CN109988792A - A method of 5-HMFA is synthesized using D. radiodurans R1 - Google Patents
A method of 5-HMFA is synthesized using D. radiodurans R1 Download PDFInfo
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- 241001003009 Deinococcus radiodurans R1 Species 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 22
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 claims abstract description 52
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 claims abstract description 52
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 16
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 14
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 10
- 235000015097 nutrients Nutrition 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims abstract description 6
- 238000011081 inoculation Methods 0.000 claims abstract description 5
- 230000003698 anagen phase Effects 0.000 claims abstract description 4
- 239000002054 inoculum Substances 0.000 claims abstract description 3
- 239000008363 phosphate buffer 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 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 239000007986 glycine-NaOH buffer Substances 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 9
- 230000003647 oxidation Effects 0.000 abstract description 5
- 238000007254 oxidation reaction Methods 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 239000011942 biocatalyst Substances 0.000 abstract description 3
- PCSKKIUURRTAEM-UHFFFAOYSA-N 5-hydroxymethyl-2-furoic acid Chemical compound OCC1=CC=C(C(O)=O)O1 PCSKKIUURRTAEM-UHFFFAOYSA-N 0.000 description 48
- 239000002609 medium Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- CHTHALBTIRVDBM-UHFFFAOYSA-N furan-2,5-dicarboxylic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)O1 CHTHALBTIRVDBM-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 241000192091 Deinococcus radiodurans Species 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000005855 radiation Effects 0.000 description 4
- DSLRVRBSNLHVBH-UHFFFAOYSA-N 2,5-furandimethanol Chemical compound OCC1=CC=C(CO)O1 DSLRVRBSNLHVBH-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- -1 furans aromatic compounds Chemical class 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- PXJJKVNIMAZHCB-UHFFFAOYSA-N 2,5-diformylfuran Chemical compound O=CC1=CC=C(C=O)O1 PXJJKVNIMAZHCB-UHFFFAOYSA-N 0.000 description 1
- QMKUAAAZHQCMKH-UHFFFAOYSA-N 2-(furan-2-yl)propan-2-ol Chemical compound CC(C)(O)C1=CC=CO1 QMKUAAAZHQCMKH-UHFFFAOYSA-N 0.000 description 1
- NSQYDLCQAQCMGE-UHFFFAOYSA-N 2-butyl-4-hydroxy-5-methylfuran-3-one Chemical compound CCCCC1OC(C)=C(O)C1=O NSQYDLCQAQCMGE-UHFFFAOYSA-N 0.000 description 1
- SHNRXUWGUKDPMA-UHFFFAOYSA-N 5-formyl-2-furoic acid Chemical compound OC(=O)C1=CC=C(C=O)O1 SHNRXUWGUKDPMA-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 150000002240 furans Chemical class 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000003863 metallic catalyst Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The present invention relates to a kind of methods using D. radiodurans R1 synthesis 5-HMFA, by D. radiodurans R1 streak inoculation in TGY solid medium, picking single colonie is inoculated in TGY fluid nutrient medium after culture, after culture to logarithmic growth phase, is accessed in fresh TGY fluid nutrient medium by 1% inoculum concentration, cultivate 48h, collect somatic cells, it is added in the buffer containing 5 hydroxymethyl furfural, 3~48h is reacted at 20~60 DEG C of temperature, obtains 5-HMFA.The present invention using D. radiodurans R1 as biocatalyst, it has height endurability to 5 hydroxymethyl furfural, high concentration substrate selective oxidation synthesis target product can be catalyzed, yield is up to 86% or more, the method of the present invention is higher with concentration of substrate, reaction efficiency is more excellent, the good feature of selectivity.
Description
Technical field
The present invention relates to a kind of methods using D. radiodurans R1 synthesis 5-HMFA, belong to microorganism and change
Learn field of engineering technology.
Background technique
Environment-friend substitution product are advocated with green energy resource substitution petroleum resources and sought in chemical industry with 21 century
Sustainable development idea is goed deep into, biomass energy and biotechnology the resource and environment problem increasingly serious present age in occupation of
More and more crucial status.5 hydroxymethyl furfural (HMF) is considered most valuable and potentiality substitute basisization in petrochemical industry
The biology base platform chemicals of product, HMF is the byproduct generated in lignin preprocessing process, by hexose (mainly Portugal
Grape sugar) dehydration generation.People are based primarily upon following two major reason to the research of 5 hydroxymethyl furfural: on the one hand, biology is pre-
HMF in processing hydrolyzate will lead to the growth inhibition of many microorganisms, and have certain toxic effect, to influence subsequent
The yield and efficiency of fermentation synthesis of chemicals and Fuel Process；On the other hand, due to having aldehyde radical and hydroxyl on HMF molecule furan nucleus
The high active function groups such as methyl can generate a series of furans aromatic compounds with catalysis oxidation, according to the position of oxidation and oxygen
Change degree can be oxidized into 5- methylol -2- furancarboxylic acid (5-hydroxymethylfuroic acid, HMFCA), 2,
5- furans dicarbaldehyde (2,5-diformylfuran, DFF), 5- formoxyl -2- furancarboxylic acid (5-formylfuroic acid,
FFCA), 2,5-furandicarboxylic acid (2,5-furandicarboxylic acid, FDCA), when aldehyde radical is reduced to hydroxyl on ring,
Obtain the reduzate 2 of HMF, 5- furyl dimethyl carbinol (2,5-Furandimethanol, BHMF).These redox derivatives
It is all crucial synthesis bridge compound, in the fields such as agricultural, the energy, medicine, macromolecule extensive application.For example, HMFCA
Itself has carboxyl and methylol, can aggregate into a variety of polyester with itself or with other compounds, while being also used as white
Interleukin inhibitor.
Currently, mainly having chemical transformation and biotransformation method by HMF preparation HMFCA.Chemical transformation have been achieved with compared with
More progress are mainly deposited with metallic catalyst catalysis HMF oxidation preparation however, chemical method reaction needs higher temperature and pressure
In the problem of selectivity of product and substrate tolerance difference.And can be with HMF under mild reaction conditions using biocatalyst
Substrate is converted into HMFCA, has high yield and highly selective, while keeping high chemical purity, low by-product, but due to 5-
For hydroxymethylfurfural to the toxic effect of microbial cell, most of microorganism is lower to the substrate tolerance concentration of HMF and is catalyzed
HMF conversion rate is slow, therefore, filters out a kind of couple of substrate HMF with height endurability, and can efficient catalytic HMF selectivity oxygen
The bacterial strain for being combined to HMFCA is necessary.
D. radiodurans Deinococcus radiodurans (DR) is a kind of red, illiquidity, does not generate spore
The spherical bacterial of son is the rotten meat still rotten after radiation sterilization for the first time by American scientist Anderson etc. in 1956
A kind of rhodobacterium separated in class can.DR is one of most radiation-resistant biology of the earth, be the damage of current researching DNA with
Ideal model organism is repaired, therefore the damage of radiation is had focused largely on to the research of DR and repairs aspect.The present invention uses
D. radiodurans R1 (Deinococcus radiodurans R1) is used as catalyst, it is found that it can efficiently, with high selectivity
Catalysis HMF is converted into target product HMFCA.
Summary of the invention
It is an object of the invention in view of the problems of the existing technology, provide a kind of utilization D. radiodurans R1 synthesis
The method of 5-HMFA, D. radiodurans R1 have height endurability to substrate HMF, and can efficient catalytic HMF selection
Property oxidative synthesis HMFCA.
Technical solution
A method of 5-HMFA being synthesized using D. radiodurans R1, is included the following steps:
(1) by D. radiodurans R1 streak inoculation after TGY solid medium, 30 DEG C of culture 72h, picking single colonie is connect
Kind is cultivated under the conditions of 30 DEG C, 180-220r/min to logarithmic growth phase in TGY fluid nutrient medium；Then, by 1% inoculation
Amount accesses in fresh TGY fluid nutrient medium, and 48h is cultivated under conditions of 30 DEG C, 180-220r/min, collects somatic cells；
(2) by step (1) collect somatic cells be added in the buffer containing 5 hydroxymethyl furfural, temperature 20~
3~48h is reacted at 60 DEG C, obtains 5-HMFA.
Further, in step (1), the formula of the TGY solid medium: yeast 0.3%, peptone 0.5%, glucose
0.1%, agar 1.8%.
Further, in step (1), the formula of the TGY fluid nutrient medium: yeast 0.3%, peptone 0.5%, glucose
0.1%.
Further, in step (2), the buffer is phosphate buffer, Tris-HCl buffer or glycine-NaOH
Any one in buffer, the pH of buffer are 6.0~10.0.
Further, in step (2), the dosage of the somatic cells is 10~200mg/mL.
Further, in step (2), concentration of the 5 hydroxymethyl furfural in buffer is 100~500mM.
Beneficial effects of the present invention:
1) present invention is used as catalyst using D. radiodurans R1 (Deinococcus radiodurans R1), can be high
Effect, catalysis HMF is converted into target product HMFCA with high selectivity, and overcomes the disagreeableness disadvantage of chemical catalyst environment.
2) the biocatalyst D. radiodurans R1 (Deinococcus radiodurans R1) that the present invention utilizes is right
HMF have height endurability, can be catalyzed high concentration substrate (300mM) selective oxidation synthesis target product, yield up to 86% with
On.Compared with reported biocatalysis technique, not only concentration of substrate is higher by the present invention, reaction efficiency is more excellent, but also selects
Property is also more preferable.
3) reaction process of the present invention is simple, without adding culture medium (addition culture medium can make reaction system more complicated),
Easily-controllable, mild condition is conducive to the separation purifying technique for simplifying succeeding target product.
4) D. radiodurans R1 (Deinococcus radiodurans R1) is to radiation, high temperature, drying, chemical reagent
The extreme environments such as toxicity have apparent tolerance, therefore can not be limited by industrial process conditions.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of D. radiodurans R1；
Fig. 2 is the liquid chromatogram of synthetic product in embodiment 1.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.Listed embodiment is only made to demonstrate it
With, and show that the spirit and scope of the present invention are not limited to the details in this and its modification case.
In following embodiments, the biomaterial D. radiodurans R1 used is answered from Xinjiang Agricultural Sciences institute microorganism
With research institute, classification naming is Deinococcus radiodurans R1, and Chinese Typical Representative training is preserved on March 14th, 2019
Object collection (abbreviation CCTCC) is supported, preservation address is the Wuhan Wuhan University, China, and deposit number is CCTCC No:M
2019141。
Embodiment 1
A method of 5-HMFA being synthesized using D. radiodurans R1, is included the following steps:
(1) by D. radiodurans R1 streak inoculation in TGY solid medium (yeast 0.3%, peptone 0.5%, grape
Sugar 0.1%, agar 1.8%), after 30 DEG C of culture 72h, the colonial morphology figure of D. radiodurans R1 is shown in Fig. 1, and picking single colonie connects
Kind is trained under the conditions of 30 DEG C, 200r/min in TGY fluid nutrient medium (yeast 0.3%, peptone 0.5%, glucose 0.1%)
It supports to logarithmic growth phase；Then, it is accessed in fresh TGY fluid nutrient medium by 1% inoculum concentration, in 30 DEG C, the item of 200r/min
48h is cultivated under part, collects somatic cells；
(2) 0.25mmol HMF (100mM) is added in 2.5mL phosphate buffer (100mM, pH 7.4), mixing is equal
After even, by step (1) collect somatic cells by 120mg/mL (based on wet cell weight) concentration addition, 30 DEG C of temperature,
Reacted under conditions of 850r/min, monitored and reacted using liquid chromatogram, after 8h, liquid chromatogram see Fig. 2 (as can be seen that
The retention time of HMFCA and HMF is respectively 2.808min, 6.038min), HMF conversion ratio is that 87.27%, HMFCA yield is
98.60%.
Wherein, it is Thermo Fisher ultimate that the method for liquid chromatographic detection and condition, which are respectively as follows: instrument,
3000, detector is UV detector；Detection wavelength is 230nm；Chromatographic column be Sepax GP-C18 column (4.6mm ×
250mm,5μm)；Mobile phase is A:20mM KH2PO4；B:100% acetonitrile；Gradient elution (0min:10%B；7min:24%；
10min:10%B)；Flow velocity is 1.0ml min-1；Column temperature is 25 DEG C；Sample volume is 5 μ l.
Embodiment 2
Reaction temperature in step (2) is changed to 40 DEG C, remaining is same as Example 1.After reacting 8h, HMF conversion ratio is
76.46%, HMFCA yield are 98.13%.
Embodiment 3
Reaction temperature in step (2) is changed to 60 DEG C, remaining is same as Example 1.After reacting 8h, HMF conversion ratio is
57.05%, HMFCA yield are 99.54%.
Embodiment 4
The pH 7.4 of phosphate buffer in step (2) is changed to pH 6.0, remaining is same as Example 1.After reacting 8h,
HMF conversion ratio is that 68.38%, HMFCA yield is 99.13%.
Embodiment 5
The pH 7.4 of phosphate buffer in step (2) is changed to pH 7.0, remaining is same as Example 1.After reacting 8h,
HMF conversion ratio is that 72.23%, HMFCA yield is 98.85%.
Embodiment 6
2.5mL phosphate buffer (100mM, pH 7.4) in step (2) is changed to 2.5mL Tris-HCl buffer
(50mM, pH 8.0), remaining is same as Example 1.After reacting 8h, HMF conversion ratio is that 76.57%, HMFCA yield is
98.38%.
Embodiment 7
2.5mL phosphate buffer (100mM, pH 7.4) in step (2) is changed to 2.5mL Tris-HCl buffer
(50mM, pH 9.0), remaining is same as Example 1.After reacting 8h, HMF conversion ratio is that 78.70%, HMFCA yield is
98.31%.
Embodiment 8
The additional amount 120mg/mL of somatic cells in step (2) is changed to 40mg/mL, remaining is same as Example 1.Reaction
After 12h, HMF conversion ratio is that 36.18%, HMFCA yield is 98.56%.
Embodiment 9
The additional amount 120mg/mL of somatic cells in step (2) is changed to 80mg/mL, remaining is same as Example 1.Reaction
After 12h, HMF conversion ratio is that 67.84%, HMFCA yield is 99.36%.
Embodiment 10
0.25mmol HMF (100mM) in step (2) is changed to 0.375mmol HMF (150mM), the addition of somatic cells
Amount 120mg/mL is changed to 200mg/mL, remaining is same as Example 1.After reaction for 24 hours, HMF conversion ratio is 91.86%, HMFCA
Yield is 99.48%.
Embodiment 11
0.25mmol HMF (100mM) in step (2) is changed to 0.5mmol HMF (200mM), the additional amount of somatic cells
120mg/mL is changed to 200mg/mL, remaining is same as Example 1.After reaction for 24 hours, HMF conversion ratio is 78.22%, HMFCA production
Rate is 99.37%.
Embodiment 12
0.25mmol HMF (100mM) in step (2) is changed to 1.5mmol HMF (300mM), the additional amount of somatic cells
120mg/mL is changed to 200mg/mL, remaining is same as Example 1.After reacting 36h, HMF conversion ratio is 58.66%, HMFCA production
Rate is 99.25%.
Embodiment 13
By 2.5mL phosphate buffer (100mM, pH 7.4) in step (2) be changed to 5mL phosphate buffer (100mM,
PH 7.4), 0.25mmol HMF (100mM) is changed to 1.5mmol HMF (300mM), and the additional amount 120mg/mL of somatic cells changes
Sodium hydroxide is added by reaction system pH and is adjusted to 7.0 when 3h for 200mg/mL, remaining is same as Example 1, reaction
After 36h, HMF conversion ratio is that 86.05%, HMFCA yield is 99.45%.
Embodiment 14
By 2.5mL phosphate buffer (100mM, pH 7.4) in step (2) be changed to 5mL phosphate buffer (100mM,
PH 7.4), 0.25mmol HMF (100mM) is changed to 2.5mmol HMF (500mM), and the additional amount 120mg/mL of somatic cells changes
Sodium hydroxide is added by reaction system pH and is adjusted to 7.0 when 3h for 200mg/mL, remaining is same as Example 1, reaction
After 48h, HMF conversion ratio is that 49.45%, HMFCA yield is 99.33%.
Claims (6)
1. a kind of method using D. radiodurans R1 synthesis 5-HMFA, which comprises the steps of:
(1) by D. radiodurans R1 streak inoculation after TGY solid medium, 30 DEG C of culture 72h, picking single colonie is inoculated in
TGY fluid nutrient medium is cultivated under the conditions of 30 DEG C, 180-220r/min to logarithmic growth phase；Then, it is connect by 1% inoculum concentration
Enter in fresh TGY fluid nutrient medium, 48h is cultivated under conditions of 30 DEG C, 180-220r/min, collects somatic cells；
(2) somatic cells that step (1) is collected are added in the buffer containing 5 hydroxymethyl furfural, at 20~60 DEG C of temperature
3~48h of lower reaction, obtains 5-HMFA.
2. utilizing the method for D. radiodurans R1 synthesis 5-HMFA as described in claim 1, which is characterized in that step
(1) in, the formula of the TGY solid medium: yeast 0.3%, peptone 0.5%, glucose 0.1%, agar 1.8%.
3. utilizing the method for D. radiodurans R1 synthesis 5-HMFA as described in claim 1, which is characterized in that step
(1) in, the formula of the TGY fluid nutrient medium: yeast 0.3%, peptone 0.5%, glucose 0.1%.
4. utilizing the method for D. radiodurans R1 synthesis 5-HMFA as described in claim 1, which is characterized in that step
(2) in, the buffer is any one in phosphate buffer, Tris-HCl buffer or glycine-NaOH buffer,
The pH of buffer is 6.0~10.0.
5. utilizing the method for D. radiodurans R1 synthesis 5-HMFA as described in claim 1, which is characterized in that step
(2) in, the dosage of the somatic cells is 10~200mg/mL.
6. utilizing the method for D. radiodurans R1 synthesis 5-HMFA, feature as described in any one of claim 1 to 5
It is, in step (2), concentration of the 5 hydroxymethyl furfural in buffer is 100~500mM.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910210440.2A CN109988792B (en) | 2019-03-20 | 2019-03-20 | Method for synthesizing 5-hydroxymethyl furoic acid by using deinococcus radiodurans R1 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910210440.2A CN109988792B (en) | 2019-03-20 | 2019-03-20 | Method for synthesizing 5-hydroxymethyl furoic acid by using deinococcus radiodurans R1 |
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| CN112899392A (en) * | 2021-03-10 | 2021-06-04 | 浙江大学 | Primer group for specific identification molecular marker of transgenic insect-resistant and glyphosate-resistant cotton and application thereof |
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