CN115433746A - Method for biotransformation of miglitol intermediate - Google Patents
Method for biotransformation of miglitol intermediate Download PDFInfo
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- CN115433746A CN115433746A CN202110624248.5A CN202110624248A CN115433746A CN 115433746 A CN115433746 A CN 115433746A CN 202110624248 A CN202110624248 A CN 202110624248A CN 115433746 A CN115433746 A CN 115433746A
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- miglitol
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- biotransformation
- hydroxide solution
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- IBAQFPQHRJAVAV-ULAWRXDQSA-N Miglitol Chemical compound OCCN1C[C@H](O)[C@@H](O)[C@H](O)[C@H]1CO IBAQFPQHRJAVAV-ULAWRXDQSA-N 0.000 title claims abstract description 52
- 229960001110 miglitol Drugs 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 48
- 230000036983 biotransformation Effects 0.000 title claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 69
- QWXYZCJEXYQNEI-OSZHWHEXSA-N intermediate I Chemical compound COC(=O)[C@@]1(C=O)[C@H]2CC=[N+](C\C2=C\C)CCc2c1[nH]c1ccccc21 QWXYZCJEXYQNEI-OSZHWHEXSA-N 0.000 claims abstract description 36
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000001301 oxygen Substances 0.000 claims abstract description 28
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 28
- 239000003513 alkali Substances 0.000 claims abstract description 23
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 65
- 230000008569 process Effects 0.000 claims description 23
- 238000009423 ventilation Methods 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 241001052560 Thallis Species 0.000 claims description 7
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 4
- 229960002442 glucosamine Drugs 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 claims description 2
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 241000589232 Gluconobacter oxydans Species 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 claims 1
- 238000005984 hydrogenation reaction Methods 0.000 abstract description 20
- 230000001105 regulatory effect Effects 0.000 abstract description 17
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 239000012528 membrane Substances 0.000 description 23
- 238000004128 high performance liquid chromatography Methods 0.000 description 18
- 239000000919 ceramic Substances 0.000 description 13
- 230000001276 controlling effect Effects 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- 238000012258 culturing Methods 0.000 description 12
- 238000001914 filtration Methods 0.000 description 12
- 239000000706 filtrate Substances 0.000 description 9
- 239000002028 Biomass Substances 0.000 description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- LXBIFEVIBLOUGU-JGWLITMVSA-N duvoglustat Chemical compound OC[C@H]1NC[C@H](O)[C@@H](O)[C@@H]1O LXBIFEVIBLOUGU-JGWLITMVSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- LXBIFEVIBLOUGU-UHFFFAOYSA-N Deoxymannojirimycin Natural products OCC1NCC(O)C(O)C1O LXBIFEVIBLOUGU-UHFFFAOYSA-N 0.000 description 3
- BGMYHTUCJVZIRP-UHFFFAOYSA-N Nojirimycin Natural products OCC1NC(O)C(O)C(O)C1O BGMYHTUCJVZIRP-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- BGMYHTUCJVZIRP-GASJEMHNSA-N nojirimycin Chemical compound OC[C@H]1NC(O)[C@H](O)[C@@H](O)[C@@H]1O BGMYHTUCJVZIRP-GASJEMHNSA-N 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 230000001603 reducing effect Effects 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 239000003888 alpha glucosidase inhibitor Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 229940122816 Amylase inhibitor Drugs 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 241000032681 Gluconacetobacter Species 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101100321409 Rattus norvegicus Zdhhc23 gene Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 239000003392 amylase inhibitor Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 239000012492 regenerant Substances 0.000 description 1
- 230000029219 regulation of pH Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of medicines, and relates to a preparation method for biotransformation of a miglitol intermediate, in particular to a method for biotransformation of a miglitol intermediate II by using a fed-batch intermediate I. After the thallus solution is put into the conversion tank, the temperature and dissolved oxygen are regulated, the pH is controlled by feeding the intermediate I and feeding the alkali liquor to obtain the intermediate II, and the miglitol is obtained by hydrogenation.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a bioconversion miglitol intermediate.
Background
Diabetes mellitus is the most common disease caused by endocrine-metabolic disorders, and is mainly classified into type I diabetes (insulin-dependent diabetes mellitus, IDD) and type II diabetes (non-insulin-dependent diabetes mellitus, NIDD). With the increase in the living standard of people and the improvement of nutritional structure, diabetes is on the epidemic trend as a chronic non-infectious disease. Among them, type II diabetes is the main factor.
Nojirimycin (nojirimycin) was first discovered in streptomyces fermentation broth in 1966, and it was found four years later to have a strong inhibitory effect on β -glucosidase, and became the first discovered amylase inhibitor. 1-deoxynojirimycin (1-deoxynojirimycin) obtained by reducing nojirimycin and a series of derivatives thereof also have glycosidase inhibiting effect, and N-substituted-1-deoxynojirimycin has the most obvious blood sugar reducing effect, and miglitol is one of the effects.
Miglitol (N-hydroxyethyl-1-deoxynojirimycin) is a parent modification of 1-deoxynojirimycin, and has a structure similar to that of glucose. As the first pseudo-monosaccharide alpha-glucosidase inhibitor, the alpha-glucosidase inhibitor has become a first choice drug for treating type II diabetes due to the obvious blood glucose reducing effect, safety, effectiveness and good general tolerance.
Most of the miglitol production at present adopts chemical synthesis and combines biological catalysis. Amination with glucose first yields N-hydroxyethyl glucosamine (intermediate I), conversion of the regenerant to 6- (substituted amino) -6-deoxy- α -L-sorbose (intermediate II), and finally hydrogenation to N-hydroxyethyl-1-deoxynojirimycin, miglitol. In the process of biotransformation of the intermediate I (N-hydroxyethyl glucosamine), most manufacturers firstly adjust the pH value of the intermediate I by acid, then add thalli, control the temperature and dissolve oxygen, add alkali liquor to adjust the pH value, collect filtrate after the reaction is finished, and then hydrogenate to finally obtain miglitol. In the biotransformation process, the reaction is violent in the initial stage, the alkali liquor adding amount is large, the medium and later stages tend to be stable, acid is used for adjusting the intermediate I, and alkali liquor is added for adjusting the pH value in the biotransformation process, so that waste is caused.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides a preparation method of a bioconversion miglitol intermediate, adopts a fed-batch intermediate I method for bioconversion, controls the solution to keep pH =5.0-5.6 by the fed-batch intermediate I and alkali liquor, saves acid for initially adjusting the intermediate I, greatly reduces the alkali liquor used in the reaction, is controllable in fed-batch, can control the reaction speed and process at any time, is suitable for industrial production, and is environment-friendly.
The first purpose of the invention is to provide a preparation method of a miglitol intermediate, which comprises the steps of putting thalli into a biotransformation tank, and carrying out biotransformation by adopting a mode of feeding the miglitol intermediate I in a fed-batch manner to obtain a miglitol intermediate II.
The miglitol intermediate I is N-hydroxyethyl glucosamine; the miglitol intermediate II is 6- (substituted amino) -6-deoxy-alpha-L-furan sorbose; the thallus is gluconacetobacter oxydans.
Further, the pH value of the miglitol intermediate I during feeding is controlled to be 5.0-5.6.
Further, alkali liquor can be fed in when the miglitol intermediate I is fed in, and the pH is regulated to 5.2-5.4 together.
Specifically, the biotransformation method is feeding the intermediate I, the pH change is related to the feeding speed, the feeding speed is properly reduced when the pH value rises rapidly, and the feeding speed is properly increased when the pH value drops.
Wherein the alkali liquor is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and calcium hydroxide solution.
Specifically, the alkali liquor is a sodium hydroxide solution, and preferably a sodium hydroxide solution with the mass fraction of 10%.
Further, the volume ratio of the bacterial cells to the miglitol intermediate I is 0.6-1.4, preferably 0.8-1.2.
In the biotransformation reaction, the temperature is controlled to be 27-32 ℃, the ventilation volume is 0.2-0.4vvm, the tank pressure is 0.04-0.06MPa, and the dissolved oxygen is always more than or equal to 30 percent.
Specifically, in the biotransformation reaction, the feeding time is 1-4h.
And the midbody II of the miglitol is judged by the biotransformation end point, and the Rf value is 0.4-0.5 by adopting TLC spot plate sampling detection.
The second purpose of the invention is to provide the application of the method in the preparation of miglitol by a biotransformation method, which comprises the following steps:
solid plate culture: coating the strain on a solid culture medium, and culturing at 30 deg.C for 3-5 days;
carrying out shake flask propagation: plating into shake flask, culturing at 30 deg.C and 180rpm for 2-3 days;
seed tank culture: inoculating into seed tank in shake flask, culturing at 30 deg.C at rotation speed of 200rpm, ventilation amount of 0.5vvm, and tank pressure of 0.04kg/cm 2 When the dissolved oxygen is more than 40 percent, the seeds can be transferred within 15 to 25 hours;
culturing in a fermentation tank: inoculating seed liquid into the fermentation liquid by pressure difference method, culturing at 30 deg.C, rotating speed of 80rpm, ventilation amount of 0.5vvm, and tank pressure of 0.04kg/cm 2 Regulating pH to 5.3-5.5 with alkali solution, culturing for 25-45 hr, and transferring to ceramic membrane for filtering;
and (3) collecting thalli: filtering the fermentation liquor by using a ceramic membrane, collecting thalli, eluting and transferring the thalli into a biological conversion tank;
and (3) biotransformation: the temperature is 30 ℃, the rotating speed is 150rpm, the air throughput is 0.3vvm, the tank pressure is 0.05MPa, the dissolved oxygen is maintained to be more than or equal to 30 percent, the intermediate I is fed in a flow manner, the pH value is regulated and controlled by alkali liquor fed in a flow manner to be 5.2-5.4, and the mixture can be placed in a tank and transferred into a ceramic membrane and a micro-membrane for filtration after 8-14 hours;
and (3) collecting an intermediate II: collecting the intermediate II by using a ceramic membrane and a micro-membrane, and transferring to a hydrogenation process;
hydrogenation: under the action of a catalyst, reacting the intermediate II with hydrogen under high pressure to generate miglitol;
compared with the prior art, the method of the invention has the following advantages:
the method for controlling the pH value of the solution by jointly feeding the intermediate I and the alkali liquor has the advantages of mild conversion process, easier control over pH regulation and dissolved oxygen, higher purity of the obtained miglitol, higher yield, reduced use amount of acid and alkali, environmental friendliness, labor and material conservation for collection and elution of subsequent conversion liquor and easiness in industrial production.
Detailed Description
In order to make the purpose and technical solution of the present invention more clear, the present invention is further described with reference to the following examples, but the scope of the present invention is not limited to these examples, and the examples are only used for explaining the present invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true scope of the invention. The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
Thallus culture-gluconacetogenic bacteria
1) Solid plate culture: the solid culture medium comprises the following components: glucose 0.1%, yeast extract 0.2%, peptone 0.1% potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, calcium chloride 0.05%, magnesium sulfate 0.05%, adjusting pH to 5.5, sterilizing at 121 deg.C for 30 min, coating the strain on the culture medium, and culturing at 30 deg.C for 3-5 days;
2) Carrying out shake flask culture expansion: plating in a shake flask, wherein the shake flask culture medium comprises the following components: glucose 0.1%, yeast extract 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, adjusting pH to 5.5, sterilizing at 121 deg.C for 30 min, and culturing at 30 deg.C and 180rpm for 2-3 days;
3) Seed tank culture: shake-flask inoculation in seed tank, culture medium identical to shake-flask culture medium, sterilizing at 121 deg.C for 30 min, culturing at 30 deg.C, rotating speed of 200rpm, ventilation amount of 0.5vvm, and tank pressure of 0.04kg/cm 2 When the dissolved oxygen is more than 40 percent, the seeds can be transferred within 15 to 25 hours;
4) Culturing in a fermentation tank: the fermentation medium comprises yeast extract 0.1%, D-sorbitol 1.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, adjusting pH to 5.5, sterilizing at 121 deg.C for 30 min, inoculating seed liquid into the fermentation liquid by pressure difference method, culturing at 30 deg.C, rotating speed of 80rpm, ventilation amount of 0.5vvm, and tank pressure of 0.04kg/cm 2 Regulating pH to 5.3-5.5 with alkali solution, culturing for 25-45 hr, and transferring to filter;
5) And (3) collecting thalli: filtering the fermentation liquid with ceramic membrane with aperture of 0.2-0.5 μm, collecting thallus, eluting until the conductivity is below 100, and transferring into a biotransformation tank;
HPLC detection of miglitol
Analytical conditions and system applicability of miglitol HPLC column are as follows:
a Lichrospher aminopropylsilane-bonded silica gel chromatography column (250lqflm 4.6mm,5 μm); mobile phase: acetonitrile-0.01 mol/L ammonium dihydrogen phosphate solution (triethylamine adjusted to pH 7.5) (65; the detection wavelength is 210nm; the flow rate is 1.0ml/min; the sample volume is 20 mul; the column temperature is 25 ℃; a detector: diode Array Detector (DAD). The theoretical plate number is not lower than 1000 calculated according to miglitol peak.
Example 1
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5, and the ventilation volume at 32 +/-3 m 3 The reaction time is 0.05MPa, the initial rotation speed is 150rpm, the intermediate I begins to flow and add, the pH value in the conversion process is controlled to be about 5.0, the dissolved oxygen is 50 percent, the flow and addition is completed within 1 hour, the conversion reaction is slight violent in the flow and addition process, the dissolved oxygen is slightly reduced, the pH value and the dissolved oxygen are regulated and controlled by flow and addition of alkali liquor (10 percent of sodium hydroxide solution), the Rf value is 0.46 through TLC point plate detection after 8 hours, the conversion is completed,transferring the conversion solution to the next working procedure;
and (3) filtering, eluting and concentrating the conversion solution by a ceramic membrane and a micro-membrane, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the miglitol concentration by HPLC (high performance liquid chromatography), wherein the yield is 94.66%. The dosage of 10 percent sodium hydroxide solution is 150L, and the addition of miglitol intermediate I is 350m 3 。
Example 2
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5, and the ventilation volume at 32 +/-3 m 3 The method comprises the following steps of/h, pressure 0.05MPa, initial rotation speed 150rpm, starting to add the intermediate I in a flowing manner, controlling pH to be about 5.3 in the conversion process, dissolving oxygen by 40%, completing the adding in the flowing manner within 2 hours, wherein the conversion reaction is mild, the fluctuation range of the dissolved oxygen is small, adding alkali liquor (10% of sodium hydroxide solution) in the flowing manner to regulate and control the pH and the dissolved oxygen, detecting an Rf value by a TLC point plate after 10 hours, and transferring the conversion solution to the next process after the conversion is finished;
and (3) filtering the conversion solution by a ceramic membrane and a micro-membrane, eluting and concentrating, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the concentration of the miglitol by HPLC (high performance liquid chromatography), wherein the yield is 98.94%. The dosage of 10 percent sodium hydroxide solution is 120L, and the addition of miglitol intermediate I is 350m 3 。
Example 3
Collecting the 280m eluted 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5 and the ventilation volume at 32 +/-3 m 3 The method comprises the following steps of (1) h, pressure of 0.05MPa, initial rotation speed of 150rpm, starting to add the intermediate I in a flowing manner, controlling pH in the conversion process to be about 5.2, dissolving oxygen by 30 percent, completing the adding in the flowing manner within 3 hours, wherein the conversion reaction is relatively gentle in the adding process, the dissolved oxygen is gradually reduced, adjusting and controlling the pH and the dissolved oxygen by adding alkali liquor in a flowing manner (10 percent of sodium hydroxide solution), detecting an Rf value of 0.47 by a TLC point plate after 11 hours, and after the conversion is finished, transferring the conversion solution to the next process;
and (3) filtering the conversion solution by a ceramic membrane and a micro-membrane, eluting and concentrating, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the concentration of the miglitol by HPLC (high performance liquid chromatography), wherein the yield is 96.92%. 116L of 10 percent sodium hydroxide solution and the addition amount of miglitol intermediate I350m 3 。
Example 4
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5 and the ventilation volume at 32 +/-3 m 3 H, pressure is 0.05MPa, initial rotation speed is 150rpm, feeding of the intermediate I is started, pH in the conversion process is controlled to be about 5.4, dissolved oxygen is 40%, feeding is completed in 4 hours, the conversion reaction is relatively gentle in the feeding process, the dissolved oxygen is gradually reduced, feeding of alkali liquor (10% of sodium hydroxide solution) is used for regulating and controlling the pH and the dissolved oxygen, a TLC point plate detects an Rf value of 0.47 after 12 hours, conversion is completed, and the conversion solution is transferred to the next process;
and (3) filtering the conversion solution by a ceramic membrane and a micro-membrane, eluting and concentrating, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the concentration of the miglitol by HPLC (high performance liquid chromatography), wherein the yield is 95.93%. The dosage of 10 percent sodium hydroxide solution is 115L, and the addition of miglitol intermediate I is 350m 3 。
Example 5
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5 and the ventilation volume at 32 +/-3 m 3 The reaction is finished in 1.5 hours, the conversion reaction is relatively stable, the dissolved oxygen is gradually reduced, the pH and the dissolved oxygen are regulated and controlled by adding alkali liquor (10 percent of sodium hydroxide solution) in a flowing manner, the Rf value is 0.46 detected by a TLC point plate after 9 hours, the conversion is finished, and the conversion solution is transferred to the next procedure;
and (3) filtering, eluting and concentrating the conversion solution by a ceramic membrane and a micro-membrane, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the miglitol concentration by HPLC (high performance liquid chromatography), wherein the yield is 95.61%. 134L of 10 percent sodium hydroxide solution and 350m of miglitol intermediate I 3 。
Example 6
Collecting the eluted 420m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5 and the ventilation volume at 32 +/-3 m 3 H, pressure 0.05MPa, initial rotation speed 150rpm, start of fed-batchThe pH value of the intermediate I is controlled to be about 5.3 in the conversion process, the dissolved oxygen is 30%, the flow addition is completed within 2.5 hours, the conversion reaction is stable in the flow addition process, the fluctuation range of the dissolved oxygen is small, the pH value and the dissolved oxygen are regulated and controlled by adding alkali liquor (10% of sodium hydroxide solution) in a flow manner, the Rf value is 0.46 through TLC point plate detection after 10 hours, and the conversion solution is transferred to the next process after the conversion is finished;
and (3) filtering the conversion solution by a ceramic membrane and a micro-membrane, eluting and concentrating, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the concentration of the miglitol by HPLC (high performance liquid chromatography), wherein the yield is 97.83%. The dosage of 10 percent sodium hydroxide solution is 118L, and the addition of miglitol intermediate I is 350m 3 。
Comparative example 1
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5, and the ventilation volume at 32 +/-3 m 3 H, the pressure is 0.05MPa, the initial rotation speed is 150rpm, the intermediate I is adjusted to pH5.5 by hydrochloric acid, the intermediate I is directly added into a conversion kettle, the pH value in the conversion process is controlled to be about 5.3, the dissolved oxygen is 30 percent, the initial conversion reaction is very violent, the dissolved oxygen is rapidly reduced, the alkali liquor (10 percent of sodium hydroxide solution) is rapidly added, and the reaction is gradually slowed after 1.2 hours. After adding base 260L for 8h, detecting Rf value of 0.46 by TLC point plate, finishing conversion, and transferring the conversion solution to the next procedure;
and (3) filtering the conversion solution by a ceramic membrane and a micro-membrane, eluting and concentrating, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the miglitol concentration by HPLC (high performance liquid chromatography), wherein the yield is 87.71%. The dosage of 10 percent sodium hydroxide solution is 260L, and the addition of miglitol intermediate I is 350m 3 。
Comparative example 2
Collecting the eluted 350m 3 Adding bacterial liquid and biomass 25% (v/v) into a conversion tank, regulating and controlling the temperature at 30 ℃, the initial pH value at 5.5 and the ventilation volume at 32 +/-3 m 3 The reaction time is 0.05MPa, the initial rotation speed is 150rpm, the intermediate I begins to flow and add, the pH value in the conversion process is controlled to be about 5.8, the dissolved oxygen is 20 percent, the flow and add is completed within 0.5 hour, the conversion reaction is very violent in the flow and add alkali liquor (10 percent of sodium hydroxide solution) for regulation and control, the Rf value is detected by a TLC point plate after 8 hours and is 0.46, the conversion is finished, and the conversion solution is transferred to the next process；
And (3) filtering, eluting and concentrating the conversion solution by a ceramic membrane and a micro-membrane, transferring the conversion solution into a hydrogenation kettle for hydrogenation reaction, and after the reaction is finished, taking filtrate to detect the miglitol concentration by HPLC (high performance liquid chromatography), wherein the yield is 90.02%. 220L of 10 percent sodium hydroxide solution and 350m of miglitol intermediate I 3 。
Claims (10)
1. A method for bioconversion of a miglitol intermediate, characterized by: and (3) adding the thalli into a biotransformation tank, and performing biotransformation by adjusting the pH value of the miglitol intermediate I in a flowing manner to obtain a miglitol intermediate II.
2. The method of claim 1, wherein the pH is controlled to be 5.0-5.6.
3. The method according to claim 1, wherein the bacterial cells are gluconobacter oxydans; the miglitol intermediate I is N-hydroxyethyl glucosamine; the miglitol intermediate II is 6- (substituted amino) -6-deoxy-alpha-L-furan sorbose.
4. The method as claimed in claim 2, wherein the pH of the miglitol intermediate I is adjusted to 5.2-5.4 by adding lye in a combined manner.
5. The method according to claim 4, wherein the alkali liquor is one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution and calcium hydroxide solution, preferably 10% sodium hydroxide solution by mass fraction.
6. The method according to claim 1, wherein the volume ratio of the microbial cells to miglitol intermediate I is 0.6 to 1.4, preferably 0.8 to 1.2.
7. The method according to claim 1, wherein in the biotransformation reaction, the temperature is controlled to be 27-32 ℃, the ventilation capacity is 0.2-0.4vvm, the tank pressure is 0.04-0.06MPa, and the dissolved oxygen is maintained to be more than or equal to 30%.
8. The method of claim 1, wherein the time for feeding in the biological conversion reaction is 1-4h.
9. The method of claim 1, wherein the miglitol intermediate II is detected by TLC spot plate sampling and has an Rf value of 0.4-0.5.
10. Use of a process according to any one of claims 1 to 9 in the preparation of miglitol by a bioconversion process.
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