WO2018227644A1 - 一种催化甾体生物转化的方法 - Google Patents

一种催化甾体生物转化的方法 Download PDF

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WO2018227644A1
WO2018227644A1 PCT/CN2017/088954 CN2017088954W WO2018227644A1 WO 2018227644 A1 WO2018227644 A1 WO 2018227644A1 CN 2017088954 W CN2017088954 W CN 2017088954W WO 2018227644 A1 WO2018227644 A1 WO 2018227644A1
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cyclodextrin
alginate
reaction
solution
beads
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PCT/CN2017/088954
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English (en)
French (fr)
Inventor
王敏
申雁冰
骆健美
牛璐璐
于子棋
郑宇�
夏梦雷
宋佳
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天津科技大学
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Priority claimed from CN201710452656.0A external-priority patent/CN107022542B/zh
Priority claimed from CN201710452644.8A external-priority patent/CN107236779B/zh
Priority claimed from CN201710452650.3A external-priority patent/CN107164360B/zh
Application filed by 天津科技大学 filed Critical 天津科技大学
Publication of WO2018227644A1 publication Critical patent/WO2018227644A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating

Definitions

  • the invention belongs to the technical field of biocatalysis, and in particular relates to a method for catalyzing the biotransformation of steroids.
  • Steroidal compounds are the second largest class of drugs after antibiotics, which have functions of regulating the metabolism and physiological functions of various substances in life.
  • the industrial production of steroidal drugs is mainly to modify the structure of natural steroidal compounds.
  • the method of microbial transformation can form a variety of steroidal active intermediates with high purity.
  • cyclodextrin can increase the solubility of hydrophobic steroids, and its special cavity structure can encapsulate the steroid substrate, thereby improving the bioavailability and yield of steroid compounds.
  • the higher price of cyclodextrin restricts its wide application in the field of biocatalysis. It is an important problem to be solved by using suitable methods to expand the application of cyclodextrin in biotransformation reaction.
  • the cyclodextrin is immobilized on a polymer carrier such as chitosan or sodium alginate by a grafting technique, and the ⁇ -cyclodextrin can be prepared from a water-soluble powder material into a water-insoluble polymer material, thereby overcoming The defect that cyclodextrin is difficult to recycle makes it possible to recycle and reduce the cost of industrial use.
  • the grafted cyclodextrin still retains the unique cavity structure and other excellent properties, and also has the good mechanical properties and stability of the polymer carrier, which greatly broadens the application space of the cyclodextrin and enhances it. Application value.
  • Sodium alginate is a natural polyanionic polysaccharide polymer compound composed of two units, G and M. When it is in contact with water, the surface is sticky, which makes the solution also sticky. It is safe, non-toxic, low in cost, biodegradable and biocompatible, and has the properties required for pharmaceutical excipients. When it encounters cations such as Ca 2+ and Zn 2+ , it will chemically react to form a gel ball. In view of this property, it can be used as a sustained release and controlled release material for drugs, as well as for immobilizing unstable substances, improving stability, and can also be used for immobilizing microorganisms, cells, enzymes, etc., to achieve recycling. At present, the application of sodium alginate mainly involves pesticides, food, biomedicine and other fields, and has also been gradually extended to the treatment of heavy metal pollution.
  • Chinese patent CN 105754984-A discloses a sodium alginate composite immobilized bacteria agent, a preparation method thereof and use thereof, and discloses a sodium alginate composite immobilized bacteria agent and a preparation method thereof, which solve the problem that the prior art is not suitable for dichloroquine. The problem of the acidification of the immobilization of Pseudomonas.
  • the present invention comprises a granule prepared by mixing a carrier medium having Pseudomonas and sodium alginate, and dropping it into calcium chloride having a concentration of 1 to 4% for 2 to 4 hours; the sodium alginate The mass concentration is 2 to 6%; the carrier medium comprises an adsorption carrier and an inorganic salt liquid medium; the weight ratio of the adsorption carrier to the sodium alginate is 1 to 3:1, and the adsorption carrier has a weight ratio of 1:1. ⁇ 2:1 corncob, bamboo charcoal and oil dry.
  • the invention effectively improves the degradation efficiency of the bacteria body; and the degradation rate of the immobilized bacteria agent of the invention is far higher than the sum of the physical adsorption efficiency of the blank ball and the biodegradation efficiency of the free strain, and can effectively achieve the mutual promotion effect.
  • sodium alginate contains two hydrophilic groups of -COOH and -OH, has strong hydrophilicity, and can be grafted with cyclodextrin by using epichlorohydrin as a crosslinking agent. The formed cyclodextrin-alginate graft can immobilize the cells to achieve co-circulation of cyclodextrin and cells.
  • the present invention provides a method for catalyzing the biotransformation of a steroid, which can realize the recycling of a reaction solution, a cyclodextrin and a microbial cell, as follows: In the steroid biocatalytic reaction, the cyclodextrin-alginate immobilized cell beads are used as a catalyst.
  • the sperm-alginate-immobilized cell beads are washed with the reaction solution for 1-8 times, and then reused for the biocatalytic reaction of the steroid, thereby realizing the cyclodextrin medium and cell recycling;
  • the reaction solution is a Tris-HCl buffer solution, physiological saline, acetic acid-sodium acetate buffer or pure water, etc., pH 5.0-pH 7.6;
  • the cyclodextrin is a natural cyclodextrin or a derived cyclodextrin; In another embodiment of the present application, the natural or derived cyclodextrin is specifically ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclo
  • the beads are further immersed in the CaCl2 solution for 0.5-6 h, filtered, and washed with the reaction solution for 1-8 times to obtain a cyclodextrin-alginate-immobilized cell beads;
  • the cyclodextrin-alginate-immobilized cell beads are prepared as follows: (1) Sodium alginate grafted cyclodextrin Sodium alginate and cyclodextrin were weighed according to sodium alginate and cyclodextrin at a molar ratio of 1:0.2-1:1.5, added to a triangular flask, and 45-55 times (mass to volume) distilled water of sodium alginate was added.
  • the beads are further soaked in the CaCl2 solution for 1-6 h, filtered, and washed with the reaction solution for 1-8 times to obtain a cyclodextrin-alginate-immobilized cell beads;
  • the cyclodextrin-alginate-immobilized cell beads are prepared as follows: (1) Sodium alginate grafted cyclodextrin Sodium alginate and cyclodextrin were weighed according to sodium alginate and cyclodextrin molar ratio 1: 0.5-1:1.5, and distilled water of 30 times (mass to volume ratio) of sodium alginate was added, and stirred in a water bath at 70 ° C.
  • the beads are further immersed in a CaCl2 solution for 0.5-2 h, filtered, and washed with the reaction solution for 1-6 times to obtain a cyclodextrin-alginate-immobilized cell beads;
  • the cyclodextrin-alginate-immobilized cell beads can be recycled more than 6 times without adding cyclodextrin and microbial cells;
  • the cyclodextrin-alginate-immobilized cell beads can be recycled more than 8 times without adding cyclodextrin and microbial cells;
  • the cyclodextrin-alginate-immobilized cell beads can prolong the number of recycling by activation, and the activation method is specifically as follows: (1) 10-40 g of cyclodextrin-alginate-immobilized cell beads reduced in catalytic efficiency, and put into 30-50 mL of fermentation medium for activation, 150-200 r/min, under a shake
  • the cyclodextrin-alginate-immobilized cell beads can prolong the number of recycling by activation, and the activation method is specifically as follows: (1) 10 g of cyclodextrin-alginate-immobilized cell beads reduced in catalytic efficiency, and put into 30 mL fermentation medium for activation, 160 r/min, shaking culture under shaking at 32 ° C for 20 h; The fermentation medium is a medium used for fermenting the cells; (2) After the completion of the culture, the fermentation broth is filtered to obtain a rubber bead, and the rubber beads are washed with the reaction solution, and then fixed in a CaCl 2 solution for 2 h, and the reaction solution is washed and collected for 1-8 times, and placed in 4 Store in the refrigerator at °C; In another embodiment of the present application, the cyclodextrin-alginate-immobilized cell beads can prolong the number of recycling by activation, and the activation method is specifically as follows: (1) 20-40g of cyclodextrin-al
  • the present invention realizes the recycling of cyclodextrin medium, cells and reaction solution for the first time, and reduces the production cost and environmental pollution while improving the efficiency of the catalytic reaction of the steroid, and achieves the purpose of biotransformation by the green method. Application value and promotion prospects.
  • the present invention can increase the initial conversion rate of the biocatalyst of the steroid substrate and increase the final conversion rate.
  • Example 1 Application of cyclodextrin-alginate-immobilized cell beads and its dehydrogenation reaction at the C1,2 position of cortisone acetate
  • the microbial strain uses Arthrobacter simplex TCCC 11037, which can realize the dehydrogenation conversion of the steroid compound at C1 and 2, and convert cortisone acetate (CA) into prednisone acetate.
  • CA cortisone acetate
  • Slant medium (g/L): glucose 10, yeast extract 10, agar 20, pH 7.2; Seed medium (g/L): glucose 10, corn syrup 10, peptone 5, dipotassium hydrogen phosphate 2.5, pH 7.2; Fermentation medium (g/L): glucose 10, corn syrup 15, peptone 5, dipotassium hydrogen phosphate 2.5, pH 7.2; Preparation of simple resting cells of Arthrobacter: TCCC 11037 was cultured at 32 °C, 160 r/min, 30/250 mL seed medium loading for 18 h, then inoculated into a 250 mL shake flask containing 30 mL of fermentation medium at 32 °C according to 5% inoculum.
  • Bacillus resting cell suspension Preparation of cyclodextrin-grafted sodium alginate immobilized cells: Accurately weigh 0.99g (5.0mmol) of sodium alginate, add ⁇ -cyclodextrin with a molar ratio of 1:0.7 to a 100mL flask, add about 50mL of distilled water, and stir it completely in a 60°C water bath.
  • Biotransformation Weigh 0.06 g of CA into a 100 mL flask, add 20 mL of Tris-HCl (pH 7.2), and add 10 g of cyclodextrin-alginate-immobilized cell beads prepared by the above procedure (grafted). The amount of ⁇ -cyclodextrin was 0.115 g), and the control group was added with sodium alginate immobilized cell beads without grafting cyclodextrin, and the added cells were controlled in the same amount as the experimental group, and transformed at 34 ° C and 180 r/min.
  • the steps are as follows: The recovered 10 g of cyclodextrin-alginate-immobilized cell beads were put into 30 mL fermentation medium for activation, and stirred at 160 r/min for 32 h under shaking at 32 °C; After the completion of the culture, the fermentation broth was filtered to obtain a bead, and the beads were washed with a pH 7.2 Tris-HCl buffer, and then fixed in a CaCl 2 solution for 2 h, washed to collect the beads, and used again for CA biocatalysis.
  • Cyclodextrin grafted sodium alginate immobilized cells Accurately weigh 0.99g (5.0mmol) of sodium alginate, add ⁇ -cyclodextrin with a molar ratio of 1:1.5 in a 100mL flask, add about 50mL of distilled water, and stir it completely in a 70°C water bath. 1 mL of epichlorohydrin was added, and 10 mL of a 0.5 mol/L NaOH solution was added dropwise thereto, and the mixture was dropped in about 10 minutes, and the reaction was stopped after about 6 hours. After the reaction solution was cooled, the cell suspension cell suspension was added to make the cell concentration 30 g/L.
  • the amount was 0.072g), and the control group was added with sodium alginate immobilized cell beads which were not grafted with cyclodextrin, and transformed at 34 °C and 180 r/min for 8 h.
  • the substrate conversion rate was determined by HPLC. After the cyclodextrin was grafted with sodium alginate, it was used for the biocatalytic reaction of the above CA. After the reaction, the cyclodextrin-alginate-immobilized cell beads were collected and washed twice with physiological saline (pH 7.5). The dosage is 100 ml per gram of cell beads, and is reused for bioconversion of cortisone acetate after washing, and the dosage is unchanged.
  • Cyclodextrin grafted sodium alginate immobilized cells Accurately weigh 0.99g (5.0mmol) of sodium alginate, add ⁇ -cyclodextrin with a molar ratio of 1:0.2 in a 100mL flask, add about 50mL of distilled water, and stir it completely in a 30°C water bath. 0.05 mL of epichlorohydrin was added, and 10 mL of 0.5 mol/L NaOH solution was added dropwise, and the mixture was dropped in about 10 minutes, and the reaction was stopped after about 1.5 hours. After the reaction solution was cooled, the cell suspension cell suspension was added so that the cell concentration was 1 g/L.
  • the amount was 0.058 g), and the control group was added with sodium alginate immobilized cell beads which were not grafted with cyclodextrin, and transformed at 34 ° C, 180 r / min for 8 h, and the substrate conversion rate was determined by HPLC;
  • the cyclodextrin was grafted with sodium alginate for the biocatalytic reaction of the above CA. After the reaction, the cyclodextrin-alginate-immobilized cell beads were collected and washed twice with Tris-HCl (pH 7.5).
  • the dosage is 100ml per gram of cell beads, and is reused for bioconversion of cortisone acetate after washing, and the dosage is unchanged.
  • Example 4 Derivatization of cyclodextrin-alginate-immobilized cell beads and its application in the dehydrogenation of C1,2 position of cortisone acetate
  • the microbial strain uses Arthrobacter simplex TCCC 11037, which can realize the dehydrogenation conversion of the steroid compound at C1 and 2, and convert cortisone acetate (CA) into prednisone acetate.
  • CA cortisone acetate
  • Slant medium (g/L): glucose 10, yeast extract 10, agar 20, pH 7.2; Seed medium (g/L): glucose 10, corn syrup 10, peptone 5, dipotassium hydrogen phosphate 2.5, pH 7.2; Fermentation medium (g/L): glucose 10, corn syrup 15, peptone 5, dipotassium hydrogen phosphate 2.5, pH 7.2; Preparation of simple resting cells of Arthrobacter: TCCC 11037 was cultured at 32 °C, 160 r/min, 30/250 mL seed medium loading for 18 h, then inoculated into a 250 mL shake flask containing 30 mL of fermentation medium at 32 °C according to 5% inoculum.
  • Biotransformation Weigh 0.06 g of CA into a 100 mL flask, add 20 mL of Tris-HCl (pH 7.2), and add 10 g of cyclodextrin-alginate-immobilized cell beads prepared by the above procedure (grafted).
  • the amount of hydroxypropyl- ⁇ -cyclodextrin was 0.043 g), and the control group was added with sodium alginate immobilized cell beads which were not grafted with cyclodextrin, and the added cells were controlled in the same amount as the experimental group, 34 ° C, 180 r /min was transformed for 8 h, and the conversion rate of the substrate was determined by HPLC; Cyclodextrin and cell recycling process: The cyclodextrin and sodium alginate were grafted to fix the cells, and then used for the biocatalytic reaction of the above CA. After the reaction, the cyclodextrin-alginate-immobilized cell beads were collected and used as Tris-HCl (pH 7.2).
  • the dosage is 50ml per gram of cell beads, and after re-use for the biotransformation of cortisone acetate, the dosage is unchanged, and the substrate conversion rate after each cycle is determined by HPLC; After 5 cycles of recycling, the conversion rate was reduced to 91%, and the cyclodextrin-alginate-immobilized cell beads were activated.
  • the steps are as follows: The recovered 10 g of the derived cyclodextrin-alginate-immobilized cell beads were put into a 30 mL fermentation medium for activation, and the mixture was shaken at 160 r/min for 20 h under a shaking at 32 °C; After the completion of the culture, the fermentation broth was filtered to obtain a bead, and the beads were washed with Tris-HCl (pH 7.2) buffer, and then fixed in a CaCl 2 solution for 2 h, washed to collect the beads, and again used for the CA organism.
  • Tris-HCl pH 7.2
  • Preparation of derivatized cyclodextrin-grafted sodium alginate immobilized cells Accurately weigh 0.99g (5.0mmol) of sodium alginate, add sulfobutyl- ⁇ -cyclodextrin with a molar ratio of 1:1.5 in a 100mL flask, add about 50mL of distilled water, and stir it in a 60°C water bath. Completely dissolved, add 1 mL of epichlorohydrin, while adding 10 mL of 0.5 mol/L NaOH solution, drip about 10 min, and stop the reaction after 6.5 h. After the reaction solution was cooled, the cell suspension cell suspension was added to make the cell concentration 30 g/L.
  • the amount of butyl- ⁇ -cyclodextrin was 0.119 g), and the control group was added with sodium alginate immobilized cell beads which were not grafted with cyclodextrin, and the added cells were controlled in the same amount as the experimental group, 34 ° C, 180 r / Min conversion for 8 h, HPLC conversion of substrate conversion; After the cyclodextrin is grafted with sodium alginate, it is used for the biocatalytic reaction of the above CA. After the reaction, the cyclodextrin-alginate-immobilized cell beads are collected and washed four times with physiological saline (pH 7.2).
  • the dosage is 80ml per gram of cell gel beads, and is reused for bioconversion of cortisone acetate after washing, and the dosage is unchanged.
  • the results showed that the initial conversion rate of grafted sulfobutyl- ⁇ -cyclodextrin-alginate-immobilized cell beads for CA biocatalysis was 1.4 in the control group (1.1 ⁇ 10-2 g/L min-1). After doubling and recycling for 5 times, the sulfobutyl- ⁇ -cyclodextrin-alginate-immobilized cells were activated, and the activation method was the same as in Example 1. After the activation, the cycle was continued for 3 times, and the final conversion rate of CA was 94. %.
  • Cyclodextrin grafted sodium alginate immobilized cells Accurately weigh 0.99g (5.0mmol) of sodium alginate, add methyl- ⁇ -cyclodextrin with a molar ratio of 1:1 to a 100mL flask, add about 50mL of distilled water, and stir it in a 70°C water bath. Completely dissolved, 0.1 mL of epichlorohydrin was added, and 10 mL of a 0.5 mol/L NaOH solution was added dropwise, and the mixture was dropped in about 10 minutes, and the reaction was stopped after about 1.5 hours.
  • the cell suspension cell suspension was added to make the cell concentration 15 g/L.
  • the suspension was stirred uniformly, and dropped into a 0.25 mol/L CaCl 2 solution by a magnetic stirring under a magnetic stirring.
  • the beads were further immersed in a CaCl 2 solution for 2 hours, filtered, and washed twice with Tris-HCl (pH 7.5).
  • Biotransformation Weigh 0.06 g of CA into a 100 mL flask, add 20 mL of Tris-HCl (pH 7.5), and add 10 g of derivatized cyclodextrin-alginate-immobilized cell beads (grafted methyl- ⁇ - The amount of cyclodextrin was 0.063 g).
  • the control group was added with sodium alginate immobilized cell beads without grafting cyclodextrin.
  • the cells were controlled to be equal to the experimental group, and transformed at 34 °C and 180 r/min for 8 h. Determination of substrate conversion by HPLC;
  • the cyclodextrin is grafted with sodium alginate for the biocatalytic reaction of the above CA. After the reaction, the cyclodextrin-alginate-immobilized cell beads are collected and washed with Tris-HCl (pH 7.5).
  • the dosage is 100 ml per gram of cell beads, and is reused for bioconversion of cortisone acetate after washing, and the dosage is unchanged.
  • Example 7 Cyclodextrin-alginate-immobilized cell beads and its application in hydroxylation of 17 ⁇ hydroxyprogesterone
  • the microbial strain uses Aspergillus ochraceus CICC41473, which can realize hydroxylation of C11 at the steroidal compound and convert 17 ⁇ hydroxyprogesterone to 11,17 ⁇ dihydroxyprogesterone;
  • Preparation of Aspergillus oryzae resting cells The Aspergillus oryzae spore suspension was prepared by the transformation of Aspergillus oryzae and inserted into the fermentation medium (50 mL medium/250 mL flask) so that the final concentration of spores in the flask was 106/mL, 28 °C, 200 r / min culture for 18
  • Biotransformation Weigh 0.06 g of 17 ⁇ -hydroxyprogesterone in a 100 mL flask, add 20 mL of Tris-HCl (pH 7.0), and add 40 g of cyclodextrin-alginate-immobilized cell beads prepared in the above procedure.
  • the control group was added with sodium alginate immobilized cell beads without grafting cyclodextrin, and the added cells were controlled in the same amount as the experimental group, and transformed at 28 °C, 200 r/min for 18 h, and the substrate conversion rate was determined by HPLC; Cyclodextrin, fungal cells and reaction solution recycling process: The cyclodextrin and the sodium alginate were grafted to the cells to be used for the hydroxylation reaction of the above 17 ⁇ hydroxyprogesterone.
  • the cyclodextrin-alginate-immobilized cell beads were collected by filtration and used for Tris-HCl ( The solution was washed 3 times with pH 7.0), and the amount of the reaction solution was 50 ml per gram of cell beads.
  • the filtrate was centrifuged at 5000 r/min for 10 min to obtain a secondary filtrate. Ethyl acetate twice the amount of the second filtrate was added, and extracted three times.
  • the raffinate phase was vacuum-dried at 45 ° C to remove residual ethyl acetate, and the mixture was filtered.
  • the sperm-alginate immobilized cell beads were re-introduced into the steroid biocatalytic reaction for recycling, and the substrate conversion rate after each cycle was determined by HPLC; The results showed that the initial conversion rate of the control group was 80%, and the initial conversion rate of the sodium alginate immobilized cell beads coated with hydroxypropyl- ⁇ -cyclodextrin was 96%. After 6 cycles of recycling, the 17 ⁇ hydroxy luteal body The final conversion of ketones is still as high as 92%, see Table 1.
  • the cyclodextrin-alginate-immobilized cell beads were activated after 6 cycles of recycling.
  • the activation method was as follows: (1) 20 g of cyclodextrin-alginate-immobilized cell beads with reduced catalytic efficiency, and put into 50 mL fermentation medium for activation, 150 r/min, shaking culture at 28 °C for 18 h; The fermentation medium is a medium used for fermenting the cells; (2) After the completion of the culture, the fermentation broth was filtered to obtain a bead. After washing the beads with Tris-HCl, it was fixed in CaCl2 solution for 1 h, and the reaction solution was washed and collected for 3 times, and placed at 4 °C. Store in the refrigerator and continue to recycle 3 times. The results are shown in Table 3.
  • Biotransformation Weigh 0.06 g of 17 ⁇ -hydroxyprogesterone in a 100 mL flask and add 20 mL of normal saline. In the experimental group, add 40 g of cyclodextrin-alginate-immobilized cell beads prepared in the above procedure. The sodium alginate immobilized cell beads were obtained by the cyclodextrin, and the added cells were controlled in the same amount as the experimental group, and transformed at 28 ° C, 200 r / min for 18 h, and the substrate conversion rate was determined by HPLC; The cyclodextrin was grafted with sodium alginate for the biocatalytic reaction of the above 17 ⁇ hydroxyprogesterone.
  • the cyclodextrin-alginate-immobilized cell beads were collected and washed with physiological saline for 4 times.
  • the amount of the solution is 80 ml per gram of cell beads, the filtrate is centrifuged at 5000 r/min for 10 min to obtain a secondary filtrate, three times the amount of the second filtrate is added to dichloromethane, extracted three times, and the raffinate phase is vacuum-screwed at 30 ° C to remove the residue.
  • Methylene chloride Methylene chloride.
  • the cell gelatin beads were immobilized on the cyclodextrin-alginate filter and re-introduced into the steroid biocatalytic reaction for recycling, and the substrate conversion rate after each cycle was determined by HPLC; The results showed that the initial conversion rate of grafted carboxymethyl- ⁇ -cyclodextrin-alginate-immobilized cell beads for 17 ⁇ -hydroxyprogesterone biocatalysis was 1.1 times that of the control group. After 7 cycles of recycling, 17 ⁇ The final conversion of hydroxyprogesterone was 91%, see Table 2.
  • the cyclodextrin-alginate-immobilized cell beads were activated after 7 cycles of use, and the activation method was as follows: (1) 40g of cyclodextrin-alginate-immobilized cell beads reduced in catalytic efficiency, put into 50 mL fermentation medium for activation, 200 r/min, shaking culture at 28 °C for 24 h; The fermentation medium is a medium used for fermenting the cells; (2) After the completion of the culture, the fermentation broth was filtered to obtain a rubber bead, and the rubber beads were washed with physiological saline, and then fixed in a CaCl 2 solution for 2 h, and the reaction solution was washed and collected for 6 times, and placed in a refrigerator at 4 ° C.
  • Example 9 The same as in the seventh embodiment except the following. Cyclodextrin grafted sodium alginate immobilized cells: Accurately weigh 1.0g of sodium alginate (about 5.0mmol), add methyl- ⁇ -cyclodextrin with a molar ratio of 1:1 in a 100mL flask, add about 30mL of distilled water, and electric stir in a 70°C water bath.
  • Biotransformation Weigh 0.06 g of 17 ⁇ -hydroxyprogesterone in a 100 mL flask, add 20 mL of sodium acetate-acetate (pH 5.5), and add 30 g of cyclodextrin-alginate-immobilized cell beads.
  • the sodium alginate immobilized cell beads were grafted with cyclodextrin, and transformed at 28 °C and 200 r/min for 18 h.
  • the substrate conversion rate was determined by HPLC.
  • the cyclodextrin was grafted with sodium alginate for the biocatalytic reaction of the above 17 ⁇ hydroxyprogesterone.
  • the cyclodextrin-alginate-immobilized cell beads were collected and treated with acetic acid-sodium acetate (pH 5. 5) Washing twice, the amount of the reaction solution is 100 ml per gram of cell beads, the filtrate is centrifuged at 5000 r/min for 10 min to obtain a secondary filtrate, and 7 times the amount of chloroform of the second filtrate is added, and the mixture is extracted three times. The residual chloroform was removed by vacuum spinning at 35 °C.
  • the cell gelatin beads were immobilized on the cyclodextrin-alginate filter and re-introduced into the steroid biocatalytic reaction for recycling, and the substrate conversion rate after each cycle was determined by HPLC; The results showed that the initial conversion rate of grafted methyl- ⁇ -cyclodextrin-alginate-immobilized cell beads for 17 ⁇ -hydroxyprogesterone biocatalysis was 1.4 times that of the control group, and after 17 cycles of recycling, 17 ⁇ -hydroxyl The final conversion rate of progesterone was 92%, see Table 5.
  • Example 10 Cyclodextrin-alginate-immobilized cell beads and its application in hydroxylation of 17 ⁇ hydroxyprogesterone
  • the microbial strain adopts Cunningpamycetes elegans CICC 40250, which can realize the hydroxylation of C11 at the steroidal compound and convert 17 ⁇ hydroxyprogesterone into 11,17 ⁇ dihydroxyprogesterone;
  • Inclined medium g/L: potato 200, glucose 20, agar 20;
  • Preparation of elegant resting cells of Agaricus blazei After the slanting surface was activated, the spore suspension was prepared and inserted into the fermentation medium (50 mL medium/250 mL flask) so that the final concentration of spores in the flask was
  • Biotransformation Weigh 0.06 g of 17 ⁇ -hydroxyprogesterone in a 100 mL flask, add 20 mL of acetic acid-sodium acetate buffer (pH 5.0), and add 30 g of the cyclodextrin-alginate-immobilized cells prepared in the above procedure. Glue beads, control group were added with sodium alginate immobilized cell beads without grafting cyclodextrin, and the added cells were controlled in the same amount as the experimental group, and transformed at 28 °C and 150 r/min for 14 h. HPLC method was used to determine substrate conversion.
  • Cyclodextrin, fungal cells and reaction solution recycling process The cyclodextrin and sodium alginate were grafted to fix the cells, and then used for the hydroxylation reaction of the above 17 ⁇ hydroxyprogesterone. After the reaction, the cyclodextrin-alginate-immobilized cell beads were collected by filtration, and the sodium acetate-acetate was used. The buffer (pH 5.0) was washed 3 times, and the acetic acid-sodium acetate buffer was used in an amount of 50 ml per gram of cell beads.
  • the filtrate was centrifuged at 5000 r/min for 10 min to obtain a secondary filtrate, and isobutyl acetate was added five times to the amount of the second filtrate, and extracted three times.
  • the raffinate phase was vacuum-dried at 45 ° C to remove residual isobutyl acetate.
  • the cell gelatin beads were immobilized on the cyclodextrin-alginate filter and re-introduced into the steroid biocatalytic reaction for recycling, and the substrate conversion rate after each cycle was determined by HPLC; The results showed that the initial conversion rate of the control group was 76%, and the initial conversion rate of the sodium alginate immobilized cell beads coated with hydroxypropyl- ⁇ -cyclodextrin was 95%.
  • the cyclodextrin-alginate-immobilized cell beads were activated after 6 cycles of recycling.
  • the activation method was as follows: (1) 30 g of cyclodextrin-alginate-immobilized cell beads with reduced catalytic efficiency, and put into 50 mL fermentation medium for activation, 180 r/min, shaking culture at 28 °C for 20 h under shaking; The fermentation medium is a medium used for fermenting the cells; (2) After the completion of the culture, the fermentation broth was filtered to obtain a rubber bead, and the rubber beads were washed with acetic acid-sodium acetate buffer, and then fixed in a CaCl 2 solution for 1.5 h, and the reaction solution was washed and collected for 3 times.

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Abstract

提供了一种催化甾体生物转化的方法，该方法采用海藻酸钠接枝环糊精固定化细胞作为催化剂，从而实现环糊精介质、细胞和反应液的循环利用，在提高甾体催化反应效率的同时降低生产成本、减少环境污染，达到用绿色方法进行生物转化的目的。

Description

一种催化甾体生物转化的方法 技术领域：
本发明属于生物催化技术领域，具体涉及一种催化甾体生物转化的方法。
背景技术：
甾体化合物作为仅次于抗生素的第二大类药物，其在生命体内具有调控各种物质代谢及生理作用的功能。甾体药物的工业化生产主要是改造天然甾体化合物的结构，与传统化学方法合成相比，微生物转化的方法可以形成多种甾体药物活性中间体且产物纯度高。环糊精作为增溶剂能增加疏水性甾体的溶解度，其特殊空腔结构可以包结甾体底物，从而提高甾体化合物的生物利用度和产率。然而环糊精的价格较高制约了其在生物催化领域中的大范围应用，采用合适的方法扩大环糊精在生物转化反应中的应用是亟待解决的重要问题。
通过接枝技术将环糊精固载到壳聚糖、海藻酸钠等高分子载体上，可以把β-环糊精从溶于水的粉体材料制备成不溶于水的高分子材料，克服环糊精难回收的缺陷，使其能够循环利用，降低工业使用的成本。接枝后的环糊精仍保留独特的空腔结构及其它优良性质，同时还拥有了高分子载体良好的机械性能、稳定性等特征，这大大拓宽了环糊精的应用空间，提升了它的应用价值。海藻酸钠是一种天然聚阴离子多糖高分子化合物，由G、M两个单元构成，遇水后表面具有粘性，会使得溶液也具有粘性。其安全无毒，成本低，生物可降解性以及生物相容性良好，具备药用辅料所需的性质，遇到Ca2+、Zn2+等阳离子时，会发生化学反应形成凝胶球，针对这种性质，可用作药物的缓释和控释材料，以及用于固定性质不稳定物质，提高稳定性，同时还可用于固定微生物、细胞、酶等，实现循环利用。目前海藻酸钠的应用主要涉及到农药、食品、生物医药等领域，也被逐渐延伸到重金属污染的救治方面。
[根据细则26改正25.08.2017]　
中国专利CN 105754984-A公布了海藻酸钠复合固定化菌剂及其制备方法以及用途，公开了海藻酸钠复合固定化菌剂及其制备方法，解决了现有技术中并没有适用于二氯喹啉酸降解用假单胞菌的固定化方式的问题。本发明包括将具有假单胞菌的载体培养基和海藻酸钠混合后，滴入浓度为1～4%的氯化钙中静置2～4h制成的颗粒剂；所述海藻酸钠的质量浓度为2～6%；所述载体培养基包括吸附载体和无机盐液体培养基；该吸附载体与海藻酸钠的重量比为1～3:1,该吸附载体由重量比为1:1～2:1的玉米芯、竹炭和油枯组成。本发明通过有效提高菌体的降解效率；且本发明固定化菌剂的降解率远远高于空白小球物理吸附效率和游离菌株的生物降解效率之和，能有效达到相互促进的效果。
海藻酸钠作为天然高分子载体，分子中含有-COOH和-OH两种亲水基团，具有较强的亲水性，并且可以环氧氯丙烷为交联剂与环糊精进行接枝，形成的环糊精-海藻酸钠接枝物可以固定化细胞，实现环糊精和细胞的共循环。目前关于以海藻酸钠作为载体，以天然高分子材料接枝环糊精的形式作为促溶剂运用于难溶化合物的生物转化，并对反应溶液、环糊精及细胞进行共循环利用的技术尚未可见。
发明内容：
为了解决上述技术问题，本发明提供一种催化甾体生物转化的方法，该方法能够实现反应溶液、环糊精及微生物细胞的循环利用，具体如下：
在甾体生物催化反应中，以环糊精-海藻酸钠固定化细胞胶珠作催化剂，反应结束后，进行过滤，分别得滤液和过滤物，滤液用于收集反应产物，过滤物即环糊精-海藻酸钠固定化细胞胶珠，使用反应溶液洗涤1-8次后，重新用于甾体生物催化反应，从而实现环糊精介质、细胞循环利用；
所述反应溶液为Tris-HCl缓冲溶液、生理盐水、乙酸-乙酸钠缓冲液或纯水等，pH5.0-pH7.6；
所述的环糊精为天然环糊精或衍生环糊精；
在本申请的另一实施方式中，所述天然或衍生环糊精具体为α-环糊精、β-环糊精、γ-环糊精、羟丙基-β-环糊精、甲基-β-环糊精、磺丁基-β-环糊精、羧甲基-β-环糊精、羟乙基-β-环糊精、磺酸基-β-环糊精、羟丙基-γ-环糊精或甲基-γ-环糊精等；
所述的细胞可以是真菌或细菌；
在本申请的另一实施方式中，所述的真菌或细菌具体为赭曲霉、雅致小克银汉霉、黑根霉、绿僵菌或简单节杆菌等甾体催化反应常用菌株；
所述洗涤细胞胶珠的反应溶液的用量为每克细胞胶珠10-100mL；
在本申请的另一实施方式中，滤液经离心、有机溶剂萃取收集反应产物后，所得的含有反应溶液的萃余相除去残留有机溶剂后备用，从而实现反应溶液的循环利用；
在本申请的另一实施方式中，所述滤液的处理方法具体如下：
（1）滤液经5000r/min离心10min后得二次滤液；
（2）按照有机溶剂与二次滤液体积比为1-10:1加入有机溶剂萃取，收集萃余相；
所述的有机溶剂为乙酸乙酯、乙酸丁酯、乙酸异丁酯、二氯甲烷或三氯甲烷；
（3）萃余相30-45℃真空旋蒸除去残留有机溶剂后，将回收的反应溶液重新用于甾体生物催化反应；
所述的回收的反应溶液可循环利用6次以上；
所述环糊精-海藻酸钠固定化细胞胶珠的制备方法如下：
（1）海藻酸钠接枝环糊精
按海藻酸钠和环糊精按摩尔比1:0.2-1:1.5称取海藻酸钠和环糊精，加入三角瓶中，并加入海藻酸钠30-55倍（质量体积比）的蒸馏水，30-70℃水浴中搅拌使之完全溶解；
按环氧氯丙烷与蒸馏水体积比0.05-1:50加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.05-1:10滴加 0.5mol/L的NaOH溶液，滴加时间10min，之后反应0.5-6.5h；
（2）固定化细胞胶珠的制备
待步骤（1）的反应液冷却后，加入终浓度为1-100g/L的菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.1-0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡0.5-6 h，过滤，用反应溶液洗涤1-8次，即得环糊精-海藻酸钠固定化细胞胶珠；
在本申请的另一实施方式中，所述环糊精-海藻酸钠固定化细胞胶珠的制备方法如下：
（1）海藻酸钠接枝环糊精
按海藻酸钠和环糊精按摩尔比1:0.2-1:1.5称取海藻酸钠和环糊精，加入三角瓶中，并加入海藻酸钠45-55倍（质量体积比）的蒸馏水，30-70℃水浴中搅拌使之完全溶解；
按环氧氯丙烷与蒸馏水体积比0.05-1:50加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.05-1:10滴加 0.5mol/L的NaOH溶液，滴加时间10min，之后反应1.5-6.5h；
（2）固定化细胞胶珠的制备
待步骤（1）的反应液冷却后，加入终浓度为1-30g/L的菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.1-0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡1-6 h，过滤，用反应溶液洗涤1-8次，即得环糊精-海藻酸钠固定化细胞胶珠；
在本申请的另一实施方式中，所述环糊精-海藻酸钠固定化细胞胶珠的制备方法如下：
（1）海藻酸钠接枝环糊精
按海藻酸钠和环糊精按摩尔比1: 0.5-1:1.5称取海藻酸钠和环糊精，并加入海藻酸钠质量30倍（质量体积比）的蒸馏水，70℃水浴中搅拌使之完全溶解；
按环氧氯丙烷与蒸馏水体积比0.1:30加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.1:10滴加 0.5mol/L的NaOH溶液，之后反应0.5-2h；
（2）固定化细胞胶珠的制备
待步骤（1）的反应液冷却后，调节反应液的pH至5.0-6.0，加入终浓度为50-100g/L的真菌菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡0.5-2h，过滤，用反应溶液洗涤1-6次，即得环糊精-海藻酸钠固定化细胞胶珠；
所述环糊精-海藻酸钠固定化细胞胶珠可在不补加环糊精及微生物细胞的条件下循环使用6次以上；
在本申请的另一实施方式中，所述环糊精-海藻酸钠固定化细胞胶珠可在不补加环糊精及微生物细胞的条件下循环使用8次以上；
所述环糊精-海藻酸钠固定化细胞胶珠可通过活化延长循环利用的次数，所述的活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠10-40g,投入到30-50 mL发酵培养基进行活化，150-200 r/min，28-32 ℃的摇床下振荡培养18-24h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定1-2 h，反应溶液洗涤收集胶珠1-8次，置于4℃冰箱中保存备用；
活化后环糊精-海藻酸钠固定化细胞胶珠可再次循环利用3-5次。
在本申请的另一实施方式中，所述环糊精-海藻酸钠固定化细胞胶珠可通过活化延长循环利用的次数，所述的活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠10g,投入到30 mL发酵培养基进行活化，160 r/min，32℃的摇床下振荡培养20 h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2 h，反应溶液洗涤收集胶珠1-8次，置于4℃冰箱中保存备用；
在本申请的另一实施方式中，所述环糊精-海藻酸钠固定化细胞胶珠可通过活化延长循环利用的次数，所述的活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠20-40g，投入到50 mL发酵培养基进行活化，150-200 r/min，28℃的摇床下振荡培养18-24 h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定1-2 h，反应溶液洗涤收集胶珠1-6次，置于4℃冰箱中保存备用。
有益效果：
（1）本发明首次实现环糊精介质、细胞及反应溶液的循环利用，在提高甾体催化反应效率的同时降低生产成本、减少环境污染，达到用绿色方法进行生物转化的目的，具有很好的应用价值和推广前景。
（2）本发明可以提高甾体底物生物催化初始转化速率，提高最终转化率。
（3）本发明所述的环糊精循环利用工艺方法简单便捷，便于实现，成本节约。
具体实施方式：
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：环糊精-海藻酸钠固定化细胞胶珠及其在醋酸可的松C1,2位脱氢反应中的应用
微生物菌种采用简单节杆菌（Arthrobacter simplex）TCCC 11037，它能实现甾体化合物的C1，2位脱氢转化，将醋酸可的松（CA）转化为醋酸泼尼松；
斜面培养基(g/L)：葡萄糖10，酵母膏10，琼脂20，pH 7.2；
种子培养基(g/L)：葡萄糖10，玉米浆10，蛋白胨5，磷酸氢二钾2.5，pH 7.2；
发酵培养基(g/L)：葡萄糖10，玉米浆15，蛋白胨5，磷酸氢二钾2.5，pH 7.2；
简单节杆菌静息细胞的制备：
TCCC 11037在32 ℃，160 r/min，30/250 mL种子培养基装量条件下培养18 h后，按5%接种量接种于装有30mL发酵培养基的250 mL摇瓶中，32 ℃，160 r/min条件下培养20 h，将发酵培养获得的细胞5000 r/min离心后用pH 7.2的Tris-HCl缓冲液洗涤三遍，重悬于pH 7.2的Tris-HCl缓冲液中得简单节杆菌静息细胞菌悬液；
环糊精接枝海藻酸钠固定化细胞的制备：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:0.7的β-环糊精于100mL三角瓶中，加入约50mL蒸馏水，60℃水浴中电动搅拌使之完全溶解，加入0.3mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，反应2.5h。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为10g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡2 h，过滤，用pH 7.2的Tris-HCl洗涤3次备用。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的Tris-HCl（pH 7.2），实验组加入10 g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠（接枝的β-环糊精量为0.115g），对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，34℃、180 r/min转化8 h，HPLC法测定底物转化率；
环糊精及细胞的循环利用工艺：
将环糊精与海藻酸钠接枝固定细胞后用于上述CA的生物催化反应，反应结束后收集环糊精-海藻酸钠固定化细胞胶珠，将其用Tris-HCl（pH 7.2）洗涤3次，用量为每次每克细胞胶珠50ml，洗涤后重新用于醋酸可的松的生物转化，用量不变，HPLC法测定每次循环后的底物转化率；
循环利用5次后，转化率降至90%，将环糊精-海藻酸钠固定化细胞胶珠进行活化，步骤如下：
将回收的每10g环糊精-海藻酸钠固定化细胞胶珠投入到30 mL发酵培养基进行活化，160 r/min，32 ℃的摇床下振荡培养20 h；
培养结束后将发酵液过滤得到胶珠，用pH 7.2的Tris-HCl缓冲液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2h，洗涤收集胶珠，再次用于CA的生物催化反应；
结果表明，对照组的初次转化率为86%，接枝β-环糊精的海藻酸钠固定化细胞胶珠的初次转化率是95%，初始转化速率是对照组（1.1×10-2g/L min-1）的1.3倍，循环利用8次后，CA的最终转化率为90%，见表1。
表1
循环次数 1 2 3 4 5 6 7 8
转化率 95% 93% 91% 90% 90% 92% 92% 90%


实施例2：环糊精-海藻酸钠固定化细胞胶珠及其在醋酸可的松C1,2位脱氢反应中的应用
除以下内容外，其他同实施例1。
环糊精接枝海藻酸钠固定化细胞：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:1.5的α-环糊精于100mL三角瓶中，加入约50mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约6h后停止反应。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为30g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.25mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡6h，过滤，用生理盐水（pH 7.5）洗涤2次。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的Tris-HCl（pH 7.2），再加入10 g环糊精-海藻酸钠固定化细胞胶珠（接枝的α-环糊精量为0.072g ），对照组加入等菌体量不接枝环糊精的海藻酸钠固定化细胞胶珠，34 ℃、180 r/min转化8 h， HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述CA的生物催化反应，反应结束后收集环糊精-海藻酸钠固定化细胞胶珠，将其用生理盐水（pH 7.5）洗涤2次，用量为每次每克细胞胶珠100ml，洗涤后重新用于醋酸可的松的生物转化，用量不变。
结果表明，接枝α-环糊精-海藻酸钠固定化细胞胶珠首次用于CA生物催化的初始转化速率是对照组（1.1×10-2g/L min-1）的1.2倍，且循环利用5次后对α-环糊精-海藻酸钠固定化细胞进行活化，活化方法同实施例1，活化后继续循环使用4次，CA的最终转化率为90%。

实施例3：环糊精-海藻酸钠固定化细胞胶珠及其在醋酸可的松C1,2位脱氢反应中的应用
除以下内容外，其他同实施例1。
环糊精接枝海藻酸钠固定化细胞：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:0.2的γ-环糊精于100mL三角瓶中，加入约50mL蒸馏水，30℃水浴中电动搅拌使之完全溶解，加入0.05mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约1.5h后停止反应。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为1g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.1mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡1h，过滤，用Tris-HCl（pH 7.5）洗涤2次。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的Tris-HCl（pH 7.5），再加入10 g环糊精-海藻酸钠固定化细胞胶珠（接枝的γ-环糊精量为0.058g ），对照组加入等菌体量不接枝环糊精的海藻酸钠固定化细胞胶珠，34℃、180 r/min转化8 h， HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述CA的生物催化反应，反应结束后收集环糊精-海藻酸钠固定化细胞胶珠，将其用Tris-HCl（pH 7.5）洗涤2次，用量为每次每克细胞胶珠100ml，洗涤后重新用于醋酸可的松的生物转化，用量不变。
结果表明，接枝γ-环糊精-海藻酸钠固定化细胞胶珠首次用于CA生物催化的初始转化速率是对照组（1.1×10-2g/L min-1）的1.1倍，且循环利用5次后对γ-环糊精-海藻酸钠固定化细胞进行活化，活化方法同实施例1，活化后继续循环使用3次，CA的最终转化率为91%。

实施例4：衍生环糊精-海藻酸钠固定化细胞胶珠及其在醋酸可的松C1,2位脱氢反应中的应用
微生物菌种采用简单节杆菌（Arthrobacter simplex）TCCC 11037，它能实现甾体化合物的C1，2位脱氢转化，将醋酸可的松（CA）转化为醋酸泼尼松；
斜面培养基(g/L)：葡萄糖10，酵母膏10，琼脂20，pH 7.2；
种子培养基(g/L)：葡萄糖10，玉米浆10，蛋白胨5，磷酸氢二钾2.5，pH 7.2；
发酵培养基(g/L)：葡萄糖10，玉米浆15，蛋白胨5，磷酸氢二钾2.5，pH 7.2；
简单节杆菌静息细胞的制备：
TCCC 11037在32 ℃，160 r/min，30/250 mL种子培养基装量条件下培养18 h后，按5%接种量接种于装有30mL发酵培养基的250 mL摇瓶中，32 ℃，160 r/min条件下培养20 h，将发酵培养获得的细胞5000 r/min离心后用pH 7.2的Tris-HCl缓冲液洗涤三遍，重悬于pH 7.2的Tris-HCl缓冲液中得简单节杆菌静息细胞菌悬液；
衍生环糊精接枝海藻酸钠固定化细胞的制备：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:0.2的羟丙基-β-环糊精于100mL三角瓶中，加入约50mL蒸馏水，30℃水浴中电动搅拌使之完全溶解，加入0.6 mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约3.5h后停止反应。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为20g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.3 mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡3 h，过滤，用Tris-HCl（pH 7.2）洗涤4次。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的Tris-HCl（pH 7.2），实验组加入10 g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠（接枝的羟丙基-β-环糊精量为0.043g），对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，34 ℃、180 r/min转化8 h，HPLC法测定底物转化率；
环糊精及细胞的循环利用工艺：
将环糊精与海藻酸钠接枝固定细胞后用于上述CA的生物催化反应，反应结束后收集衍生环糊精-海藻酸钠固定化细胞胶珠，将其用Tris-HCl（pH 7.2）洗涤3次，用量为每次每克细胞胶珠50ml，洗涤后重新用于醋酸可的松的生物转化，用量不变，HPLC法测定每次循环后的底物转化率；
循环利用5次后，转化率降至91%，将衍生环糊精-海藻酸钠固定化细胞胶珠进行活化，步骤如下：
将回收的10g衍生环糊精-海藻酸钠固定化细胞胶珠投入到30 mL发酵培养基进行活化，160 r/min，32 ℃的摇床下振荡培养20 h；
培养结束后将发酵液过滤得到胶珠，用Tris-HCl（pH 7.2）缓冲液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2h，洗涤收集胶珠，再次用于CA的生物催化反应；
结果表明，对照组的初次转化率为86%，接枝羟丙基-β-环糊精的海藻酸钠固定化细胞胶珠的初次转化率是95%，初始转化速率是对照组（1.1×10-2g/L min-1）的1.3倍，循环利用8次后，CA的最终转化率为92%，见表2。
表2
循环次数 1 2 3 4 5 6 7 8
转化率 95% 96% 95% 94% 91% 94% 92% 92%


实施案例5：
除以下内容外，其他同实施例4。
衍生环糊精接枝海藻酸钠固定化细胞的制备：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:1.5的磺丁基-β-环糊精于100mL三角瓶中，加入约50mL蒸馏水，60℃水浴中电动搅拌使之完全溶解，加入1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，6.5h后停止反应。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为30g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡6 h，过滤，用生理盐水（pH 7.2）洗涤3次备用。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的生理盐水（pH 7.2），实验组加入10 g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠（接枝的磺丁基-β-环糊精量为0.119g），对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，34℃、180 r/min转化8 h，HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述CA的生物催化反应，反应结束后收集衍生环糊精-海藻酸钠固定化细胞胶珠，将其用生理盐水（pH 7.2）洗涤4次，用量为每次每克细胞胶珠80ml，洗涤后重新用于醋酸可的松的生物转化，用量不变。
结果表明，接枝磺丁基-β-环糊精-海藻酸钠固定化细胞胶珠首次用于CA生物催化的初始转化速率是对照组（1.1×10-2g/L min-1）的1.4倍，且循环利用5次后对磺丁基-β-环糊精-海藻酸钠固定化细胞进行活化，活化方法同实施例1，活化后继续循环使用3次，CA的最终转化率为94%。

实施案例6：
除以下内容外，其他同实施例4。
环糊精接枝海藻酸钠固定化细胞：
准确称取海藻酸钠0.99g(5.0mmol)，加入与之摩尔比为1:1的甲基-β-环糊精于100mL三角瓶中，加入约50mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入0.1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约1.5h后停止反应。待反应液冷却后，加入菌体静息细胞悬液，使得菌体浓度为15g/L。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.25mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡2h，过滤，用Tris-HCl（pH 7.5）洗涤2次。
生物转化：
称取0.06 g的CA于100 mL三角瓶中，加入20 mL的Tris-HCl（pH 7.5），再加入10g衍生环糊精-海藻酸钠固定化细胞胶珠（接枝的甲基-β-环糊精量为0.063g），对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，34 ℃、180 r/min转化8 h，HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述CA的生物催化反应，反应结束后收集衍生环糊精-海藻酸钠固定化细胞胶珠，将其用Tris-HCl（pH 7.5）洗涤2次，用量为每次每克细胞胶珠100ml，洗涤后重新用于醋酸可的松的生物转化，用量不变。
结果表明，接枝甲基-β-环糊精-海藻酸钠固定化细胞胶珠首次用于CA生物催化的初始转化速率是对照组（1.1×10-2g/L min-1）的1.5倍，且循环利用5次后对甲基-β-环糊精-海藻酸钠固定化细胞进行活化，活化方法同实施例1，活化后继续循环使用4次，CA的最终转化率为92%。

实施例7：环糊精-海藻酸钠固定化细胞胶珠及其在17α羟基黄体酮羟基化反应中的应用
微生物菌种采用赭曲霉（Aspergillus ochraceus) CICC41473，它能实现甾体化合物的C11位羟基化，将17α羟基黄体酮转化为11，17α二羟基黄体酮；
斜面培养基(g/L)：土豆200，葡萄糖20，琼脂20；
发酵培养基(g/L)：葡萄糖15，玉米浆40，蚕蛹粉2，硫酸铵1.5，pH4.5；
赭曲霉静息细胞的制备：
赭曲霉经斜面活化后制备赭曲霉孢子悬浮液，并将其接入发酵培养基（50 mL培养基/250 mL 三角瓶）中，使得三角瓶中的孢子的终浓度为106 个/mL，28℃，200 r/min培养18h后加入终浓度为0.1g/L的底物进行诱导，继续培养6h后，将发酵液过滤，并用pH 7.0的Tris-HCl缓冲液洗涤三遍后收集菌体；
环糊精-海藻酸钠固定化细胞胶珠的制备：
准确称取海藻酸钠1.0g（约5.0mmol），加入与之摩尔比为1:0.5的羟丙基-β-环糊精于100mL三角瓶中，加入约30mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入0.1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，2h后停止反应。待反应液冷却后，调节反应液的pH至5.5，加入3g菌体静息细胞。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5 mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡2h，过滤，用Tris-HCl洗涤4次。
生物转化：
称取0.06 g的17α羟基黄体酮于100 mL三角瓶中，加入20 mL的Tris-HCl（pH7.0），实验组加入40 g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠，对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，28 ℃、200 r/min转化18 h， HPLC法测定底物转化率；
环糊精、真菌细胞及反应溶液的循环利用工艺：
将环糊精与海藻酸钠接枝固定细胞后用于上述17α羟基黄体酮的羟基化反应，反应结束后过滤收集环糊精-海藻酸钠固定化细胞胶珠，将其用Tris-HCl（pH 7.0）洗涤3次，反应溶液的用量为每次每克细胞胶珠50ml。滤液5000r/min离心10min得二次滤液，加入两倍于二次滤液量的乙酸乙酯，萃取三次，萃余相45 ℃真空旋蒸除去残留的乙酸乙酯，将其和过滤物即环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用，HPLC法测定每次循环后的底物转化率；
结果表明，对照组的初次转化率为80%，接枝羟丙基-β-环糊精的海藻酸钠固定化细胞胶珠的初次转化率是96%，循环利用6次后，17α羟基黄体酮的最终转化率仍高达92%，见表1。循环使用6次后对环糊精-海藻酸钠固定化细胞胶珠进行活化，活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠20g，投入到50 mL发酵培养基进行活化，150 r/min，28 ℃的摇床下振荡培养18 h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用Tris-HCl将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定1 h，反应溶液洗涤收集胶珠3次，置于4℃冰箱中保存并继续循环使用3次，结果见表3。
表3
循环次数 1 2 3 4 5 6 7 8 9
转化率 96% 94% 93% 92% 91% 92% 95% 92% 93%

实施例8：
除以下内容外，其他同实施例7。
环糊精-海藻酸钠固定化细胞的制备：
准确称取海藻酸钠1.0g（约5.0mmol），加入与之摩尔比为1:1.5的羧甲基-β-环糊精于100mL三角瓶中，加入约30mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入0.1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，1.5h后停止反应。待反应液冷却后，加入3g菌体静息细胞。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡1.5 h，过滤，用生理盐水洗涤3次备用。
生物转化：
称取0.06 g的17α羟基黄体酮于100 mL三角瓶中，加入20 mL的生理盐水，实验组加入40 g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠，对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，28℃、200 r/min转化18 h，HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述17α羟基黄体酮的生物催化反应，反应结束后收集环糊精-海藻酸钠固定化细胞胶珠，将其用生理盐水洗涤4次，反应溶液的用量为每次每克细胞胶珠80ml，滤液5000r/min离心10min得二次滤液，加入三倍于二次滤液量的二氯甲烷，萃取三次，萃余相30 ℃真空旋蒸除去残留的二氯甲烷。将其和过滤物即环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用，HPLC法测定每次循环后的底物转化率；
结果表明，接枝羧甲基-β-环糊精-海藻酸钠固定化细胞胶珠首次用于17α羟基黄体酮生物催化的初始转化速率是对照组的1.1倍，循环利用7次后，17α羟基黄体酮的最终转化率为91%，见表2。循环使用7次后对环糊精-海藻酸钠固定化细胞胶珠进行活化，活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠40g，投入到50 mL发酵培养基进行活化，200 r/min，28 ℃的摇床下振荡培养24 h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用生理盐水将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2 h，反应溶液洗涤收集胶珠6次，置于4℃冰箱中保存并继续循环使用3次，结果见表4。
表4
循环次数 1 2 3 4 5 6 7 8 9 10
转化率 93% 92% 95% 91% 90% 90% 91% 95% 94% 90%

实施例9：
除以下内容外，其他同实施例7。
环糊精接枝海藻酸钠固定化细胞：
准确称取海藻酸钠1.0g（约5.0mmol），加入与之摩尔比为1:1的甲基-β-环糊精于100mL三角瓶中，加入约30mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入0.1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约1.5h后停止反应。待反应液冷却后，加入3g菌体静息细胞。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡2h，过滤，用乙酸-乙酸钠（pH5.5）洗涤2次。
生物转化：
称取0.06 g的17α羟基黄体酮于100 mL三角瓶中，加入20 mL的乙酸-乙酸钠（pH5.5），再加入30g环糊精-海藻酸钠固定化细胞胶珠，对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，28 ℃、200 r/min转化18 h，HPLC法测定底物转化率；
将环糊精与海藻酸钠接枝后用于上述17α羟基黄体酮的生物催化反应，反应结束后收集环糊精-海藻酸钠固定化细胞胶珠，将其用乙酸-乙酸钠（pH5.5）洗涤2次，反应溶液的用量为每次每克细胞胶珠100ml，滤液5000r/min离心10min得二次滤液，加入7倍于二次滤液量的三氯甲烷，萃取三次，萃余相35 ℃真空旋蒸除去残留的三氯甲烷。将其和过滤物即环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用，HPLC法测定每次循环后的底物转化率；
结果表明，接枝甲基-β-环糊精-海藻酸钠固定化细胞胶珠首次用于17α羟基黄体酮生物催化的初始转化速率是对照组的1.4倍，循环利用7次后，17α羟基黄体酮的最终转化率为92%，见表5。
表5
循环次数 1 2 3 4 5 6 7
转化率 91% 93% 93% 94% 91% 94% 92%


实施例10：环糊精-海藻酸钠固定化细胞胶珠及其在17α羟基黄体酮羟基化反应中的应用
微生物菌种采用雅致小克银汉霉（Cunningpamycetes elegans）CICC 40250，它能实现甾体化合物的C11位羟基化，将17α羟基黄体酮转化为11，17α二羟基黄体酮；
斜面培养基(g/L)：土豆200，葡萄糖20，琼脂20；
发酵培养基(g/L)：葡萄糖20，酵母膏5，蛋白胨5，氯化钠 5，磷酸氢二钾 0.5，pH6.5；
雅致小克银汉霉静息细胞的制备：
雅致小克银汉霉经斜面活化后，制备孢子悬浮液，并接入发酵培养基（50 mL培养基/250 mL 三角瓶）中，使得三角瓶中的孢子的终浓度为106 个/mL，28 ℃，150 r/min培养24h后，将发酵液过滤后用pH5.0乙酸-乙酸钠缓冲液洗涤三遍后收集菌体；
环糊精-海藻酸钠固定化细胞的制备：
准确称取海藻酸钠1.0g（约5.0mmol），加入与之摩尔比为1:1.2的羟丙基-β-环糊精于100mL三角瓶中，加入约30mL蒸馏水，70℃水浴中电动搅拌使之完全溶解，加入0.1mL的环氧氯丙烷，同时滴加10mL 0.5mol/L的NaOH溶液，约10min滴完，约1h后停止反应。待反应液冷却后，调节反应溶液的pH至5.0，加入2.5g菌体静息细胞。将混悬液搅拌均匀，在磁力搅拌下用注射器滴加到0.5 mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡2h，过滤，用乙酸-乙酸钠缓冲液（pH5.0）洗涤4次。
生物转化：
称取0.06 g的17α羟基黄体酮于100 mL三角瓶中，加入20 mL的乙酸-乙酸钠缓冲液（pH5.0），实验组加入30g上述步骤制备的环糊精-海藻酸钠固定化细胞胶珠，对照组加入不接枝环糊精的海藻酸钠固定化细胞胶珠，控制加入的菌体与实验组等量，28 ℃、150 r/min转化14 h， HPLC法测定底物转化率；
环糊精、真菌细胞及反应溶液的循环利用工艺：
将环糊精与海藻酸钠接枝固定细胞后用于上述17α羟基黄体酮的羟基化反应，反应结束后过滤收集环糊精-海藻酸钠固定化细胞胶珠，将其用乙酸-乙酸钠缓冲液（pH5.0）洗涤3次，乙酸-乙酸钠缓冲液的用量为每次每克细胞胶珠50ml。滤液5000r/min离心10min得二次滤液，加入五倍于二次滤液量的乙酸异丁酯，萃取三次，萃余相45 ℃真空旋蒸除去残留的乙酸异丁酯。将其和过滤物即环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用，HPLC法测定每次循环后的底物转化率；
结果表明，对照组的初次转化率为76%，接枝羟丙基-β-环糊精的海藻酸钠固定化细胞胶珠的初次转化率是95%，循环利用6次后，17α羟基黄体酮的最终转化率仍高达91%，见表6。循环使用6次后对环糊精-海藻酸钠固定化细胞胶珠进行活化，活化方法具体如下：
（1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠30g，投入到50 mL发酵培养基进行活化，180 r/min，28 ℃的摇床下振荡培养20 h；
所述的发酵培养基为发酵培养菌体时使用的培养基；
（2）培养结束后将发酵液过滤得到胶珠，用乙酸-乙酸钠缓冲液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定1.5 h，反应溶液洗涤收集胶珠3次，置于4℃冰箱中保存并继续循环使用3次。
表6
循环次数 1 2 3 4 5 6 7 8 9
转化率 95% 95% 93% 94% 91% 91% 93% 92% 90%

虽然上文已经用一般性说明、具体实施方式及实验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (17)

  1. [根据细则26改正25.08.2017]一种催化甾体生物转化的方法，其特征在于，具体如下：在甾体生物催化反应中，以环糊精-海藻酸钠固定化细胞胶珠作催化剂，反应结束后，进行过滤，分别得滤液和过滤物，滤液用于收集反应产物，过滤物即环糊精-海藻酸钠固定化细胞胶珠，使用反应溶液洗涤1-8次后，重新用于甾体生物催化反应，实现环糊精介质和细胞的循环利用。
  2. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述滤液经离心、有机溶剂萃取收集反应产物后，所得的含有反应溶液的萃余相除去残留有机溶剂后备用，实现反应溶液的循环利用。
  3. [根据细则26改正25.08.2017]如权利要求2所述的一种催化甾体生物转化的方法，其特征在于，所述滤液的处理方法具体如下： （1）滤液经5000r/min离心10min后得二次滤液； （2）按照有机溶剂与二次滤液体积比为1-10:1加入有机溶剂萃取，收集萃余相； （3）萃余相30-45℃真空旋蒸除去残留有机溶剂后，将回收的反应溶液重新用于甾体生物催化反应。
  4. [根据细则26改正25.08.2017]如权利要求3所述的一种催化甾体生物转化的方法，其特征在于，所述的有机溶剂为乙酸乙酯、乙酸丁酯、乙酸异丁酯、二氯甲烷或三氯甲烷。
  5. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述环糊精-海藻酸钠固定化细胞胶珠的制备方法如下： （1）海藻酸钠接枝环糊精 按海藻酸钠和环糊精按摩尔比1:0.2-1:1.5称取海藻酸钠和环糊精，加入三角瓶中，并加入海藻酸钠30-55倍的蒸馏水，30-70℃水浴中搅拌使之完全溶解； 按环氧氯丙烷与蒸馏水体积比0.05-1:50加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.05-1:10滴加 0.5mol/L的NaOH溶液，滴加时间10min，之后反应0.5-6.5h； （2）固定化细胞胶珠的制备 待步骤（1）的反应液冷却后，加入终浓度为1-100g/L的菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.1-0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡0.5-6 h，过滤，用反应溶液洗涤1-8次，即得环糊精-海藻酸钠固定化细胞胶珠。
  6. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述环糊精-海藻酸钠固定化细胞胶珠可通过活化延长循环利用的次数，所述的活化方法具体如下： （1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠10g,投入到30 mL发酵培养基进行活化，160 r/min，32℃的摇床下振荡培养20 h； 所述的发酵培养基为发酵培养菌体时使用的培养基； （2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2 h，反应溶液洗涤收集胶珠1-8次，置于4℃冰箱中保存备用。
  7. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述反应溶液为Tris-HCl缓冲溶液、生理盐水、乙酸-乙酸钠缓冲液或纯水，pH5.0-pH7.6。
  8. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述的环糊精为天然环糊精或衍生环糊精； 所述天然或衍生环糊精具体为α-环糊精、β-环糊精、γ-环糊精、羟丙基-β-环糊精、甲基-β-环糊精、磺丁基-β-环糊精、羧甲基-β-环糊精、羟乙基-β-环糊精、磺酸基-β-环糊精、羟丙基-γ-环糊精或甲基-γ-环糊精。
  9. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，所述的细胞可以是真菌或细菌； 所述的真菌或细菌具体为赭曲霉、雅致小克银汉霉、黑根霉、绿僵菌或简单节杆菌等甾体催化反应常用菌株。
  10. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，在甾体生物催化反应中，以天然环糊精-海藻酸钠固定化细胞胶珠作催化剂，反应结束后，进行过滤，滤液用于收集反应产物，过滤物即环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用； 所述天然环糊精-海藻酸钠固定化细胞胶珠的制备方法如下： （1）海藻酸钠接枝环糊精 按海藻酸钠和天然环糊精按摩尔比1:0.2-1:1.5称取海藻酸钠和环糊精，加入三角瓶中，并加入海藻酸钠45-55倍的蒸馏水，30-70℃水浴中搅拌使之完全溶解； 按环氧氯丙烷与蒸馏水体积比0.05-1:50加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.05-1:10滴加 0.5mol/L的NaOH溶液，之后反应1.5-6.5h； （2）固定化细胞胶珠的制备 待步骤（1）的反应液冷却后，加入终浓度为1-30g/L的菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.1-0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡1-6 h，过滤，用反应溶液洗涤1-8次，即得环糊精-海藻酸钠固定化细胞胶珠； 所述反应溶液为Tris-HCl缓冲溶液、生理盐水或纯水，pH7.0-pH7.6。
  11. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，具体如下：在甾体生物催化反应中，以衍生环糊精-海藻酸钠固定化细胞胶珠作催化剂，反应结束后，进行过滤，滤液用于收集反应产物，过滤物即衍生环糊精-海藻酸钠固定化细胞胶珠，重新投入甾体生物催化反应，进行循环利用； 所述衍生环糊精-海藻酸钠固定化细胞胶珠的制备方法如下： （1）海藻酸钠接枝环糊精 按海藻酸钠和衍生环糊精按摩尔比1:0.2-1:1.5称取海藻酸钠和环糊精，加入三角瓶中，并加入海藻酸钠45-55倍的蒸馏水，30-70℃水浴中搅拌使之完全溶解； 按环氧氯丙烷与蒸馏水体积比0.05-1:50加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.05-1:10滴加 0.5mol/L的NaOH溶液，滴加时间10min，之后反应1.5-6.5h； （2）固定化细胞胶珠的制备 待步骤（1）的反应液冷却后，加入终浓度为1-30g/L的菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到0.1-0.5mol/L的CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡1-6 h，过滤，用反应溶液洗涤1-8次，即得衍生环糊精-海藻酸钠固定化细胞胶珠； 所述反应溶液为Tris-HCl缓冲溶液、生理盐水或纯水等，pH7.0-pH7.6。
  12. [根据细则26改正25.08.2017]如权利要求10或11所述的一种催化甾体生物转化的方法，其特征在于，所述固定化细胞胶珠通过活化延长循环利用的次数，所述的活化方法具体如下： （1）将催化效率降低的天然环糊精-海藻酸钠固定化细胞胶珠10g，投入到30 mL发酵培养基进行活化，160 r/min，32 ℃的摇床下振荡培养20 h； 所述的发酵培养基为发酵培养菌体时使用的培养基； （2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定2 h，反应溶液洗涤收集胶珠1-8次，置于4℃冰箱中保存备用。
  13. [根据细则26改正25.08.2017]如权利要求1所述的一种催化甾体生物转化的方法，其特征在于，在真菌催化的甾体生物转化反应中，以环糊精-海藻酸钠固定化细胞胶珠作催化剂，反应结束后进行过滤，分别得滤液和过滤物，滤液经离心、有机溶剂萃取后，所得的含有反应溶液的萃余相除去残留有机溶剂后备用，过滤物使用反应溶液洗涤1-6次后，重新用于甾体生物催化反应，从而实现环糊精介质、真菌细胞和反应溶液的循环利用。
  14. [根据细则26改正25.08.2017]如权利要求13所述的一种催化甾体生物转化的方法，其特征在于，所述滤液的处理方法具体如下： （1）滤液经离心后得二次滤液； （2）按照有机溶剂与二次滤液体积比为1-10:1加入有机溶剂萃取，收集萃余相； 所述的有机溶剂为乙酸乙酯、乙酸丁酯、乙酸异丁酯、二氯甲烷或三氯甲烷等； （3）萃余相30-45℃真空旋蒸除去残留有机溶剂后，将回收的反应溶液重新用于甾体生物催化反应。
  15. [根据细则26改正25.08.2017]如权利要求13所述的一种催化甾体生物转化的方法，其特征在于，所述环糊精-海藻酸钠固定化细胞胶珠可通过活化延长循环利用的次数，所述的活化方法具体如下： （1）将催化效率降低的环糊精-海藻酸钠固定化细胞胶珠20-40g，投入到50 mL发酵培养基中进行活化，150-200 r/min，28 ℃的摇床下振荡培养18-24 h； 所述的发酵培养基为发酵培养菌体时使用的培养基； （2）培养结束后将发酵液过滤得到胶珠，用反应溶液将胶珠洗涤干净后，将其置于CaCl2溶液中再次固定1-2 h，反应溶液洗涤收集胶珠1-6次，置于4℃冰箱中保存备用。
  16. [根据细则26改正25.08.2017]如权利要求13所述的一种催化甾体生物转化的方法，其特征在于，所述环糊精-海藻酸钠固定化细胞胶珠的制备方法如下： （1）海藻酸钠接枝环糊精 按海藻酸钠和环糊精按摩尔比1: 0.5-1:1.5称取海藻酸钠和环糊精，并加入海藻酸钠质量30倍的蒸馏水，水浴中搅拌使之完全溶解； 按环氧氯丙烷与蒸馏水体积比0.1:30加入环氧氯丙烷，同时按环氧氯丙烷与NaOH溶液体积比0.1:10滴加NaOH溶液，之后反应0.5-2h； （2）固定化细胞胶珠的制备 待步骤（1）的反应液冷却后，调节反应液的pH至5.0-6.0，加入终浓度为50-100g/L的真菌菌体静息细胞，搅拌均匀，在磁力搅拌下用注射器滴加到CaCl2溶液中，将胶珠在CaCl2溶液中继续浸泡0.5-2h，过滤，用反应溶液洗涤1-6次，即得环糊精-海藻酸钠固定化细胞胶珠。
  17. [根据细则26改正25.08.2017]如权利要求13所述的一种催化甾体生物转化的方法，其特征在于，其特征在于，所述反应溶液为Tris-HCl缓冲液、乙酸-乙酸钠缓冲液或生理盐水，pH5.0-pH7.0。
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