thalline concentration is 20 g/L, uniformly stirring the suspension, and dropwise adding 0.3 mol/L mol of CaCl by using an injector under the condition of magnetic stirring2In solution, the gel beads are placed in CaCl2The solution was further soaked for 3h, filtered and washed 4 times with Tris-HCl (pH 7.2).
And (3) biotransformation:
weighing 0.06g of CA in a 100m L triangular flask, adding 20m L of Tris-HCl (pH 7.2), adding 10g of the cyclodextrin-sodium alginate immobilized cell gel beads (the grafted hydroxypropyl- β -cyclodextrin is 0.043g), adding sodium alginate immobilized cell gel beads without grafting cyclodextrin in a control group, controlling the added thallus to be equal to the experimental group, converting at 34 ℃ and 180r/min for 8h, and determining the substrate conversion rate by an HP L C method;
the cyclic utilization process of cyclodextrin and cells comprises the following steps:
grafting and fixing cells by using cyclodextrin and sodium alginate, then using the cells for biocatalytic reaction of CA, collecting derivative cyclodextrin-sodium alginate immobilized cell colloidal beads after the reaction is finished, washing the cell colloidal beads for 3 times by using Tris-HCl (pH 7.2), wherein the using amount is 50ml per gram of cell colloidal beads, washing the cell colloidal beads, and then using the cell colloidal beads for bioconversion of cortisone acetate again, wherein the using amount is unchanged, and determining the substrate conversion rate after each cycle by using an HP L C method;
after 5 times of recycling, the conversion rate is reduced to 91%, and the derivative cyclodextrin-sodium alginate immobilized cell gel beads are activated, and the steps are as follows:
putting 10g of the recovered derivatized cyclodextrin-sodium alginate immobilized cell gel beads into a 30m L fermentation medium for activation, and carrying out shake culture at 160r/min and 32 ℃ for 20h on a shaking table;
filtering the fermentation liquid to obtain gel beads after the culture is finished, washing the gel beads with Tris-HCl (pH 7.2) buffer solution, and placing the gel beads into CaCl2Fixing the solution for 2 hours again, washing and collecting the rubber beads, and using the rubber beads for the biological catalytic reaction of CA again;
the results showed that the primary conversion was 86% in the control group, 95% in the sodium alginate-immobilized cell gel beads grafted with hydroxypropyl- β -cyclodextrin, and the initial conversion rate was that of the control group (1).1×10-2g/L min-1) 1.3 times of that of the final conversion of CA after 8 cycles, 92%, see table 1.
TABLE 1
Number of cycles 1 2 3 4 5 6 7 8
Conversion rate 95％ 96％ 95％ 94％ 91％ 94％ 92％ 92％
Example 2:
the procedure was as in example 1 except for the following.
Preparing a derivative cyclodextrin grafted sodium alginate immobilized cell:
accurately weighing 0.99g (5.0mmol) of sodium alginate, adding sulfobutyl- β -cyclodextrin with the molar ratio of 1:1.5 into a 100m L triangular flask, adding 50m L distilled water, electrically stirring in a water bath at 60 ℃ to completely dissolve the sodium alginate, adding 1m L of epoxy chloropropane, simultaneously dropwise adding 10m L0.5/0.5 mol/L of NaOH solution, stopping the reaction after about 10min, cooling the reaction solution, adding a thalli resting cell suspension to ensure that the thalli concentration is 30 g/L, uniformly stirring the suspension, dropwise adding 0.5 mol/L of CaCl by using an injector under the condition of magnetic stirring2In solution, the gel beads are placed in CaCl2The solution was further soaked for 6h, filtered and washed 3 times with physiological saline (pH 7.2) for further use.
And (3) biotransformation:
weighing 0.06g of CA in a 100m L triangular flask, adding 20m L of normal saline (pH 7.2), adding 10g of the cyclodextrin-sodium alginate immobilized cell colloid beads (the grafted sulfobutyl- β -cyclodextrin is 0.119g), adding sodium alginate immobilized cell colloid beads without grafting cyclodextrin in a control group, controlling the added thallus to be equal to the experimental group, converting at 34 ℃ and 180r/min for 8 hours, and determining the substrate conversion rate by an HP L C method;
grafting cyclodextrin and sodium alginate, and performing biocatalytic reaction on the CA, collecting the derivative cyclodextrin-sodium alginate immobilized cell colloidal beads after the reaction is finished, washing the cell colloidal beads with physiological saline (pH 7.2) for 4 times, wherein the dosage is 80ml per gram of the cell colloidal beads, and reusing the cell colloidal beads after washing for biotransformation of cortisone acetate, wherein the dosage is unchanged.
The results show that the initial conversion rate of the grafted sulfobutyl- β -cyclodextrin-sodium alginate immobilized cell gel beads for the first time in CA biocatalysis is that of the control group (1.1 × 10)-2g/L min-1) 1.4 times of the total amount of the active components, activating sulfobutyl- β -cyclodextrin-sodium alginate immobilized cells after 5 times of cyclic utilization, wherein the activation method is the same as that of example 1, the cells are continuously and cyclically utilized for 3 times after activation, and the final conversion rate of CA is 94%.
Example 3:
the procedure was as in example 1 except for the following.
Cyclodextrin grafting sodium alginate immobilized cells:
accurately weighing 0.99g (5.0mmol) of sodium alginate, adding methyl- β -cyclodextrin with the molar ratio of 1:1 into a 100m L triangular flask, adding 50m L distilled water, electrically stirring in a 70 ℃ water bath to completely dissolve the sodium alginate, adding 0.1m L of epoxy chloropropane, simultaneously dropwise adding 10m L0.5 mol/L of NaOH solution, stopping the reaction after about 10min, cooling the reaction solution, adding a thalli resting cell suspension to ensure that the thalli concentration is 15 g/L, uniformly stirring the suspension, dropwise adding 0.25 mol/L of CaCl by using a syringe under the magnetic stirring condition2In solution, the gel beads are placed in CaCl2The solution was further soaked for 2h, filtered and washed 2 times with Tris-HCl (pH 7.5).
And (3) biotransformation:
weighing 0.06g of CA in a 100m L triangular flask, adding 20m L of Tris-HCl (pH 7.5), adding 10g of derivative cyclodextrin-sodium alginate immobilized cell gel beads (the amount of grafted methyl- β -cyclodextrin is 0.063g), adding sodium alginate immobilized cell gel beads without grafting cyclodextrin in a control group, controlling the added thallus to be equal to the experimental group, converting at 34 ℃ and 180r/min for 8h, and determining the substrate conversion rate by an HP L C method;
grafting cyclodextrin and sodium alginate, and then using the grafted cyclodextrin and sodium alginate for biocatalytic reaction of CA, collecting the derivative cyclodextrin-sodium alginate immobilized cell colloidal beads after the reaction is finished, washing the derivative cyclodextrin-sodium alginate immobilized cell colloidal beads for 2 times by using Tris-HCl (pH 7.5), wherein the using amount is 100ml per gram of cell colloidal beads, and reusing the washed cell colloidal beads for biotransformation of cortisone acetate without changing the using amount.
The results show that the initial conversion rate of the grafted methyl- β -cyclodextrin-sodium alginate immobilized cell gel beads for the first time in CA biocatalysis is the control group (1.1 × 10)-2g/L min-1) 1.5 times of the total amount of the active component, and activating the methyl- β -cyclodextrin-sodium alginate immobilized cells after 5 times of cyclic utilization, wherein the activation method is the same as that of example 1, the cells are continuously recycled for 4 times after activation, and the final conversion rate of CA is 92%.
Although the present invention has been described in detail above with reference to the general description, the specific embodiments and experiments, it can be modified or improved based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. A method for deriving a cyclodextrin medium and catalyzing cells to be recycled in steroid biocatalysis reaction is characterized by comprising the following steps: in the steroid biocatalysis reaction, derivative cyclodextrin-sodium alginate immobilized cell gel beads are used as a catalyst, after the reaction is finished, filtration is carried out, filtrate is used for collecting reaction products, and the filtrate, namely the derivative cyclodextrin-sodium alginate immobilized cell gel beads, is put into the steroid biocatalysis reaction again for recycling;
the preparation method of the derivative cyclodextrin-sodium alginate immobilized cell gel bead comprises the following steps:
(1) sodium alginate graft derivative cyclodextrin
Weighing sodium alginate and derivative cyclodextrin according to the mol ratio of 1:0.2-1:1.5, adding into a triangular flask, adding 45-55 times of distilled water, and stirring in water bath at 30-70 deg.C to dissolve completely;
adding epoxy chloropropane according to the volume ratio of the epoxy chloropropane to the distilled water of 0.05-1:50, simultaneously dropwise adding 0.5 mol/L NaOH solution according to the volume ratio of the epoxy chloropropane to the NaOH solution of 0.05-1:10, dropwise adding for 10min, and then reacting for 1.5-6.5 h;
(2) preparation of immobilized cell gel beads
After the reaction liquid in the step (1) is cooled, adding thalline resting cells with the final concentration of 1-30 g/L, stirring uniformly, and dripping 0.1-0.5 mol/L of CaCl by using a syringe under the magnetic stirring2In solution, the gel beads are placed in CaCl2Continuously soaking in the solution for 1-6h, filtering, and washing with the reaction solution for 1-8 times to obtain derivative cyclodextrin-sodium alginate immobilized cell gel beads;
the reaction solution is Tris-HCl buffer solution, normal saline or pure water, and the pH value is 7.0-7.6.
2. The method of claim 1, wherein the number of times of recycling of the derivatized cyclodextrin medium and the catalyzed cell gel bead is 8 or more.
3. The method of claim 1, wherein the derivatized cyclodextrin is hydroxypropyl- β -cyclodextrin, methyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin, carboxymethyl- β -cyclodextrin, hydroxyethyl- β -cyclodextrin, sulfo- β -cyclodextrin, hydroxypropyl- γ -cyclodextrin or methyl- γ -cyclodextrin.
4. The method for deriving cyclodextrin media and catalyzing cell recycling in steroid biocatalytic reactions as claimed in claim 1, wherein said derived cyclodextrin-sodium alginate immobilized cell gel beads are activated for extended recycling times, said activation method specifically comprising:
(1) adding 10g of the derivative cyclodextrin-sodium alginate immobilized cell gel beads with reduced catalytic efficiency into a fermentation medium of 30m L for activation, and carrying out shake culture at 160r/min and 32 ℃ for 20h in a shaking table;
the fermentation medium is used for fermenting and culturing the thalli;
(2) filtering the fermentation liquid to obtain rubber beads after the culture is finished, washing the rubber beads with the reaction solution, and placing the rubber beads into CaCl2Fixing the solution for 2h again, washing the reaction solution to collect the rubber beads for 1-8 times, and storing in a refrigerator at 4 ℃ for later use.
5. The method of claim 4, wherein the activated cyclodextrin-sodium alginate-derived immobilized cell gel beads can be recycled 3-5 times.
6. The method for derivatizing cyclodextrin media and recycling catalytic cells in steroid biocatalytic reactions as claimed in claim 1, wherein the filtered cell gel beads are washed 1-8 times with a reaction solution, and reused for steroid biocatalytic reactions, wherein the reaction solution is Tris-HCl buffer solution, physiological saline or pure water, ph7.0-ph 7.6.
7. The method for derivatizing cyclodextrin media and recycling catalytic cells in steroid biocatalytic reactions as claimed in claim 6, wherein said reaction solution for washing cell gel beads is used in an amount of 10-100m L per gram of cell gel beads.
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