CN105886563A - Method for producing hydroxyl sulfydryl benzoic acid by microorganisms - Google Patents
Method for producing hydroxyl sulfydryl benzoic acid by microorganisms Download PDFInfo
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- CN105886563A CN105886563A CN201610464860.XA CN201610464860A CN105886563A CN 105886563 A CN105886563 A CN 105886563A CN 201610464860 A CN201610464860 A CN 201610464860A CN 105886563 A CN105886563 A CN 105886563A
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- 244000005700 microbiome Species 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title abstract description 5
- OAYIYKBNCJLBAT-UHFFFAOYSA-N 3-hydroxy-2-sulfanylbenzoic acid Chemical compound OC=1C(=C(C(=O)O)C=CC=1)S OAYIYKBNCJLBAT-UHFFFAOYSA-N 0.000 title abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- 238000000855 fermentation Methods 0.000 claims abstract description 42
- 230000004151 fermentation Effects 0.000 claims abstract description 42
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 21
- 238000003756 stirring Methods 0.000 claims abstract description 14
- 241000222173 Candida parapsilosis Species 0.000 claims abstract description 13
- 229940055022 candida parapsilosis Drugs 0.000 claims abstract description 13
- 230000001804 emulsifying effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 12
- QTCQXOXUIPNFNH-UHFFFAOYSA-N 2-hydroxy-5-sulfanylbenzoic acid Chemical compound OC(=O)C1=CC(S)=CC=C1O QTCQXOXUIPNFNH-UHFFFAOYSA-N 0.000 claims abstract description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 19
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 18
- 239000000376 reactant Substances 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 15
- 230000002210 biocatalytic effect Effects 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 14
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 11
- 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 claims description 10
- 239000012531 culture fluid Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 9
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 9
- 239000000839 emulsion Substances 0.000 claims description 9
- 208000035126 Facies Diseases 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 230000015556 catabolic process Effects 0.000 claims description 8
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 7
- 230000001133 acceleration Effects 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 210000000582 semen Anatomy 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 241000589220 Acetobacter Species 0.000 claims description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims 1
- 239000011260 aqueous acid Substances 0.000 claims 1
- 239000007809 chemical reaction catalyst Substances 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 210000005253 yeast cell Anatomy 0.000 claims 1
- 239000000047 product Substances 0.000 description 16
- 230000000813 microbial effect Effects 0.000 description 11
- 229940093499 ethyl acetate Drugs 0.000 description 10
- 235000019439 ethyl acetate Nutrition 0.000 description 10
- 239000012295 chemical reaction liquid Substances 0.000 description 9
- 239000000284 extract Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 5
- 208000012839 conversion disease Diseases 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000006473 carboxylation reaction Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- ALSTYHKOOCGGFT-KTKRTIGZSA-N (9Z)-octadecen-1-ol Chemical compound CCCCCCCC\C=C/CCCCCCCCO ALSTYHKOOCGGFT-KTKRTIGZSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- BQODPTQLXVVEJG-UHFFFAOYSA-N [O].C=C Chemical group [O].C=C BQODPTQLXVVEJG-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005354 coacervation Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940055577 oleyl alcohol Drugs 0.000 description 1
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000007086 side reaction Methods 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
- C12P11/00—Preparation of sulfur-containing organic compounds
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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 provides a method for producing hydroxyl sulfydryl benzoic acid by microorganisms, and particularly relates to a method for preparing 2-hydroxyl-5-sulfydryl-benzoic acid as a medical intermediate in a biological catalysis manner by candida parapsilosis. The method comprises the following steps: adding a substrate in microorganism fermentation liquor; stirring and emulsifying; and carrying out biological catalysis reaction. The yield of the product is high, reaction conditions are mild, the method is operated at normal temperature and under normal pressure, and difficulty of harsh production conditions of an existing method is solved.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to Candida parapsilosis living things catalysis and prepare in medicine
The technology of mesosome 2-hydroxyl-5-sulfydryl-benzoic acid.
Background technology
Living things catalysis has the outstanding advantages such as catalytic efficiency high, selectivity strong, mild condition, environmental friendliness, is sustainable
Evolution substitutes and expands the important method of tradition organic chemical synthesis.Wherein microorganism catalysis is that living things catalysis is most inhaled
One of application of gravitation, plays key player in industrial biocatalytic always.
The carboxylation reaction of aromatic hydrocarbons is the reaction that a class is important, and its product is aromatic hydrocarbons hydroxy carboxylic acid, is important pharmaceutical synthesis
Initiation material.But, present producing uses traditional chemosynthesis, with aromatic hydrocarbons and CO2Reaction, reaction pressure is up to 10MPa,
Temperature is up to 140-380 DEG C, and reaction selectivity is poor, and by-product is many.Use microorganism catalysis, then may change this situation.Adopt
With microorganism catalysis, can react production aromatic hydrocarbons hydroxy carboxylic acid, mild condition under room temperature, normal pressure, selectivity is high, side reaction
Few, productivity is high.
The present invention will use microorganism catalysis to react, production aromatic hydrocarbons hydroxy carboxylic acid: 2-hydroxyl-5-sulfydryl-benzoic acid.
Summary of the invention
The present invention uses Candida parapsilosis catalysis preparation 2-hydroxyl-5-sulfydryl-benzoic acid, and reaction equation is as follows:
Substrate p-phenolic group sulfonic acid (1), through Candida parapsilosis catalytic reaction, obtains product 2-hydroxyl-5-sulfydryl-benzoic acid (2).
There is multiple-microorganism can be catalyzed this reaction, screen through great many of experiments, finally determine that employing Candida parapsilosis is as urging
Agent, because the effect of its catalytic reaction is best, by-product is few, and reaction yield is high.
Many candida mycodermas can be carried out this reaction of living things catalysis, but, its effect is different, differs greatly, through experiment,
The present invention selects Candida parapsilosis bacterial strain to be DSM-70125, its catalytic reaction best results.
Owing to substrate solubility is relatively low, so, add surfactant to increase the dissolubility of substrate.Although surface activity
Agent adds substrate solubility, and substrate still can not dissolve very well, belongs to liquid-liquid 2 phase reaction, substrate, the interphase mass transfer of product
For reaction limiting element, so, also need to take measures, to improve response speed.Increase the reaction interface of inhomogeneous reaction, be
Improve one of effective measures of inhomogeneous reaction.After adding surfactant, under vigorous stirring, reactant liquor becomes emulsion, makes
Obtain catalytic reaction interface to increase considerably, thus increase substantially response speed.Particularly, the surfactant of addition, it is necessary to
Bio-compatible, does not suppress the catalysis activity of microorganism, so, kinds of surfactants and the selection of concentration, be very important.
Great many of experiments shows, for this reaction, adds paregal O (PerogolO, the oxygen ethylene of 15 units and the condensation substance of oleyl alcohol)
19-20g/L is optimal.
After reaction terminates, extract product with organic solvent.Need breakdown of emulsion, have the multiple method can be with breakdown of emulsion, including salting out method:
Add sodium chloride, ammonium sulfate or calcium chloride, magnesium chloride, calcium sulfate, magnesium sulfate；Coacervation: add aluminium polychlorid, Alumen etc.；
Or above-mentioned 2 class mixing are added；Heating etc..Different products, method used is different, and experiment shows, for this reaction, adds
Calcium chloride+0.05% aluminium polychlorid of 1.6% is optimal.
In carboxylation reaction, use NaHCO3CO is provided2, it practice, there is many kinds of substance can provide CO2But, by greatly
Amount experiment, result shows, for the present invention, uses NaHCO3It is optimal.Owing to adding NaHCO3The pH liter of reactant liquor can be made
Height, and suitable pH, of crucial importance for microorganism catalysis reaction, so, it is impossible to once add, it is necessary to use fed-batch mode to add
NaHCO3.But, even if stream adds NaHCO3, the pH of reactant liquor still raises, so, it is necessary to take measures, maintain at reactant liquor
In suitable pH, microorganism is enable normally to play biocatalysis.Being shown by great many of experiments, stream adds aqueous hydrochloric acid solution and is
Suitable method, regulates NaHCO3With the stream rate of acceleration of aqueous hydrochloric acid solution, the pH of solution can be made to be in optimum range, make biology
Catalytic reaction is normally carried out.
It is apparent that microbial strains involved in the present invention selects, surfactant selects, demulsifier selects,
CO2Donor seletion, also reaction temperature, in the response time, react pH, concentration of substrate, and biocatalytic reaction liquid composition determines, many
Multifactor being both needed to considers, and, they influence each other.Particularly, need from laboratory lab scale, pilot scale, until commercial scale is anti-
Should, increase considerably work difficulty, workload especially.So, experimental design is important to managing, so many factor, and biology is urged
Change kinetics theory analysis and experimental work amount is huge.
Culture medium:
1, seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6.2;Join
The agar powder of 15g/L is added during solid medium processed.
2, culture fluid composition: glucose 32-36 g/L, xylose 7-8g/L, corn plumule powder 13-15 g/L, yeast carries
Take thing 6-8 g/L, (NH4 ) 2HPO4 12-15 g/L, KH2PO 4 6.5-7.5 g/L. MgSO4 .7H2O 0.7-0.8
g/L, NaCl 0.1-0.12 g/L; pH6.4。
2, solid medium composition: add the agar powder of 2% in liquid medium within.
Prepared by microbial fermentation solution.Candida parapsilosis DSM-70125 is planted through inclined-plane, shaking flask, seed tank culture
Sub-liquid；Fermentation tank adds culture fluid, and coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, is cooled to 32-33 DEG C, will
Candida parapsilosis seed liquor is seeded to fermentation tank, and inoculative proportion is 7.3-7.9%, and ventilation ratio is that 1.2-1.4V/V divide
Clock, cultivates 40-44 hour for 32-33 DEG C, and in fermentation liquid, wet barm cell concentration is 92-96g/L, and this fermentation liquid is used as living things catalysis
Catalysts.
Surveying product yield and purity, C18 post, UV-detector, wavelength 254nm with HPLC, flowing is that the phosphoric acid of pH3 delays mutually
Rush liquid: methanol=90:10.
Biocatalytic reaction: fermentation tank, i.e. prepares microbial fermentation solution fermentation tank, adds substrate p-in its fermentation liquid
Phenolic group sulfonic acid, making concentration is 92-95g/L, adds following composition, makes the concentration be: paregal O 19-20g/L, glucose 18-
22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation pH is 6.1-6.4, and stirring makes reactant liquor
Emulsifying, stirring and emulsifying, carry out biocatalytic reaction, reaction temperature 35 DEG C, the response time is 36-40 hour；When reaction starts,
Stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the aqueous hydrochloric acid solution that concentration is 1% simultaneously, regulates NaHCO3With hydrochloric acid
The stream rate of acceleration of aqueous solution, maintains pH6.1-6.4, and the material added is all through sterilization treatment；Ventilation ratio is 0.6-0.65V/
V minute, ventilation the most per minute was 0.6-0.65 times of reactant liquor volume, and after reaction terminates, breakdown of emulsion, with 0.2 times of reactant liquor volume
Ethyl acetate extractive reaction liquid extracts 3 times, merges organic facies, and organic facies steams ethyl acetate, obtains product 2-hydroxyl-5-mercapto
Base-benzoic acid, reaction conversion ratio 97.5-99.3%, product yield 95.5-97.2%.
Embodiment 1
Culture fluid composition: glucose 32-36 g/L, xylose 7-8g/L, corn plumule powder 13-15 g/L, yeast extract 6-
8 g/L, (NH4 ) 2HPO4 12-15 g/L, KH2PO 4 6.5-7.5 g/L. MgSO4 .7H2O 0.7-0.8 g/L,
NaCl 0.1-0.12 g/L; pH6.4。
Prepared by microbial fermentation solution.Candida parapsilosis DSM-70125 is planted through inclined-plane, shaking flask, seed tank culture
Sub-liquid；Fermentation tank adds culture fluid, and coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, is cooled to 32-33 DEG C, will
Candida parapsilosis seed liquor is seeded to fermentation tank, and inoculative proportion is 7.3-7.9%, and ventilation ratio is that 1.2-1.4V/V divide
Clock, cultivates 40-44 hour for 32-33 DEG C, and in fermentation liquid, wet barm cell concentration is 92-96g/L, and this fermentation liquid is used as living things catalysis
Catalysts.
Biocatalytic reaction: 15L fermentation tank, i.e. prepares microbial fermentation solution fermentation tank, adds substrate in its fermentation liquid
P-phenolic group sulfonic acid, making concentration is 90-95g/L, adds following composition, makes the concentration be: paregal O 19-20g/L, glucose
18-22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation pH is 6.1-6.4, and stirring makes reaction
Liquid emulsifying, stirring and emulsifying, carry out biocatalytic reaction, reaction temperature 35 DEG C, the response time is 36-40 hour；Reaction starts
Time, stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the aqueous hydrochloric acid solution that concentration is 1% simultaneously, regulates NaHCO3With
The stream rate of acceleration of aqueous hydrochloric acid solution, maintains pH6.1-6.4, and the material added is all through sterilization treatment；Ventilation ratio is 0.6-
0.65V/V minute, ventilation the most per minute was 0.6-0.65 times of reactant liquor volume, and after reaction terminates, breakdown of emulsion, with 0.2 times of reaction
Liquid volume of ethylacetate extractive reaction liquid extract 3 times, merge organic facies, organic facies steams ethyl acetate, obtain product 2-hydroxyl-
5-sulfydryl-benzoic acid, reaction conversion ratio 97.5-99.3%, product yield 95.5-97.2%.
Embodiment 2
Culture fluid composition and microbial fermentation solution are prepared with embodiment 1.
Biocatalytic reaction: 150L fermentation tank, i.e. prepares microbial fermentation solution fermentation tank, adds substrate in its fermentation liquid
P-phenolic group sulfonic acid, making concentration is 90-95g/L, adds following composition, makes the concentration be: paregal O 19-20g/L, glucose 18-
22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation pH is 6.1-6.4, and stirring makes reactant liquor
Emulsifying, stirring and emulsifying, carry out biocatalytic reaction, reaction temperature 35 DEG C, the response time is 36-40 hour；When reaction starts,
Stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the aqueous hydrochloric acid solution that concentration is 1% simultaneously, regulates NaHCO3With hydrochloric acid
The stream rate of acceleration of aqueous solution, maintains pH6.1-6.4, and the material added is all through sterilization treatment；Ventilation ratio is 0.6-0.65V/
V minute, ventilation the most per minute was 0.6-0.65 times of reactant liquor volume, reaction terminate after, breakdown of emulsion, respectively with 0.2 times, 0.15
Times, the ethyl acetate extractive reaction liquid of 0.1 times of reactant liquor volume respectively extract 1 time, merge organic facies, steam ethyl acetate, obtain
Product 2-hydroxyl-5-sulfydryl-benzoic acid, reaction conversion ratio 97.5%, product yield 95.5%.
Embodiment 3
Culture fluid composition and microbial fermentation solution are prepared with embodiment 1.
Biocatalytic reaction: 1000L fermentation tank, i.e. prepares microbial fermentation solution fermentation tank, adds at the end in its fermentation liquid
Thing p-phenolic group sulfonic acid, making concentration is 90-95g/L, adds following composition, makes the concentration be: paregal O 19-20g/L, glucose
18-22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation pH is 6.1-6.4, and stirring makes reaction
Liquid emulsifying, stirring and emulsifying, carry out biocatalytic reaction, reaction temperature 35 DEG C, the response time is 36-40 hour；Reaction starts
Time, stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the aqueous hydrochloric acid solution that concentration is 1% simultaneously, regulates NaHCO3With
The stream rate of acceleration of aqueous hydrochloric acid solution, maintains pH6.1-6.4, and the material added is all through sterilization treatment；Ventilation ratio is 0.6-
0.65V/V minute, ventilation the most per minute was 0.6-0.65 times of reactant liquor volume, and after reaction terminates, breakdown of emulsion, respectively with 0.2
Times, the ethyl acetate extractive reaction liquid of 0.15 times, 0.1 times reactant liquor volume respectively extract 1 time, merge organic facies, steam acetic acid second
Ester, obtains product 2-hydroxyl-5-sulfydryl-benzoic acid, reaction conversion ratio 98.5%, product yield 96.5%.
Embodiment 4
Culture fluid composition and microbial fermentation solution are prepared with embodiment 1.
Biocatalytic reaction: 15000L fermentation tank, i.e. prepares microbial fermentation solution fermentation tank, adds at the end in its fermentation liquid
Thing p-phenolic group sulfonic acid, making concentration is 90-95g/L, adds following composition, makes the concentration be: paregal O 19-20g/L, glucose
18-22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation pH is 6.1-6.4, and stirring makes reaction
Liquid emulsifying, stirring and emulsifying, carry out biocatalytic reaction, reaction temperature 35 DEG C, the response time is 36-40 hour；Reaction starts
Time, stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the aqueous hydrochloric acid solution that concentration is 1% simultaneously, regulates NaHCO3With
The stream rate of acceleration of aqueous hydrochloric acid solution, maintains pH6.1-6.4, and the material added is all through sterilization treatment；Ventilation ratio is 0.6-
0.65V/V minute, ventilation the most per minute was 0.6-0.65 times of reactant liquor volume, after reaction terminates, and breakdown of emulsion, use counter-current extraction
Machine extracts, and extract is ethyl acetate, steams the ethyl acetate of organic facies, steams ethyl acetate, obtains product 2-hydroxyl-5-mercapto
Base-benzoic acid, reaction conversion ratio 99.3%, product yield 97.2%.
Claims (2)
1. the method that a Candida parapsilosis living things catalysis prepares 2-hydroxyl-5-sulfydryl-benzoic acid, it is characterised in that the most smooth
Adding substrate p-phenolic group sulfonic acid in candida mycoderma fermentation liquid, making concentration is 90-95g/L, adds following composition, makes the concentration be: flat
Put down and add O 19-20g/L, glucose 18-22g/L, xylose 45-50g/L, sucrose 25-28g/L, Semen phaseoli radiati powder 18-22 g/L；Regulation
PH is 6.1-6.4, and stirring makes reactant liquor emulsifying, stirring and emulsifying, carries out biocatalytic reaction, reaction temperature 35 DEG C, response time
For 36-40 hour；When reaction starts, stream adds the NaHCO that concentration is 5% immediately3Aqueous solution, stream adds the salt that concentration is 1% simultaneously
Aqueous acid, regulates NaHCO3With the stream rate of acceleration of aqueous hydrochloric acid solution, maintaining pH6.1-6.4, the material added is all through going out
Bacterium processes；Ventilation ratio is 0.6-0.65V/V minute, and ventilation the most per minute is 0.6-0.65 times of reactant liquor volume, and reaction terminates
After, breakdown of emulsion, it is extracted with ethyl acetate reactant liquor, organic facies steams ethyl acetate, obtains product 2-hydroxyl-5-sulfydryl-benzoic acid, instead
Answering conversion ratio 97.5-99.3%, product yield 95.5-97.2%, described fermentation liquid is obtained by culture fluid fermentative microorganism.
2. the method described in claim 1, it is characterised in that Candida parapsilosis fermentation liquid preparation process is as follows: nearly smooth vacation
Silk yeast obtains seed liquor through inclined-plane, shaking flask, seed tank culture；Fermentation tank adds culture fluid, and coefficient is 0.6-0.7, and 121
DEG C autoclaving 30 minutes, is cooled to 32-33 DEG C, Candida parapsilosis seed liquor is seeded to fermentation tank, and inoculative proportion is
7.3-7.9%, ventilation ratio is 1.2-1.4V/V minute, cultivates 40-44 hour for 32-33 DEG C, and in fermentation liquid, wet yeast cells is dense
Degree is 92-96g/L, and this fermentation liquid is used as biocatalytic reaction catalyst；In fermentation tank, culture fluid composition is: glucose 32-36
G/L, xylose 7-8g/L, corn plumule powder 13-15 g/L, yeast extract 6-8 g/L, (NH4 ) 2HPO4 12-15 g/
L, KH2PO 4 6.5-7.5 g/L. MgSO4 .7H2O 0.7-0.8 g/L, NaCl 0.1-0.12 g/L; pH6.4。
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