CN111440779A - Method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase - Google Patents
Method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase Download PDFInfo
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- CN111440779A CN111440779A CN202010328405.3A CN202010328405A CN111440779A CN 111440779 A CN111440779 A CN 111440779A CN 202010328405 A CN202010328405 A CN 202010328405A CN 111440779 A CN111440779 A CN 111440779A
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- JWUBBDSIWDLEOM-UHFFFAOYSA-N 25-Hydroxycholecalciferol Natural products C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2C1=CC=C1CC(O)CCC1=C JWUBBDSIWDLEOM-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 101000919178 Streptomyces griseolus Vitamin D3 dihydroxylase Proteins 0.000 title claims abstract description 33
- JWUBBDSIWDLEOM-DCHLRESJSA-N 25-Hydroxyvitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C/C=C1\C[C@@H](O)CCC1=C JWUBBDSIWDLEOM-DCHLRESJSA-N 0.000 title claims abstract description 31
- JWUBBDSIWDLEOM-NQZHSCJISA-N 25-hydroxy-3 epi cholecalciferol Chemical compound C1([C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=CC=C1C[C@H](O)CCC1=C JWUBBDSIWDLEOM-NQZHSCJISA-N 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 45
- 238000000855 fermentation Methods 0.000 claims abstract description 35
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 5
- 230000006698 induction Effects 0.000 claims abstract description 4
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 21
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 claims description 21
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 20
- 235000005282 vitamin D3 Nutrition 0.000 claims description 20
- 239000011647 vitamin D3 Substances 0.000 claims description 20
- 229940021056 vitamin d3 Drugs 0.000 claims description 20
- 239000003960 organic solvent Substances 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 239000000758 substrate Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 229910017604 nitric acid Inorganic materials 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 6
- 230000008025 crystallization Effects 0.000 claims description 6
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 5
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000013078 crystal Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 241000186361 Actinobacteria <class> Species 0.000 claims description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 3
- 229920000858 Cyclodextrin Polymers 0.000 claims description 3
- 102000018832 Cytochromes Human genes 0.000 claims description 3
- 108010052832 Cytochromes Proteins 0.000 claims description 3
- 229910001424 calcium ion Inorganic materials 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- 159000000014 iron salts Chemical class 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 102000008109 Mixed Function Oxygenases Human genes 0.000 claims 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 claims 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 4
- 239000000047 product Substances 0.000 abstract description 3
- 239000007795 chemical reaction product Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 13
- 235000021318 Calcifediol Nutrition 0.000 description 5
- JWUBBDSIWDLEOM-DTOXIADCSA-N calcidiol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)CCC1=C JWUBBDSIWDLEOM-DTOXIADCSA-N 0.000 description 5
- 229960004361 calcifediol Drugs 0.000 description 5
- 241001052560 Thallis Species 0.000 description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000013038 Hypocalcemia Diseases 0.000 description 1
- 208000029725 Metabolic bone disease Diseases 0.000 description 1
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- 238000001631 haemodialysis Methods 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 230000000705 hypocalcaemia Effects 0.000 description 1
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Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
-
- 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
- C12P33/06—Hydroxylating
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13013—Calcidiol 1-monooxygenase (1.14.13.13), i.e. 25-hydroxyvitamin D-1-alpha-hydroxylase
Abstract
A method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase, which comprises the following steps: a. fermentation: inoculating the genetically engineered bacteria producing vitamin D3 hydroxylase into a fermentation culture medium, and adding IPTG (isopropyl-beta-D-thiogalactoside) for inducing enzyme production when the culture is carried out until OD600 is 1-5, wherein the final concentration of the IPTG is 1.2-2mM, the induction temperature is 28 ℃, and the fermentation culture is carried out for 16-24h to obtain fermentation liquor; b. wall breaking: centrifuging the fermentation liquor at 11000-; and the steps shorten the conversion period from the flow, directly introduce hydroxyl into the reaction product in a short time to generate an active product, and the conversion condition is mild.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase.
Background
Calcifediol, also known as 25-hydroxyvitamin D3[25(OH) VD3], is a biologically active derivative of vitamin D3, vitamin D3 is not active in humans and must be converted to active biological components by biological enzymes to function. Animal experiments show that the calcifediol has obvious effect on metabolic bone diseases such as osteoporosis, rickets, osteomalacia and the like, and can be used for treating hypocalcemia caused by hemodialysis. Can also be used as raw materials of health food and feed additives, and has wide application in health food, medicine field, feed additives and other fields.
25-hydroxyvitamin D3[25(OH) VD3] can be synthesized by a chemical method, but most of the methods used in the chemical synthesis are multi-step reactions such as ring opening, bond breaking, ring combination, reduction, coupling, oxidation, purification and the like, and the chemical synthesis method has the defects of multiple reaction steps, low conversion rate, complex separation and purification process and the like, and is not beneficial to large-scale industrial production. In recent years, with the screening of enzyme-producing strains and the development of genetically engineered bacteria, the production of the calcifediol by converting vitamin D3 through microorganisms becomes a research hotspot, and the production of the calcifediol through a microbial conversion method has the advantages of low production cost, high reaction specificity, mild reaction conditions, environmental friendliness and the like, and is a research focus for producing the calcifediol in the future.
Disclosure of Invention
The invention aims to provide a method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase, so as to solve the problems in the background technology. The process is simplified, the time is saved, the hydroxyl is directly introduced in a short time to generate an active product, and the catalysis effect of the free enzyme is good.
The purpose of the invention is realized by the following technical scheme: a method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase, which comprises the following steps:
a. fermentation: inoculating the genetically engineered bacteria producing vitamin D3 hydroxylase into a fermentation culture medium, and adding IPTG (isopropyl-beta-D-thiogalactoside) for inducing enzyme production when the culture is carried out until OD600 is 1-5, wherein the final concentration of the IPTG is 1.2-2mM, the induction temperature is 28 ℃, and the fermentation culture is carried out for 16-24h to obtain fermentation liquor;
b. wall breaking: centrifuging the fermentation liquor at 11000-;
c. crude extraction: centrifuging the wall breaking liquid obtained in the step b at 6000-;
d. substrate configuration: dissolving vitamin D3 in organic solvent, shaking, filtering substrate, refrigerating for 4 hr in refrigerator, pre-embedding cyclodextrin and substrate, shaking, and culturing for 3D;
e. and (3) transformation: dissolving vitamin D3 in an organic solvent, shaking up, slowly adding into the secondary enzyme solution obtained in the step c, simultaneously adding 0.1-0.5% of the cofactor of vitamin D3 hydroxylase, and carrying out free enzyme conversion under the shaking condition of 400-700 rpm;
f. and e, after the conversion in the step e, carrying out reduced pressure concentration, collecting the organic solvent, and carrying out high-temperature or low-temperature crystallization treatment on the concentrated solution to obtain a 25-hydroxy vitamin D3 crystal substance.
Further, the genetically engineered bacterium producing vitamin D3 hydroxylase in the step a is actinomycetes or escherichia coli.
Further, the main components and the contents of the fermentation medium in the step a are peptone 1%, maltose 1.5%, yeast extract 0.6%, NaCl 0.2%, and NaF 0.005%, the pH value is 7.0, and the conversion temperature is 30 ℃.
Further, the mixed buffer solution of phosphoric acid and nitric acid in the step b is 0.4 mol/L phosphoric acid and nitric acid buffer solution (pH5.8) with the volume of the fermentation liquor being 30-45%.
Further, the wall breaking condition of the high-pressure homogenizer in the step b is 800-1500bar, and the treatment time is 15-40 min.
Further, the organic solvent in the step D comprises one or a combination of two of absolute ethyl alcohol, tween, ethyl acetate, ethyl caproate and diethyl ether, and the concentration of the vitamin D3 in the organic solvent is 25-30%.
And further, adding acetone into the fermentation liquor obtained in the step b before wall breaking, adding a proper amount of ethyl acetate into a centrifuge tube, shaking uniformly, and centrifuging for 7-10min at 13000 r/min.
Further, the vitamin D3 in step c is enzyme liquid in the first enzyme liquid, wherein the final concentration is controlled to be 0.2-5 g/L.
Further, the cofactor of the vitamin D3 hydroxylase in the step e is one of NAD + and NADP +, cytochrome C, copper salt, iron salt and calcium ion.
Further, the conversion temperature of the step d is 30-45 ℃, and the conversion period is 24-48 h.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method adopts the free enzyme method to produce the 25-hydroxy vitamin D3 for the first time, greatly increases the enzyme conversion efficiency, has the conversion rate of more than 80 percent, and shortens the conversion period from the flow.
(2) After the wall is broken, the barrier action of cell walls and cell membranes is removed, the substrate can be more efficiently combined on the combination site of the vitamin D3 hydroxylase, and the substrate concentration in a conversion system is improved.
(3) The free enzyme conversion system does not need to add a surfactant additionally, so that the influence of the free enzyme conversion system on the activity of the vitamin D3 hydroxylase is avoided, and the cost of the subsequent extraction process is reduced.
(4) The invention adopts the microorganism to convert the 25-hydroxy vitamin D3, the conversion condition is mild, and a new process route is provided for industrial preparation.
(5) The invention adopts a method that the microorganism directly introduces hydroxyl at the 25-position of 25-hydroxy vitamin D3, solves the problem that organic synthesis is difficult to realize, and directly introduces hydroxyl to generate an active product in a short time.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
A method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase, which comprises the following steps:
a. fermentation: inoculating the genetically engineered bacteria producing vitamin D3 hydroxylase into a fermentation culture medium, and adding IPTG (isopropyl-beta-D-thiogalactoside) for inducing enzyme production when the culture is carried out until OD600 is 1-5, wherein the final concentration of the IPTG is 1.2-2mM, the induction temperature is 28 ℃, and the fermentation culture is carried out for 16-24h to obtain fermentation liquor;
b. wall breaking: centrifuging the fermentation liquor at 11000-;
c. crude extraction: centrifuging the wall breaking liquid obtained in the step b at 6000-;
d. substrate configuration: dissolving vitamin D3 in organic solvent, shaking, filtering substrate, refrigerating for 4 hr in refrigerator, pre-embedding cyclodextrin and substrate, shaking, and culturing for 3D;
e. and (3) transformation: dissolving vitamin D3 in an organic solvent, shaking up, slowly adding into the secondary enzyme solution obtained in the step c, simultaneously adding 0.1-0.5% of the cofactor of vitamin D3 hydroxylase, and carrying out free enzyme conversion under the shaking condition of 400-700 rpm;
f. and e, after the conversion in the step e, carrying out reduced pressure concentration, collecting the organic solvent, and carrying out high-temperature or low-temperature crystallization treatment on the concentrated solution to obtain a 25-hydroxy vitamin D3 crystal substance.
In this embodiment, the genetically engineered bacterium producing vitamin D3 hydroxylase in step a is actinomycetes or escherichia coli.
In this example, the fermentation medium of step a comprises, as main components, peptone 1%, maltose 1.5%, yeast extract 0.6%, NaCl 0.2%, and NaF 0.005%, pH7.0, and conversion temperature 30 ℃.
In this example, the mixed buffer of phosphoric acid and nitric acid in step b is 0.4 mol/L phosphoric acid and nitric acid buffer (pH5.8) with a volume of 30-45% of the fermentation broth.
In this embodiment, the wall-breaking condition of the high-pressure homogenizer in step b is 800-1500bar for 15-40 min.
In this embodiment, the organic solvent in step D includes any one or a combination of two of anhydrous ethanol, tween, ethyl acetate, ethyl caproate and diethyl ether, and the concentration of vitamin D3 in the organic solvent is 25-30%.
In this embodiment, acetone is added into the fermentation liquid in the step b before wall breaking, a proper amount of ethyl acetate is added into a centrifuge tube, the mixture is shaken up and then centrifuged for 7-10min at 13000 r/min.
In this embodiment, the vitamin D3 in step c is an enzyme solution in which the final concentration is controlled to be 0.2-5 g/L.
In this embodiment, the cofactor of the vitamin D3 hydroxylase in the step e is one of NAD + and NADP +, cytochrome C, copper and iron salts, and calcium ions.
In the embodiment, the conversion temperature of the step d is 30-45 ℃ and the conversion period is 24-48 h.
Example 1:
fermentation culture of Escherichia coli
Slant culture medium composed of peptone 10 g/L, yeast extract 5 g/L, maltose 1.5%, NaCl 10 g/L, and pH 7.0;
fermentation medium: peptone 1%, maltose 1.5%, yeast extract 0.6% -NaCl 0.2%, 0.005% NaF, pH to 7.0;
when the cells were cultured in a medium at 37 ℃ and 200rpm until OD600 became 4, IPTG (filtration sterilization) was added to the cells to a final concentration of 1.2mM as an inducer, and the cells were cultured at 28 ℃ for 24 hours to produce an enzyme, thereby obtaining an Escherichia coli solution.
Example 2:
production of 25-hydroxy vitamin D3 by enzyme conversion
Taking 400m L fermentation liquor, centrifuging at 11000rpm for 5min, collecting thalli, adding deionized water with the volume of 3 times of the fermentation liquor to wash the thalli for 2 times, adding 0.4 mol/L phosphoric acid and nitric acid buffer solution (pH7.0) with the volume of 80m L to prepare bacterial suspension, performing wall breaking treatment at 900bar pressure for 25min by a high-pressure homogenizer, centrifuging the wall breaking liquid at 10000rpm for 6min, and collecting supernatant, namely crude enzyme liquid of vitamin D3 hydroxylase.
Dissolving 20mg of vitamin D3 in 1m L ethyl acetate solution, slowly adding the solution into the crude enzyme solution, respectively adding 5mg of NADP +, uniformly mixing, performing enzyme conversion under the shaking condition of 400rpm, wherein the conversion temperature is 35 ℃, the conversion time is 48 hours, performing reduced pressure concentration after conversion, collecting an organic solvent, and performing high-temperature or low-temperature crystallization treatment on the concentrated solution to obtain a 25-hydroxy vitamin D3 crystal substance.
After the conversion by the method, the yield of 25-hydroxyvitamin D3 is 430 mg/L and the conversion rate of vitamin D3 is 83.7% by detection of an HP L C method.
Example 3:
the 25-hydroxy vitamin D3 is produced by enzymatic conversion.
Taking 0.8L fermentation liquor, centrifuging at 12000rpm for 8min, collecting thalli, adding deionized water with 6 times of the volume of the fermentation liquor to wash the thalli for 3 times, adding 0.4 mol/L phosphoric acid and nitric acid buffer solution (pH7.0)100m L to prepare bacterial suspension, performing wall breaking treatment for 40min at 1200bar pressure by a high-pressure homogenizer, centrifuging the wall breaking liquid at 12000rpm for 5min, and collecting supernatant, namely crude enzyme liquid of vitamin D3 hydroxylase.
Dissolving 40mg of vitamin D3 in 1m L ethanol or ether solution, slowly adding the solution into the crude enzyme solution, adding 10mg of NADP +, uniformly mixing, carrying out enzyme conversion under the shaking condition of 600rpm, wherein the conversion temperature is 35 ℃, the conversion time is 24 hours, carrying out reduced pressure concentration after conversion, collecting an organic solvent, and carrying out high-temperature or low-temperature crystallization treatment on the concentrated solution to obtain a 25-hydroxy vitamin D3 crystal substance.
After the conversion by the method, the yield of 25-hydroxy vitamin D3 is 458 mg/L and the conversion rate of vitamin D3 is 86.8% by detecting by an HP L C method.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase, which is characterized by comprising the following steps:
a. fermentation: inoculating the genetically engineered bacteria producing vitamin D3 hydroxylase into a fermentation culture medium, and adding IPTG (isopropyl-beta-D-thiogalactoside) for inducing enzyme production when the culture is carried out until OD600 is 1-5, wherein the final concentration of the IPTG is 1.2-2mM, the induction temperature is 28 ℃, and the fermentation culture is carried out for 16-24h to obtain fermentation liquor;
b. wall breaking: centrifuging the fermentation liquor at 11000-;
c. crude extraction: centrifuging the wall breaking liquid obtained in the step b at 6000-;
d. substrate configuration: dissolving vitamin D3 in organic solvent, shaking, filtering substrate, refrigerating for 4 hr in refrigerator, pre-embedding cyclodextrin and substrate, shaking, and culturing for 3D;
e. and (3) transformation: dissolving vitamin D3 in an organic solvent, shaking up, slowly adding into the secondary enzyme solution obtained in the step c, simultaneously adding 0.1-0.5% of the cofactor of vitamin D3 hydroxylase, and carrying out free enzyme conversion under the shaking condition of 400-700 rpm;
f. and (3) crystallization: and e, after the conversion in the step e, carrying out reduced pressure concentration, collecting the organic solvent, and carrying out high-temperature or low-temperature crystallization treatment on the concentrated solution to obtain a 25-hydroxy vitamin D3 crystal substance.
2. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase according to claim 1, wherein the genetically engineered bacterium producing vitamin D3 hydroxylase in the step a is actinomycetes or Escherichia coli.
3. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase of claim 1, wherein the fermentation medium of step a comprises peptone 1%, maltose 1.5%, yeast extract 0.6%, NaCl 0.2%, NaF 0.005%, pH7.0, and conversion temperature 30 ℃.
4. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase into the vitamin D3 as claimed in claim 1, wherein the mixed buffer of phosphoric acid and nitric acid in the b step is 0.4 mol/L mol of phosphoric acid and nitric acid buffer (pH5.8) which is 30-45% of the volume of the fermentation broth in a 1.5:1 ratio.
5. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase as claimed in claim 1, wherein the wall breaking condition of the high pressure homogenizer in the step b is 800-1500bar for 15-40 min.
6. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase according to claim 1, wherein the organic solvent in the step D comprises any one or a combination of two of absolute ethyl alcohol, tween, ethyl acetate, ethyl caproate and diethyl ether, and the concentration of vitamin D3 in the organic solvent is 25-30%.
7. The method for producing 25-hydroxyvitamin D3 through conversion of vitamin D3 hydroxylase according to claim 1, wherein acetone is added into a centrifuge tube before wall breaking of the fermentation liquid in the step b, a proper amount of ethyl acetate is added into the centrifuge tube, the mixture is shaken up and then centrifuged for 7-10min at 13000 r/min.
8. The method for producing 25-hydroxyvitamin D3 from vitamin D3 by hydroxylase conversion according to claim 1, wherein the vitamin D3 in the step c is an enzyme solution in one time, and the final concentration is controlled to be 0.2-5 g/L.
9. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase according to claim 1, wherein the cofactor of the vitamin D3 hydroxylase in the step e is one of NAD + and NADP +, cytochrome C, copper and iron salts, and calcium ions.
10. The method for producing 25-hydroxyvitamin D3 by converting vitamin D3 hydroxylase according to claim 1, wherein the conversion temperature in the D step is 30-45 ℃ and the conversion period is 24-48 h.
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