CN106554978A - A kind of method that enzyme process prepares 2,5- furandicarboxylic acids - Google Patents
A kind of method that enzyme process prepares 2,5- furandicarboxylic acids Download PDFInfo
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- CN106554978A CN106554978A CN201510644672.0A CN201510644672A CN106554978A CN 106554978 A CN106554978 A CN 106554978A CN 201510644672 A CN201510644672 A CN 201510644672A CN 106554978 A CN106554978 A CN 106554978A
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- lipase
- hydroxymethyl furfural
- fdca
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Abstract
The present invention relates to bioanalysis prepares bulk chemical field, the method that 5 hydroxymethyl furfural (HMF) prepares FDCA (FDCA) is aoxidized using biological enzyme specifically.The present invention adopts two kinds of enzymes, including the lipase and 5 hydroxymethyl furfural oxidizing ferment concerted catalysis oxidation HMF in " one kettle way " to prepare FDCA, realizes the green preparation process of FDCA.Technical solutions according to the invention are aoxidized using enzyme law catalysis, are carried out at a lower temperature, and energy consumption is little, efficiency high；Alkali need not be added in conversion process, process is simpler, green；Air or oxygen are adopted for oxidant, it is not necessary to add hydrogen peroxide, economy is more preferable.Patent of the present invention has good industrial prospect.
Description
Technical field
The present invention relates to bioanalysis prepares bulk chemical field, 5- is aoxidized using biological enzyme specifically
The method that hydroxymethylfurfural prepares 2,5- furandicarboxylic acids.
Background technology
FDCA (2,5-Furandicarboxylic acid, FDCA) is that carbohydrate is dehydrated
To 5 hydroxymethyl furfural (5-Hydroxymethylfurfural, HMF), what then oxidation was prepared has
The binary acid of cyclic conjugated structure, its structure are quite similar with terephthalic acid (TPA) (p-Phthalic acid, PTA),
Alternative terephthalic acid (TPA) prepares the materials such as polyester, polyamide, and its market scale is several ten-million-ton scales, and by
Year increases.
In terms of FDCA preparations, main at present to be prepared using heterogeneous catalytic oxidation HMF, catalyst is main
For Au [Catalysis Today, 2011,160 (1):55-60]、Pt[Journal of catalysis,2014,315:
67-74] and its loaded catalyst, reacting under alkaline solution or neutrallty condition, yield can be more than 90%.
Research finds that yield is higher in the basic conditions, its reason be FDCA in alkaline environment into salt, from
Activity without affecting solid catalyst.But, this strategy is but increased while yield is improved
Follow-up separation is difficult, is acidified into again FDCA after needing to separate FDCA salt.
Bioconversion is relative to chemical conversion, gentle with reaction condition, the advantages of selective high.Enzymatic turns
Change HMF preparation FDCA and gradually cause concern.
Koopmand etc. will be expressed in HMF oxidase gene pseudomonas putidas, constructs whole-cell catalyst,
With HMF as raw material, FDCA yields up to 97% [Bioresource Technology, 2010,101:6291-629],
But there are problems that concentration of substrate is relatively low, the reaction time.Dijkman etc. is from Methylovorus sp
The enzyme that can aoxidize HMF is found that, by gene in expression in escherichia coli, it is found that the enzyme can be by HMF Gao Xuan
Selecting property oxidation 5- formoxyl -2- furancarboxylic acids (5-formyl-2-furancarboxylic acid, FFA), yield reaches
95%, and FDCA yields it is relatively low [Applied and Environmental Microbiology, 2014,
80(3):1082-1090].When Dijkman etc. further study show that HMFO catalysis oxidations HMF, mainly
Generate intermediate, 2,5- Diformylfurans (2,5-diformylfuran, DFF) and FFA.DFF is then auxiliary
In the case that enzyme FAD is participated in, it is hydrated into together with glycol, then Jing enzymatic oxidations add FAD into FDCA,
FDCA yields are up to 95%.When FDA is added without, DFF and FFA changes into the efficiency of FDCA very
Low [Angewandte Chemie International Edition, 2014,53:6515-6518].
In order to overcome above-mentioned problem, the present invention that HMF oxidizing ferment is optimized expression first, then
Aoxidized using two kinds of enzymes, including the concerted catalysis in " one kettle way " of lipase and 5 hydroxymethyl furfural oxidizing ferment
HMF prepares FDCA, realizes the green preparation process of FDCA.
The content of the invention
It is an object of the invention to provide a kind of enzyme law catalysis oxidation HMF prepares the green method of FDCA.
In order to realize above-mentioned target, the technical solution used in the present invention is：
A kind of method that enzyme process prepares 2,5 furandicarboxylic acids, 5 hydroxymethyl furfural is dissolved in a solvent, with 5-
Hydroxymethylfurfural oxidizing ferment and lipase are catalyst, under oxygen and/or air prepare FDCA.
Solvent is that water, Organic Alcohol and ester are constituted, and their volume ratio is：10:1-10:1-5；Wherein Organic Alcohol is
Methyl alcohol, ethanol, propyl alcohol, isopropanol, butanol, sec-butyl alcohol, isobutanol, 1,3- propane diols, 1,2- propane diols
In one or two or more kinds；Ester is ethyl acetate, Ethyl formate, methyl acetate, ethyl lactate, lactic acid
One kind in methyl esters, single-ethyl succinate, monomethyl succinate, dimethyl succinate, diethyl succinate
Or more than two kinds.
Concentration of the 5 hydroxymethyl furfural in solvent is 0.001-10mol/L.
Described 5 hydroxymethyl furfural oxidizing ferment is recombinant expression protein, and its gene order is shown in sequence table SEQ ID
NO：Base sequence in 1；Preparation process includes gene connection carrier, conversion Escherichia coli, positive gram of screening
Grand, plasmid amplification and purifying are reclaimed, plasmid electricity transformed yeast, the culture of yeast-positive colony screening and induction,
Collect recombinant expression protein.
In reaction system, the oxidasic addition of 5 hydroxymethyl furfural is：1-100umol/L.
Described lipase be preferably Lipozyme RMIM, Lipozyme TLIM, Novozym 435,
Lipase PS, lipase AS, lipase AK, one or two or more kinds in lipase AYS；
The addition of lipase is：0.1-100mg/mL.
The temperature of reaction is 20 DEG C -40 DEG C.
The reaction need to be carried out under aerobic conditions, i.e., carry out under oxygen and/or air existence condition.
Described 5 hydroxymethyl furfural prepares as follows with preparation process：
By 5 hydroxymethyl furfural oxidase gene optimum synthesis, with restriction enzyme XhoI and XbaI by HMF
Oxidase gene is connected on pICZa-A expression vectors, is converted to Escherichia coli Top10, by bacterium colony PCR,
Digestion Screening and Identification positive colony；Recombinant plasmid is expanded in Escherichia coli, is returned with plasmid purification kit
Receive plasmid；
Yeast electricity step of converting is as follows：(1) electricity conversion cup is taken out from the alcohol of volumetric concentration 75%, super-clean bench
Air-dry, while ultraviolet irradiation 20 minutes, is put into ice chest precooling conversion cup afterwards；(2) 80 μ L are taken above-mentioned
Competence bacteria, is mixed with the linearizing recombinant expression plasmids of 5-10 μ g, moves into 0.2cm electricity conversion cups, ice bath
5 minutes；(3) steam is dried, is put on electroporation, select yeast parameter (shock parameters：1500V, 200V,
25 μ F) electricity conversion；(4) the 1mol/L sorbierites of 1mL ice baths are added after shocking by electricity immediately, 15mL is proceeded to
In centrifuge tube, 30 degree of standing 1h are subsequently adding 2ml YPD culture mediums (w/v, 1% yeast extract, 2% egg
White peptone, 2% glucose) 30 degree shake bacterium 3h；(5) YPD plates (w/v, 1% yeast are applied after low-speed centrifugal
Extract, 2% peptone, 2% glucose, 100 μ g/ml bleomycins)；Select Pichia pastoris positive colony
Transformant, before abduction delivering, culture medium is BMGY (w/v, 1% yeast extract, 2% peptone, 100mM
Kaliumphosphate buffer, pH6.0,1.34% yeast nitrogens (YNB), 4 × 10-5% biotins, 1% glycerine)；
After culture 24 hours, centrifugation removes BMGY culture mediums, and (w/v, 1% yeast are carried to change BMMY culture mediums
Take liquid, 2% peptone, 100mM kaliumphosphate buffers, pH6.0,1.34%YNB, 4 × 10-5% biotins,
0.5-1% methyl alcohol), into the abduction delivering stage, methyl alcohol is added to end every 24h in BMMY culture mediums
Concentration (V/V) carries out abduction delivering, continuous expression 72-144h for 0.5-1%；Zymotic fluid centrifugation is taken simultaneously, is received
Collection supernatant is 5 hydroxymethyl furfural oxidation enzyme liquid.
The invention has the advantages that：
1. technical solutions according to the invention are aoxidized using enzyme law catalysis, are carried out at a lower temperature, and energy consumption is little,
Efficiency high.
2. technical solutions according to the invention need not add alkali, and process is simpler, green.
3. technical solutions according to the invention adopt molecular oxygen, i.e. air or oxygen is oxidant, it is not necessary to plus
Enter hydrogen peroxide, economy is more preferable.
4., compared with the technology that other biological method prepares FDCA, the yield of technology FDCA of the present invention is more
Height, the meaning with industrialization.
Description of the drawings
Fig. 1 .HMF oxidizing ferment SDS-PAGE schemes.
Specific embodiment
With reference to embodiment, the present invention will be further described：
Embodiment 1
1. 5 hydroxymethyl furfural oxidase gene (sequence is shown in HMF oxidase gene sequences) is synthesized, with restricted interior
After enzyme cutting XhoI and XbaI is by HMF oxidase gene digestions, it is connected to same enzyme double digestion
On pICZa-A expression vectors.Jing T4 ligases overnight connect (8 μ l matter of each material composition in reaction system
Grain, 1 μ l reaction bufferings, 1 μ l T4 ligases).Take 5 μ l connection liquids to convert to Escherichia coli Top10,
It is coated in incubated overnight in the resistant panel containing bleomycin；Take 20mL less salts (0.5%NaCl, V/W)
LB cultures are based in 50mL conical flasks, plus 5 μ L100mg/ml bleomycin, access conversion bacterial classification (E.
Coli TOP10, Invitrogen company), 28 DEG C, shake bacterium 16-18h.
2. plasmid extraction：Bacterium solution 5000r obtained in step 1 is centrifuged into 5min.Remove supernatant, precipitation plasmid
Kit (Axygen companies, model 05114kai) extracts plasmid.
3. the conversion of plasmid：By the plasmid extracted in step 2,250 μ L in centrifuge tube are transferred to, add 30 μ
LbufferL, plus 20 μ LsacI enzymes, mechanical oscillation shake up, and 0.5min is centrifuged, and sealed membrane is sealed, 37 DEG C
Incubator places 5-10h.Take out after 5h, 4 DEG C of placement 10min of refrigerator, plus 600 μ L absolute ethyl alcohols, obtain
Linearizing recombinant plasmid.Take 1-5 μ L and be added to 80 μ L Pichia pastoris competent cell X-33 bacterium solutions
(OD600nm=50) in, ice bath 5min, electric shock, add the 1M sorbierites of 4 DEG C of 400 μ L immediately, turn
Move in 1.5mL centrifuge tubes, 30 DEG C of incubation 1-2h.Take 200 μ L coated plates and (add 1M sorbierites and 1ug/mL
The solid YPD culture mediums of bleomycin)
4. treat that the bacterium in step 3 grows up to, respectively 10 single bacterium colonies of picking (big and disperse) in 10 μ L sterilized waters,
Bacterium colony PCR and agarose gel electrophoresis are done, the bacterium colony that can be grown on antibiotic flat board is selected.
5. Induction Transformation：The most bright corresponding bacterium colony of band in selection agarose gel electrophoresis, picking single bacterium colony connect
Plant in 200mLBMGY culture mediums, 28 DEG C of shaking tables shake 20-24h, to OD600=2-6.By the bacterium for growing up to
Liquid 1500rpm is centrifuged 5min, removes supernatant, with the common 200mL of BMMY culture medium re-suspended cells, is divided in
In the conical flask of four 500mL, 26 DEG C of shaking table cultures 4 days.Every 24h plus methyl alcohol (volumetric concentration 50%)
0.5mL, and sample 100 μ L.Stay and be SDS-PAGE
6. concentrate：After cultivating 4 days, bacterium solution 8000rpm centrifugation 20min, is concentrated with 30000 super filter tube 5000rpm
Obtain HMF oxidizing ferment.
Embodiment 2
Take 23.8mg HMF to implement in 30mL 100mM buffer solution of potassium phosphate (pH7.16) plus 200uL
HMFO prepared by example 1, blowing air, 27 DEG C, oil bath, stirring reaction 30h, HPLC detections show HMF
Conversion ratio is 99%.The above-mentioned reactant liquors of 16mL are taken, (40 DEG C) eliminating water is rotated, to yellow oily drop,
Plus the 10mL tert-butyl alcohols, 10mL ethyl acetate is miscible for homogeneous, plus 200mg lipase Novozym 435,
Reaction 0,1, adds 165uL H after 2,3,4,5h2O2(30%), reaction 24h under 40 DEG C of stirrings.Liquid phase
Detection, FDCA yields are 75%.
Embodiment 3
Take 23.1mg HMF and be dissolved in 30mL 100mM buffer solution of potassium phosphate (pH7.16), add embodiment
The 1 HMFO 200uL for preparing, blowing air, 27 DEG C, oil bath, stirring reaction 30h, HPLC detections show
HMF conversion ratios are 99%.The above-mentioned reactant liquors of 15mL, plus the 6mL tert-butyl alcohols are taken, 1.5mL ethyl acetate is mixed
It is molten for homogeneous, then plus 225mg lipase Novozym 435, reaction 0,1, after 3,4,5h plus 185uLH2O2
(30%), reaction 24h under 40 DEG C of stirrings.Liquid phase detection FDCA yields are 77%.
Embodiment 4
Take 11.9mgHMF and be dissolved in 15mL 100mM buffer solution of potassium phosphate (pH7.16), add plus 6mL
The tert-butyl alcohol, 2mL ethyl acetate, the H MFO 200uL for adding embodiment 1 to prepare, plus 200mg lipase
Novozym 435, under air atmosphere.Stirring reaction 12 hours at 30 DEG C, liquid phase detection FDCA yields
For 99%.
Embodiment 5
Take 11.9mgHMF to be dissolved in 15mL water, plus the 6mL tert-butyl alcohols, 2mL ethyl acetate, add real
The H MFO 200uL of the preparation of example 1, plus 225mg lipase Novozym 435 are applied, under oxygen atmosphere.
Stirring reaction 12 hours at 30 DEG C, liquid phase detection FDCA yields are 99.5%.
Embodiment 6
Take 17.9mgHMF to be dissolved in 15mL water, plus 6mL butanol, 1.5mL ethyl lactates, add real
The H MFO 200uL of the preparation of example 1, plus 250mg Lipozyme RMIM are applied, under air atmosphere.
Stirring reaction 18 hours at 30 DEG C, liquid phase detection FDCA yields are 97.5%.
Embodiment 7
Take 11.9mgHMF to be dissolved in 15mL water, plus the 6mL tert-butyl alcohols, 1.5mL ethyl acetate, add
H MFO 200uL prepared by embodiment 1, plus 225mg lipase lipase AS, under air atmosphere.30℃
Lower stirring reaction 12 hours, liquid phase detection FDCA yields are 98%.
Embodiment 8
Take 23.8mgHMF to be dissolved in 15mL water, plus 5mL isopropanols, 1.5mL monomethyl succinates,
The H MFO 200uL for adding embodiment 1 to prepare, plus 250mg lipase lipase AYS, in air atmosphere
Under.Stirring reaction 12 hours at 30 DEG C, liquid phase detection FDCA yields are 98.5%.
Embodiment 9
Take 11.9mgHMF to be dissolved in 15mL water, plus 5mL glycerine, 1.5mL monomethyl succinates, plus
Enter the H MFO 200uL of the preparation of embodiment 1, plus 250mg Lipozyme TLIM, in air atmosphere
Under enclosing.Stirring reaction 12 hours at 30 DEG C, liquid phase detection FDCA yields are 97%.
Embodiment 10
According to the method for embodiment 1, by GenBank No.WP_013440946.1 (i.e. described in technical background
The gene order that Dijkman et al. is delivered) transformed yeast expressed, obtains HMF oxidizing ferment, be designated as HMF
Oxidizing ferment -1.
Take 11.9mgHMF and be dissolved in 15mL 100mM buffer solution of potassium phosphate (pH7.16), plus the tertiary fourths of 6mL
Alcohol, 2mL ethyl acetate add H MF oxidizing ferment -1200uL, plus 200mg lipase Novozym 435,
Under air atmosphere.Stirring reaction 12 hours at 30 DEG C, liquid phase detection FDCA yields are 65%.
Embodiment 11
Take 11.9mgHMF to be dissolved in 15mL water, add plus the 6mL tert-butyl alcohols, 2mL ethyl acetate, add
H MF oxidizing ferment -1200uL, plus 250mg Lipozyme TLIM, under oxygen atmosphere.30℃
Lower stirring reaction 12 hours, liquid phase detection FDCA yields are 62%.
Embodiment 12
Take 11.9mgHMF to be dissolved in 15mL water, add plus the 6mL tert-butyl alcohols, 2mL ethyl acetate, add
H MF oxidizing ferment -1200uL, plus 250mg lipase lipase AYS, under air atmosphere.Stir at 30 DEG C
Reaction 12 hours is mixed, liquid phase detection FDCA yields are 67%.
HMF oxidase gene sequences tables (information of SEQ ID NO.1)：
ATGACCGACACCATCTTCGATTACGTGATCGTCGGCGGAGGCACCGCCGG
CAGCGTGCTGGCTAATAGGCTGAGCGCCCGGCCTGAGAACAGGGTGCTG
CTGATCGAGGCTGGCATCGATACCCCCGAGAACAACATCCCCCCCGAGAT
CCATGACGGCCTCAGGCCCTGGCTCCCTAGGCTGTCCGGAGACAAGTTCT
TCTGGCCCAACCTCACCATTCACAGGGCCGCTGAGCATCCCGGCATCACC
AGGGAGCCTCAGTTCTACGAACAGGGCAGGCTGCTGGGCGGCGGATCCT
CCGTCAACATGGTGGTGTCCAACCGGGGCCTCCCCAGGGACTACGATGAG
TGGCAGGCTCTGGGAGCCGACGGCTGGGATTGGCAGGGAGTGCTGCCCT
ACTTCATCAAGACCGAGAGGGATGCCGACTACGGAGATGATCCCCTGCAT
GGCAACGCCGGCCCTATCCCTATTGGCAGGGTGGACAGCAGGCACTGGTC
CGACTTCACAGTGGCTGCTACCCAAGCTCTGGAGGCCGCTGGCCTGCCCA
ATATCCACGACCAGAACGCCAGGTTTGACGATGGCTATTTCCCCCCCGCT
TTTACCCTGAAGGGCGAGGAGCGGTTTAGCGCCGCTAGGGGCTACCTGGA
TGCCTCCGTGAGGGTGCGGCCTAACCTGAGCCTCTGGACCGAGAGCCGGG
TCCTGAAGCTCCTGACCACCGGAAACGCCATCACCGGCGTGAGCGTCCTG
AGGGGCAGGGAAACCCTGCAGGTCCAGGCCAGGGAGGTGATCCTGACAG
CCGGAGCCCTGCAGAGCCCTGCTATCCTGCTGCGGACCGGCATCGGCCCT
GCTGCCGACCTGCATGCTCTCGGCATTCCTGTGCTCGCTGATAGGCCTGGC
GTGGGACGGAACCTGTGGGAGCACAGCTCCATCGGCGTGGTGGCTCCCCT
GACAGAGCAGGCTAGGGCTGACGCTAGCACCGGAAAGGCCGGAAGCAGG
CACCAGCTCGGAATCCGGGCTTCCTCCGGAGTGGACCCTGCTACCCCCTC
CGATCTGTTCCTGCACATCGGCGCCGATCCTGTGTCCGGCCTCGCTAGCGC
TGTGTTCTGGGTGAACAAGCCTAGCAGCACCGGCTGGCTGAAGCTCAAGG
ACGCTGACCCCTTCAGCTACCCCGATGTGGACTTCAACCTGCTGTCCGATC
CCCGGGATCTGGGAAGGCTGAAGGCTGGCCTGAGGCTGATCACACACTA
CTTCGCCGCCCCTAGCCTGGCTAAGTATGGCCTGGCTCTGGCCCTGAGCA
GGTTTGCTGCTCCTCAGCCTGGCGGCCCCCTCCTGAATGACCTGCTCCAGG
ACGAGGCCGCCTTAGAAAGGTACCTGAGGACCAACGTGGGCGGAGTGTG
GCATGCTTCCGGCACCGCTAGGATCGGCAGGGCCGACGATAGCCAGGCT
GTGGTGGACAAGGCTGGCAGGGTCTACGGCGTGACAGGCCTGAGGGTGG
CCGATGCCTCCATCATGCCTACCGTCCCTACCGCTAACACAAACCTGCCT
ACCCTGATGCTCGCCGAAAAGATCGCTGACGCTATCCTGACCCAGGCTTG
A
The information (referring to sequence table) of SEQ ID NO.1
(a) sequence signature：
* length：1596 base-pairs
* type：Nucleic acid
* chain：Double-strand
* topological structure：Linearly
(b) molecule type：Oligonucleotide chain
C () is assumed：It is no
(d) antisense：It is no
E () is initially originated：It is artificial synthesized.
Claims (8)
1. a kind of method that enzyme process prepares 2,5 furandicarboxylic acids, it is characterised in that：5 hydroxymethyl furfural is dissolved
In a solvent, with 5 hydroxymethyl furfural oxidizing ferment and lipase as catalyst, prepare under oxygen and/or air
2,5- furandicarboxylic acids.
2. method according to claim 1, it is characterised in that：Solvent is water, Organic Alcohol and ester composition,
Their volume ratio is：10:1-10:1-5；Wherein Organic Alcohol be methyl alcohol, ethanol, propyl alcohol, isopropanol, butanol,
One or two or more kinds in sec-butyl alcohol, isobutanol, 1,3- propane diols, 1,2- propane diols；Ester be ethyl acetate,
Ethyl formate, methyl acetate, ethyl lactate, methyl lactate, single-ethyl succinate, monomethyl succinate,
One or two or more kinds in dimethyl succinate, diethyl succinate.
3. method according to claim 1 and 2, it is characterised in that：5 hydroxymethyl furfural is in solvent
In concentration be 0.001-10mol/L.
4. method according to claim 1, it is characterised in that：Described 5 hydroxymethyl furfural oxidizing ferment
For recombinant expression protein, its gene order is shown in sequence table SEQ ID NO：Base sequence in 1；
In reaction system, the oxidasic addition of 5 hydroxymethyl furfural is：1-100umol/L.
5. method according to claim 1, it is characterised in that：Described lipase is for preferably
Lipozyme RMIM, Lipozyme TLIM, Novozym 435, lipase PS, lipase AS, lipase AK,
One or two or more kinds in lipase AYS；
The addition of lipase is：0.1-100mg/mL.
6. method according to claim 1, it is characterised in that：The temperature of reaction is 20 DEG C -40 DEG C.
7. method according to claim 1, it is characterised in that：The reaction need to be carried out under aerobic conditions,
Carry out under oxygen and/or air existence condition.
8. method according to claim 4, it is characterised in that：Prepared by described 5 hydroxymethyl furfural has
Preparation process is as follows：
By 5 hydroxymethyl furfural oxidase gene optimum synthesis, with restriction enzyme XhoI and XbaI by HMF
Oxidase gene is connected on pICZa-A expression vectors, is converted to Escherichia coli Top10, by bacterium colony PCR,
Digestion Screening and Identification positive colony；Recombinant plasmid is expanded in Escherichia coli, is returned with plasmid purification kit
Receive plasmid；
Yeast electricity step of converting is as follows：(1) electricity conversion cup is taken out from the alcohol of volumetric concentration 75%, surpasses
Net typhoon is done, while ultraviolet irradiation 20 minutes, is put into ice chest precooling conversion cup afterwards；(2) 80 μ L are taken
Above-mentioned competence bacteria, is mixed with the linearizing recombinant expression plasmids of 5-10 μ g, moves into 0.2cm electricity conversion cups,
Ice bath 5 minutes；(3) steam is dried, is put on electroporation, select yeast parameter (shock parameters：1500V,
200V, 25 μ F) electricity conversion；(4) the 1mol/L sorbierites of 1mL ice baths are added after shocking by electricity immediately, is proceeded to
In 15mL centrifuge tubes, 30 degree standing 1h, be subsequently adding 2mLYPD culture mediums (w/v, 1% yeast extract,
2% peptone, 2% glucose) 30 degree shake bacterium 3h；(5) after low-speed centrifugal apply YPD plates (w/v, 1%
Yeast extract, 2% peptone, 2% glucose, 100 μ g/mL bleomycins)；Select Pichia pastoris positive
Clonal transformants, before abduction delivering culture medium be BMGY (w/v, 1% yeast extract, 2% peptone, 100
MM kaliumphosphate buffers, pH6.0,1.34% yeast nitrogens (YNB), 4 × 10-5% biotins, 1% is sweet
Oil)；After culture 24 hours, centrifugation removes BMGY culture mediums, changes BMMY culture mediums (w/v, 1% ferment
Female extract, 2% peptone, 100mM kaliumphosphate buffers, pH6.0,1.34%YNB, 4 × 10-5% gives birth to
Thing element, 0.5-1% methyl alcohol), into the abduction delivering stage, first is added every 24h in BMMY culture mediums
Alcohol carries out abduction delivering, continuous expression 72-144h for 0.5-1% to final concentration (V/V)；Simultaneously take zymotic fluid from
The heart, collects supernatant and is 5 hydroxymethyl furfural oxidation enzyme liquid.
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| CN108977472A (en) * | 2017-06-02 | 2018-12-11 | 中国科学院大连化学物理研究所 | A kind of method that tandem enzyme method prepares 2,5- furandicarboxylic acid |
| CN110724654A (en) * | 2019-11-22 | 2020-01-24 | 南京科技职业学院 | Pseudomonas aeruginosa for producing 5-hydroxymethyl-2-furancarboxylic acid and application thereof |
| CN116355974A (en) * | 2023-03-22 | 2023-06-30 | 山东金城医药研究院有限公司 | Production process for synthesizing furan ammonium salt by enzyme-chemical method |
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| CN108977472A (en) * | 2017-06-02 | 2018-12-11 | 中国科学院大连化学物理研究所 | A kind of method that tandem enzyme method prepares 2,5- furandicarboxylic acid |
| CN108977472B (en) * | 2017-06-02 | 2021-06-29 | 中国科学院大连化学物理研究所 | Method for preparing 2,5-furandicarboxylic acid by tandem enzyme method |
| CN107739354A (en) * | 2017-10-09 | 2018-02-27 | 中国科学院过程工程研究所 | The method that one pot of one-step method is prepared 2,5 furandicarboxylic acids by fructose |
| CN107739354B (en) * | 2017-10-09 | 2020-08-07 | 中国科学院过程工程研究所 | Method for preparing 2, 5-furandicarboxylic acid from fructose by one-pot one-step method |
| CN110724654A (en) * | 2019-11-22 | 2020-01-24 | 南京科技职业学院 | Pseudomonas aeruginosa for producing 5-hydroxymethyl-2-furancarboxylic acid and application thereof |
| CN116355974A (en) * | 2023-03-22 | 2023-06-30 | 山东金城医药研究院有限公司 | Production process for synthesizing furan ammonium salt by enzyme-chemical method |
| CN116355974B (en) * | 2023-03-22 | 2023-08-18 | 山东金城医药研究院有限公司 | Production process for synthesizing furan ammonium salt by enzyme-chemical method |
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