CN105177065B - A kind of method of biotransformation method synthesis α-ketoglutaric acid - Google Patents
A kind of method of biotransformation method synthesis α-ketoglutaric acid Download PDFInfo
- Publication number
- CN105177065B CN105177065B CN201510579133.3A CN201510579133A CN105177065B CN 105177065 B CN105177065 B CN 105177065B CN 201510579133 A CN201510579133 A CN 201510579133A CN 105177065 B CN105177065 B CN 105177065B
- Authority
- CN
- China
- Prior art keywords
- culture
- conversion
- ketoglutaric acid
- atcc36534
- kluyveromyces marxianus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 title claims abstract description 157
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 59
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 43
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 37
- 230000036983 biotransformation Effects 0.000 title claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 88
- 239000001963 growth medium Substances 0.000 claims abstract description 45
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 claims abstract description 41
- 244000253911 Saccharomyces fragilis Species 0.000 claims abstract description 41
- 235000018368 Saccharomyces fragilis Nutrition 0.000 claims abstract description 41
- 229940031154 kluyveromyces marxianus Drugs 0.000 claims abstract description 41
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims abstract description 23
- 239000002609 medium Substances 0.000 claims abstract description 15
- 229940124186 Dehydrogenase inhibitor Drugs 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 13
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 57
- 229960000485 methotrexate Drugs 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 31
- 238000011218 seed culture Methods 0.000 claims description 23
- 239000006228 supernatant Substances 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 17
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 8
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 8
- 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 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 229910021529 ammonia Inorganic materials 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000013028 medium composition Substances 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 3
- 230000000996 additive effect Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000006837 decompression Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 239000003054 catalyst Substances 0.000 claims 1
- 239000003610 charcoal Substances 0.000 claims 1
- 238000006356 dehydrogenation reaction Methods 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 229940125532 enzyme inhibitor Drugs 0.000 claims 1
- 239000002532 enzyme inhibitor Substances 0.000 claims 1
- 239000012530 fluid Substances 0.000 abstract description 21
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 210000005253 yeast cell Anatomy 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 description 10
- 230000010355 oscillation Effects 0.000 description 10
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000002253 acid Substances 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 230000002085 persistent effect Effects 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- 230000007704 transition Effects 0.000 description 4
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 3
- 238000011953 bioanalysis Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000222178 Candida tropicalis Species 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000235015 Yarrowia lipolytica Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 150000003628 tricarboxylic acids Chemical class 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- FECNVDHIIJYRIA-UHFFFAOYSA-N 2-oxopentanedioic acid Chemical compound OC(=O)CCC(=O)C(O)=O.OC(=O)CCC(=O)C(O)=O FECNVDHIIJYRIA-UHFFFAOYSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- ZJKLSPIEPZUTNW-UHFFFAOYSA-N C(=O)=C(C(=O)O)CCC(=O)O Chemical compound C(=O)=C(C(=O)O)CCC(=O)O ZJKLSPIEPZUTNW-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 241001363490 Monilia Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000228390 Sporobolomyces johnsonii Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001773 anti-convulsant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- WYACBZDAHNBPPB-UHFFFAOYSA-N diethyl oxalate Chemical compound CCOC(=O)C(=O)OCC WYACBZDAHNBPPB-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- -1 ferment 32h Chemical compound 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 230000007659 motor function Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 239000012744 reinforcing agent Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a kind of method of biotransformation method synthesis α-ketoglutaric acid, the method is using Pidolidone as substrate, in the conversion culture solution that the inverted culture medium culture of kluyveromyces marxianus (Kluyveromyces marxianus) ATCC36534 obtains, ketoglurate dehydrogenase inhibitor is added, synthesis conversion culture is carried out under 25~30 DEG C, 200~250r/min oscillating condition, after synthesis conversion culture, synthesis conversion fluid is through isolating and purifying to obtain the α-ketoglutaric acid；Pidolidone is converted α-ketoglutaric acid by the yeast cells of growth conditions, and ketoglurate dehydrogenase inhibitor is added, has blocked it by further metabolic exhaustion, so that α-ketoglutaric acid largely accumulates in the medium；When substrate Pidolidone feed concentrations are 50g/L, molar yield is up to 83.2%；The present invention the α-ketoglutaric acid of the Pidolidone of low value conversion high value, can heavy industrialization application, production technology has many advantages, such as that short period, high conversion rate and environmental pollution are small.
Description
(1) technical field
The invention belongs to technical field of biochemical industry, specifically, being to synthesize α -one penta 2 about a kind of biotransformation method
The method of acid.
(2) background technique
α-ketoglutaric acid (α-ketoglutaric acid) also known as alpha-carbonyl glutaric acid, a-KG or α-glue ketone
Acid, No. CAS is 328-50-7.α-ketoglutaric acid is one of intermediate product important in tricarboxylic acids (TCA) circulation, in cellular material
It plays an important role in metabolism and energetic supersession.It, as important intermediate, is the weight of a variety of amino acid of synthesis, vitamin
Want precursor.α-ketoglutaric acid has important application prospect in fields such as medicine, organic synthesis, nutrition fortifiers, it is led at present
Field to be applied has: the matched reagent as intermediate, Liver function grade in the ingredient of motor function beverage, organic synthesis
With physique reinforcing agent；Reduce the body loss of postoperative patient and patients on long-term；There are also studies have shown that α-ketoglutaric acid has anti-cyanogen
Effect has anticonvulsant action with sodium nitrite, sodium thiosulfate with the use of anti-cyanogen ability can be improved.
The production technology of α-ketoglutaric acid is mainly chemical synthesis at present.Chemical synthesis is with succinic acid and oxalic acid diethyl
Ester is that raw material is prepared through chemical reaction, since cost is excessively high, seriously polluted and face and be eliminated.The production of α-ketoglutaric acid can also
To use biological fermentation process or biotransformation method, relative to chemical synthesis, bioanalysis mild, processing step with working condition
Simply, the advantages that environmentally friendly.In recent years, the research of bioanalysis synthesis α-ketoglutaric acid has made great progress, ferment
Unit or conversion ratio are improved largely, but production cost is still higher than chemical synthesis, and industrial applications still have certain difficulty.
It is domestic in recent years to there is more research to report about bioanalysis synthesis α-ketoglutaric acid, and applied for some patents.
Chen Jian such as Southern Yangtze University et al. utilizes recombination Yarrowia lipolytica (Yarrowia lipolytica) fermenting and producing α -one penta
Diacid, ferment 144h, and the yield of α-ketoglutaric acid reaches 47.2g/L (Chinese invention patent ZL201210066444.6)；Tianjin
Chen Ning of University of Science and Technology et al. is using to Corynebacterium glutamicum (Corynebacterium glutamicum) fermenting and producing α -one
Glutaric acid, ferment 32h, and α-ketoglutaric acid yield reaches as high as 47.2g/L (Chinese invention patent ZL201110392778.8).But
That fermentation method also has its disadvantage, if the production cycle is long, product is mixed with Multiple components in fermentation liquid, cause extract with
Process for refining is complicated, and totle drilling cost is higher.And biotransformation method perhaps can overcome the defect of fermentation method, can be improved product design,
Simplify extraction process, reduce cost.As Tao Rongsheng et al. carries out biology to Pidolidone or its salt using L-GLOD
Conversion, the concentration for generating α-ketoglutaric acid may be up to 138.5g/L, and molar yield is up to 80% or more (Chinese invention patent application number
201310134674.6).But the invention needs to prepare L-GLOD, and needs to add commodity mistake when being catalyzed reaction
Hydrogen oxide enzyme, thus production in enzyme higher cost.Chen Jian of Southern Yangtze University et al. also reported a kind of using resting cell
L-amino acid deaminase gene is transformed by fallibility PCR or fixed point saturation mutation in the method that Pidolidone produces α-ketoglutaric acid,
Conversion Pidolidone generates α-ketoglutaric acid after bacillus subtilis expression, and molar yield is up to 85% or more, but substrate is dense
Degree is only 15g/L, and yield is to be improved.
(3) summary of the invention
Kluyveromyces marxianus (Kluyveromyces marxianus) is utilized it is an object of the present invention to provide a kind of
The method that ATCC36534 Whole Cell Bioconversion Pidolidone synthesizes α-ketoglutaric acid, using yeast cells as biocatalyst, L-
Glutamic acid is raw material, and full cell method bioconversion synthesizes α-ketoglutaric acid.In the low nitrogen culture medium of high sugar, addition dehydrogenase inhibits
Agent prevents α-ketoglutaric acid from being further metabolized, thus promote the accumulation of α-ketoglutaric acid, it so can be by cheap L- paddy
Propylhomoserin is converted into the higher α-ketoglutaric acid of price, solve current fermentation method, enzyme process and resting cell method production cost compared with
High problem, industrial application value with higher.
The full cell method bioconversion of the present invention synthesizes α-ketoglutaric acid reaction equation:
The technical solution adopted by the present invention is that:
The present invention provides a kind of method of biotransformation method synthesis α-ketoglutaric acid, and the method is using Pidolidone the bottom of as
Object is obtained in the inverted culture medium culture of kluyveromyces marxianus (Kluyveromyces marxianus) ATCC36534
Convert culture solution in, be added ketoglurate dehydrogenase inhibitor, under 25~30 DEG C, 200~250r/min oscillating condition into
Row synthesis culture, after synthesis culture, synthesis culture solution is through isolating and purifying to obtain the α-ketoglutaric acid；The α -one penta
Two dehydrogenase inhibitors are hydrogen peroxide (H2O2) or one or both of methotrexate (MTX) mixing；The conversion culture medium
Group become (reagent is the pure or biological reagent of commercially available analysis): 50~100g/L of sucrose, 10~20g/L of yeast extract powder,
KH2PO43~5g/L, K2HPO45~6.5g/L, MgSO40.5~1.0g/L, solvent are water, and pH value is natural.
Further, additive amount of the substrate in conversion culture solution is 20~50g/L.The substrate Pidolidone
It (CAS:56-86-0) is marketable material, purity >=98%.
Further, the ketoglurate dehydrogenase inhibitor additive amount to convert nutrient solution volume be calculated as 0.0005~
50mmol/L.The ketoglurate dehydrogenase inhibitor is H2O2When, the H2O2With the H of mass concentration 30%2O2It is water-soluble
The form of liquid is added, H2O2H in aqueous solution2O2Additional amount be calculated as 10~50mmol/L to convert nutrient solution volume.The α-
When ketoglutaric dehydrogenase inhibitor is methotrexate (MTX), the methotrexate (MTX) is in the form of the methotrexate (MTX) aqueous solution of 1mmol/L
It is added, methotrexate (MTX) additional amount is calculated as 0.5~1 μm of ol/L to convert nutrient solution volume in the methotrexate (MTX) aqueous solution.It is described
Ketoglurate dehydrogenase inhibitor be H2O2When with the mixing of methotrexate (MTX), the H2O2With the H of mass concentration 30%2O2Water
The form of solution is added, H2O2H in aqueous solution2O2Additional amount be calculated as 40mmol/L, the first ammonia butterfly to convert nutrient solution volume
Purine is added in the form of the methotrexate (MTX) aqueous solution of 1mmol/L, and methotrexate (MTX) additional amount is in the methotrexate (MTX) aqueous solution to turn
Change nutrient solution volume and is calculated as 0.8 μm of ol/L.The effect of the ketoglurate dehydrogenase inhibitor is to inhibit α-ketoglutaric acid
The activity of dehydrogenase blocks α-ketoglutaric acid to be further metabolized, so that α-ketoglutaric acid is accumulated.
Kluyveromyces marxianus ATCC36534 of the present invention in the form of the conversion fluid of 6~10g/L of dry mycelium concentration with
Substrate carries out conversion reaction, and the conversion fluid is that kluyveromyces marxianus ATCC36534 inclined-plane thalline is seeded to conversion training
It supports base or the seed liquor of 5~7g/L of dry mycelium concentration is seeded to conversion culture medium warp with the inoculum concentration of percentage by volume 5-10%
Conversion culture obtains.
Specifically, the step of biotransformation method synthesis α-ketoglutaric acid of the present invention, is as follows:
(1) thallus for the kluyveromyces marxianus ATCC36534 that refrigerator saves is inoculated in fresh slant medium, in
24~36h, the kluyveromyces marxianus ATCC36534 strain after being activated are cultivated in 25~30 DEG C of constant incubators.Institute
The slant medium composition stated are as follows: 10~20g/L of glucose, 5~10g/L of peptone, 3~5g/L of yeast extract powder, agar 15
~20g/L, solvent are water, pH natural (actual measurement 6.3~6.5), 121 DEG C of 15~20min of sterilizing of high steam.
(2) with kluyveromyces marxianus ATCC36534 inclined-plane thalline 2~3 after oese picking step (1) activation culture
Ring is seeded to seed culture medium.Seed culture after inoculation is based on 25~30 DEG C, cultivates under 200~250r/min oscillating condition
20~for 24 hours, obtain the seed liquor that dry mycelium concentration is 5~7g/L.The seed culture medium forms in addition to agar is not added, other at
Slant medium in point same step (1), the loading amount in triangular flask are the 20%~40% of its volume, 8 layers of tying of triangular flask,
121 DEG C of 15~20min of sterilizing of high steam.
(3) with kluyveromyces marxianus ATCC36534 inclined-plane thalline 3~5 after oese picking step (1) activation culture
Ring or step (2) preparation seed liquor, by percentage by volume 5%~10% amount access conversion culture medium, in 25~30 DEG C,
Culture 20 under 200~250r/min oscillating condition~for 24 hours, acquisitions dry mycelium concentration are 6~10g/L, addition is final concentration of 10~
The H of 50mmol/L2O2Or the methotrexate (MTX) of final concentration of 0.5~1.0 μm of ol/L, or both add simultaneously；Add final concentration
For the Pidolidone of 20~50g/L.Triangular flask carry out under the same conditions synthesis culture 20~for 24 hours, obtain α-ketoglutaric acid concentration
Up to the synthesis culture solution of 17.3~42.9g/L.
The composition of the conversion culture medium are as follows: 50~100g/L of sucrose, yeast extract powder 10~20g/L, KH2PO43~
5g/L, K2HPO45~6.5g/L, MgSO40.5~1.0g/L, solvent are tap water, pH natural (actual measurement 6.5), in triangular flask
In loading amount be its volume 20%~40%.8 layers of tying of triangular flask, 121 DEG C of 15~20min of sterilizing of high steam.
(4) synthesis culture solution prepared by step (3) is obtained after 3000~4000g, 5~10min are centrifuged off thallus
Supernatant a is obtained, active carbon is added, filters out active carbon after stirring 30~60min, filtrate decompression is concentrated into the 1/2~1/ of original volume
4, it is centrifuged off sediment again, obtains supernatant b, α-ketoglutaric acid crystal seed is added, be slowly stirred and be cooled to 4 DEG C, stand 8~
12h, collected by suction α-ketoglutaric acid crystal are dried in vacuo in 45 DEG C, obtain lenticular α-ketoglutaric acid；The active carbon is added
Amount is calculated as 5~10g/L with supernatant a volume, and the α-ketoglutaric acid additional amount is calculated as 10~20g/L with supernatant b volume.
Yeast strain used in the present invention is kluyveromyces marxianus ATCC36534, is protected purchased from American Type culture
Hiding center (American Type Culture Collection, ATCC).The bacterial strain colony characteristics: in wort agar plate
On culture medium, bacterium colony is creamy white, is glossy, protuberance, neat in edge, wet, surface is smooth, homogeneous.
The measuring method of dry mycelium concentration of the present invention is: 10mL culture solution taken, abandons supernatant after 4000g is centrifuged,
It is suspended again the thallus of precipitating with 10mL distilled water, centrifugation again discards supernatant liquid, and the thallus of centrifuge tube and precipitating is dried in 105 DEG C
Claim to obtain gross weight (W after dry 12hAlways), gross weight subtracts centrifuge tube bare weight (WIt is empty), cell concentration is calculated as follows:
Dry mycelium concentration (g/L)=(WAlways-WIt is empty)×100。
Supernatant a and supernatant b of the present invention are supernatant, for the ease of distinguishing the supernatant that different step obtains
Different and name, letter itself is without meaning.
Compared with prior art, the beneficial effects are mainly reflected as follows:
The present invention provides a kind of utilization kluyveromyces marxianus (Kluyveromyces marxianus) ATCC36534
The method that Whole Cell Bioconversion Pidolidone synthesizes α-ketoglutaric acid ties up ferment in Marx's Crewe using Pidolidone as substrate
Mother (Kluyveromyces marxianus) ATCC36534 is converted in culture solution, and ketoglurate dehydrogenase inhibitor is added,
Pidolidone is converted α-ketoglutaric acid by the yeast cells of the method for carrying out synthesis culture, growth conditions, and α -one penta 2 is added
Dehydrogenase inhibitor has blocked it by further metabolic exhaustion, so that α-ketoglutaric acid largely accumulates in the medium；The bottom of at
When object feed concentrations are 50g/L, the α-ketoglutaric acid concentration in culture solution is synthesized up to 41.3g/L, molar yield is
83.2%, the α-ketoglutaric acid of crystallized method separation, purity 96.7%, yield 81.4%；Compared to existing fermentation method, enzyme
Or resting cell method synthesizes α-ketoglutaric acid technology, has been had the advantage that using whole yeast cells conversion: yeast cells nutrition
It is required that it is low, incubation time is short, contamination resistance is strong；Growth is fast, yield is high, enzymatic activity is strong；Nuisance is not generated in conversion process
Matter, by-product concentration are low；The present invention, can heavy industrialization the α-ketoglutaric acid of the Pidolidone conversion high value of low value
Have the period short using, production technology, the advantages that high conversion rate, environmental pollution is small.
(4) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1: the screening of transformed yeast bacterial strain
Commercially available 7 yeast strains, including 2 plants of saccharomyces cerevisiae (Saccharomyces cerevisiae), Pasteur finish it is red
1 plant of yeast (Pichia pastoris), 1 plant of monilia guilliermondii (Candida guilliermond), Johnson throw spore ferment
1 plant of female (Sporidiobolus johnsonii), 1 plant of kluyveromyces marxianus (Kluyveromyces marxianus) and
1 plant of candida tropicalis (Candida tropicalis).Wherein kluyveromyces marxianus (Kluyveromyces
Marxianus American Type Culture Collecti, deposit number ATCC36534) are purchased from.
The above-mentioned yeast slant strains picking 1 that refrigerator saves first is expired into ring thallus, is inoculated in fresh slant medium, inclined-plane
36h is cultivated in 30 DEG C of biochemical cultivation cases, obtains the yeast strain inclined-plane of activation.Picking 4 expires ring bacterium from the strain inclined plane of activation
Body is inoculated into the triangular flask of the 250mL equipped with 50mL conversion culture medium, and triangular flask is trained in 30 DEG C, 200r/min transition
After supporting for 24 hours (the dry mycelium concentration of different yeast strain culture solutions differs between 6.03g/L~6.35g/L), every bottle of culture solution
The H of the 30wt% of the middle Pidolidone that 0.80g (final concentration of 20g/L) is added and 31 μ L2O2Aqueous solution (final concentration of 10mmol/
L), triangular flask carries out synthesis culture for 24 hours after persistent oscillation at identical conditions.
After synthesis culture, take the synthesis culture solution of 40mL in 50mL centrifuge tube, 4000g is centrifuged 5min, is poured out
Supernatant, supernatant analyze the α-ketoglutaric acid concentration in filtrate through 0.22 μm of filtering with microporous membrane, HPLC.
The above method is used to compare 7 saccharomycetes conversion Pidolidone for the ability of α-ketoglutaric acid, as a result
Show: the transformation efficiency highest of kluyveromyces marxianus ATCC36534 bacterial strain, the α-ketoglutaric acid concentration in conversion fluid are
8.3g/L, molar yield 41.8%, so selecting the bacterial strain as the microorganism of biotransformation method synthesis α-ketoglutaric acid
Strain.
The slant medium composition are as follows: glucose 10g/L, peptone 5g/L, yeast extract powder 3g/L, agar 20g/
L, solvent are water, pH natural (actual measurement 6.5), 121 DEG C of sterilizing 15min of high steam.
The conversion culture medium composition are as follows: sucrose 50g/L, yeast extract powder 10g/L, KH2PO43g/L, K2HPO4
5g/L, MgSO40.5g/L, solvent are tap water, pH natural (actual measurement 6.5).50mL converts culture medium and is loaded on 250mL triangular flask,
8 layers of gauze tying, 121 DEG C of sterilizing 15min of high steam.
The method of the HPLC method analysis α-ketoglutaric acid concentration are as follows: Shimadzu LC-20 type HPLC instrument, Agilent
Zorbax Extend C18 reverse-phase chromatographic column (5 μm, 250mm × 4.6mm), Detection wavelength are UV 210nm, sampling volume 20
μ L, column temperature are 25 DEG C, and mobile phase is containing 0.1% phosphoric acid ultrapure water, flow velocity 1.0mL/min.
Embodiment 2:H2O2The selection of concentration
It determines and uses kluyveromyces marxianus ATCC36534 as conversion Pidolidone for the strain of α-ketoglutaric acid
Afterwards, H is added in conversion culture medium2O2As ketoglurate dehydrogenase inhibitor, the present embodiment optimum choice H2O2Most
Good addition concentration.
The kluyveromyces marxianus ATCC36534 slant strains picking 1 that refrigerator saves first is expired into ring thallus, is inoculated in new
The kluyveromyces marxianus ATCC36534 for obtaining activation for 24 hours is cultivated on fresh slant medium, inclined-plane in 30 DEG C of biochemical cultivation cases
Bacterial strain inclined-plane.From 2 ring thallus of picking in the strain inclined plane of activation to equipped with (the triangle loaded on 250mL in 50mL seed culture medium
Bottle), seed culture is based in 30 DEG C of constant temperature oscillation shaking tables, and for 24 hours, obtain dry mycelium concentration is 5.35g/L to 200r/min shaken cultivation
Seed liquor.The seed liquor 5mL of ATCC36534 is drawn into the triangular flask equipped with 45mL conversion culture medium with Sterile pipette
(triangular flask loaded on 250mL), 30 DEG C in constant temperature oscillation shaking table, the culture of 200r/min transition for 24 hours, obtain dry mycelium concentration
For the conversion fluid of 7.17g/L.
In above-mentioned ATCC36534 conversion fluid, be added final concentration difference 0mmol/L, 10mmol/L, 20mmol/L,
The H of 30mmol/L, 40mmol/L, 50mmol/L2O2(the i.e. H of mass concentration 30%2O2L/ bottles of 0 μ of aqueous solution, L/ bottles of 31 μ, 62 μ
L/ bottles of L/ bottles, L/ bottles of 93 μ, L/ bottles of 124 μ and 155 μ) and 20g/L (0.8g/ bottles) Pidolidone, each experimental setup 3
It repeats, triangular flask carries out synthesis culture for 24 hours after persistent oscillation at identical conditions.
After synthesis culture, by 1 the method for embodiment, with the α-ketoglutaric acid concentration in HPLC analysis conversion fluid.
The result shows that: after kluyveromyces marxianus ATCC36534 is grown for 24 hours in conversion culture solution, if do not added in transformation system
Add H2O2, the molar yield of α-ketoglutaric acid only has 11.6%, adds H2O2The conversion of α-ketoglutaric acid can be remarkably promoted
Rate.In H2O2When to add concentration be 40mmol/L, the α-ketoglutaric acid concentration highest in conversion fluid reaches 15.6g/L, mole turns
Rate is 78.5%.
The composition and preparation method of the slant medium, seed culture medium and conversion culture medium are same as Example 1.
Embodiment 3: the selection of Concentration of Methotrexate
Determine use kluyveromyces marxianus ATCC36534 as convert glutamic acid for the strain of α-ketoglutaric acid after,
In the medium addition methotrexate (MTX) is as ketoglurate dehydrogenase inhibitor, this implementation of class optimum choice methotrexate (MTX)
Best addition concentration.
The kluyveromyces marxianus ATCC36534 slant strains picking 1 that refrigerator saves first is expired into ring thallus, is inoculated in new
The kluyveromyces marxianus ATCC36534 for obtaining activation for 24 hours is cultivated on fresh slant medium, inclined-plane in 30 DEG C of biochemical cultivation cases
Bacterial strain inclined-plane.It (is loaded on from 2 ring thallus of picking in the strain inclined plane of activation into the triangular flask equipped with 50mL seed culture medium
The triangular flask of 250mL), seed culture is based in 30 DEG C of constant temperature oscillation shaking tables, and 200r/min shaken cultivation for 24 hours, it is dense to obtain dry mycelium
Degree is the seed liquor of 5.43g/L.Culture medium is converted to equipped with 45mL with the seed liquor 5mL that Sterile pipette draws ATCC36534
Triangular flask in (triangular flask loaded on 250mL), triangular flask is 30 DEG C in constant temperature oscillation shaking table, 200r/min transition culture
For 24 hours, the conversion fluid that dry mycelium concentration is 7.24g/L is obtained.
In above-mentioned ATCC36534 conversion fluid, final concentration is added and distinguishes 0 μm of ol/L, 0.5 μm of ol/L, 0.6 μm of ol/L, 0.7 μ
Mol/L, 0.8 μm of ol/L, 0.9 μm of ol/L and 1 μm of ol/L methotrexate (MTX) (i.e. L/ bottles of 0 μ of methotrexate (MTX) aqueous solution of 1mmol/L,
L/ bottles of 20 L/ bottles of μ, L/ bottles of 24 μ, L/ bottles of 28 μ, L/ bottles of 32 μ, L/ bottles of 36 μ and 40 μ) and 20g/L (0.8g/ bottles) L- paddy ammonia
Acid, 3 repetitions of each experimental setup, triangular flask carry out synthesis culture for 24 hours after persistent oscillation at identical conditions.
After synthesis culture, by 1 the method for embodiment, with the α-ketoglutaric acid concentration of HPLC analysis conversion fluid.Knot
Fruit shows: after kluyveromyces marxianus ATCC36534 is grown for 24 hours in conversion culture, addition methotrexate (MTX) can significantly promote
Into the conversion ratio of α-ketoglutaric acid.α-ketoglutaric acid concentration when methotrexate (MTX) addition concentration is 0.8 μm of ol/L, in conversion fluid
Highest reaches 14.8g/L, molar yield 74.5%.
The composition and preparation method of the slant medium, seed culture medium and conversion culture medium are same as Example 1.
Embodiment 5: while adding H2O2And methotrexate (MTX)
The present embodiment adds H in conversion culture medium simultaneously2O2Inhibit with methotrexate (MTX) as ketoglurate dehydrogenase
Agent.
The kluyveromyces marxianus ATCC36534 slant strains picking 1 that refrigerator saves first is expired into ring thallus, is inoculated in new
The kluyveromyces marxianus ATCC36534 for obtaining activation for 24 hours is cultivated on fresh slant medium, inclined-plane in 30 DEG C of biochemical cultivation cases
Bacterial strain inclined-plane.From 2 ring thallus of picking in the strain inclined plane of activation to equipped with (the triangle loaded on 250mL in 50mL seed culture medium
Bottle), seed culture is based in 30 DEG C of constant temperature oscillation shaking tables, and for 24 hours, obtain dry mycelium concentration is 5.38g/L to 200r/min shaken cultivation
Seed liquor.The seed liquor 5mL of ATCC36534 is drawn into the triangular flask equipped with 45mL conversion culture medium with Sterile pipette
(triangular flask of 250mL), conversion culture based on 30 DEG C in constant temperature oscillation shaking table, the culture of 200r/min transition for 24 hours, obtain dry bacterium
Bulk concentration is the conversion fluid of 7.21g/L.
In above-mentioned ATCC36534 conversion fluid, while the H of final concentration of 40mmol/L is added2O2(with mass concentration 30%
H2O2The form of aqueous solution is added) and the methotrexate (MTX) of 0.8 μm of ol/L (added in the form of the methotrexate (MTX) aqueous solution of 1mmol/L
Enter) and 20g/L (0.8g/ bottles) Pidolidone, triangular flask at identical conditions after persistent oscillation carry out synthesis culture for 24 hours.
After synthesis culture, by 1 the method for embodiment, with the α-ketoglutaric acid concentration of HPLC analysis conversion fluid.Knot
Fruit shows: after kluyveromyces marxianus ATCC36534 is grown for 24 hours in conversion culture, while adding the H of 40mmol/L2O2With
The methotrexate (MTX) of 0.8 μm of ol/L, relatively add it is one such compare, the conversion ratio of α-ketoglutaric acid significantly improves, in conversion fluid
α-ketoglutaric acid concentration be 17.3g/L, molar yield reaches 87.1%.
The composition and preparation method of the slant medium, seed culture medium and conversion culture medium are same as Example 1.
Embodiment 6: preferred conversion process
It is conversion strain with kluyveromyces marxianus ATCC36534, on the basis of embodiment 5, optimizes seed training
It supports conditions, the preferred biotransformation methods such as base composition, conversion culture medium composition, concentration of substrate, conversion incubation time and synthesizes α -one
The processing step of glutaric acid is as follows:
(1) thallus for the ATCC36534 that refrigerator saves is inoculated in fresh slant medium, in 30 DEG C of constant incubators
It cultivates for 24 hours, the ATCC36534 strain after being activated；The slant medium composition are as follows: glucose 10g/L, peptone
5g/, yeast extract powder 3g/L, agar 20g/L, solvent are water, pH natural (actual measurement 6.5), 121 DEG C of sterilizing 15min of high steam.
(2) with 2 ring of ATCC36534 inclined-plane thalline after oese picking step (1) activation culture, it is seeded to seed culture
Base.For seed culture after inoculation based on 20h is cultivated under 30 DEG C, 200r/min oscillating condition, obtaining dry mycelium concentration is 7.03g/L's
Seed liquor；The seed culture medium composition are as follows: glucose 20g/L, peptone 10g/, yeast extract powder 5g/L, solvent are water,
PH natural (actual measurement 6.3).The seed culture medium of 50mL is fitted into the triangular flask of 250mL, 8 layers of gauze tying, and 121 DEG C of high steam
Sterilize 15min.
(3) press percentage by volume 5% inoculum concentration, by step (2)) preparation seed liquor be transferred to conversion culture medium, turn
Change culture based on 20h is cultivated under 30 DEG C, 200r/min oscillating condition, obtains the conversion fluid that dry mycelium concentration is 9.87g/L, be added eventually
Concentration is the H of 40mmol/L2O2(with the H of mass concentration 30%2O2The form of aqueous solution is added) and 0.8 μm of ol/L first ammonia butterfly
Purine (is added) in the form of the methotrexate (MTX) aqueous solution of 1mmol/L, adds the L- paddy ammonia of final concentration of 50g/L (2.5g/ bottles)
Acid, triangular flask carry out synthesis culture 20h under the same conditions.
The composition of the conversion culture medium are as follows: sucrose 100g/L, yeast extract powder 20g/L, KH2PO45g/L, K2HPO4
6.5g/L, MgSO41.0g/L, solvent are tap water, pH natural (actual measurement 6.5).The conversion culture medium of 50mL is packed into 250mL's
In triangular flask, 8 layers of gauze tying, 121 DEG C of sterilizing 15min of high steam.
After synthesis culture, by 1 the method for embodiment, with the α-ketoglutaric acid concentration of HPLC analysis conversion fluid.Knot
Fruit shows: α-ketoglutaric acid is synthesized by the above selection process biotransformation method, when substrate feed concentrations are 50g/L, and conversion fluid
In α-ketoglutaric acid concentration up to 42.9g/L, molar yield 86.4%.
Embodiment 7: amplifying and isolates and purifies
It is that conversion strain puts shake flask scale on the basis of embodiment 6 with kluyveromyces marxianus ATCC36534
To 250mL, steps are as follows:
(1) thallus for the ATCC36534 that refrigerator saves is inoculated in fresh slant medium, in 30 DEG C of constant incubators
It cultivates for 24 hours, the ATCC36534 strain after being activated；The slant medium composition are as follows: glucose 10g/L, peptone
5g/, yeast extract powder 3g/L, agar 20g/L, solvent are water, pH natural (actual measurement 6.5), 121 DEG C of sterilizing 15min of high steam.
(2) with 2 ring of ATCC36534 inclined-plane thalline after oese picking step (1) activation culture, it is seeded to seed culture
Base.For seed culture after inoculation based on 20h is cultivated under 30 DEG C, 250r/min oscillating condition, obtaining dry mycelium concentration is 6.96g/L kind
Sub- liquid；The seed culture medium composition are as follows: glucose 20g/L, peptone 10g/, yeast extract powder 5g/L, solvent are water, pH
Natural (actual measurement 6.3).The seed culture medium of 100mL is fitted into the triangular flask of 500mL, 8 layers of gauze tying, and 121 DEG C of high steam
Sterilize 20min.
(3) seed liquor prepared by step (2) is transferred to conversion culture medium, converted by the inoculum concentration for pressing percentage by volume 5%
Culture obtains the conversion fluid that dry mycelium concentration is 10.23g/L, is added eventually based on 20h is cultivated under 30 DEG C, 250r/min oscillating condition
Concentration is the H of 40mmol/L2O2(with the H of mass concentration 30%2O2The form of aqueous solution is added) and 0.8 μm of ol/L first ammonia butterfly
Purine (is added) in the form of the methotrexate (MTX) aqueous solution of 1mmol/L, adds the L- paddy ammonia of final concentration of 50g/L (12.5g/ bottles)
Acid, triangular flask carry out synthesis culture 20h under the same conditions, obtain synthesis culture solution.
The composition of the conversion culture medium are as follows: sucrose 100g/L, yeast extract powder 20g/L, KH2PO45g/L, K2HPO4
6.5g/L, MgSO41.0g/L, solvent are tap water, pH natural (actual measurement 6.5).The conversion culture medium of 250mL is packed into the three of 1L
In the bottle of angle, 8 layers of gauze tying, 121 DEG C of sterilizing 20min of high steam.
(4) it by 5 bottles of the synthesis culture solution merging of step (3) preparation, after 3000g, 10min bactofugation body, is remained
The active carbon of 12g is added in remaining supernatant 1.2L, filters out active carbon after stirring 60min.Filtrate decompression is concentrated into 300mL, again from
The heart removes sediment, and the remaining supernatant (volume is based on 300mL) of acquisition is added lenticular α-ketoglutaric acid 6g, is slowly stirred cold
But to 4 DEG C, after standing overnight, collected by suction α-ketoglutaric acid crystal is dried in vacuo in 45 DEG C, obtains lenticular α -one penta 2
Acid.α-ketoglutaric acid is synthesized by process above biotransformation method, the α -one when substrate feed concentrations are 50g/L, in conversion fluid
Glutaric acid concentration is up to 41.3g/L, molar yield 83.2%, the α-ketoglutaric acid of crystallized method separation, and purity is
96.7%, yield 81.4%.
Claims (4)
1. a kind of method of biotransformation method synthesis α-ketoglutaric acid, it is characterised in that the method is with kluyveromyces marxianus
(Kluyveromyces marxianus) ATCC36534 is catalyst, using Pidolidone as substrate, ties up ferment in Marx's Crewe
In the conversion culture solution that female inverted culture medium culture of ATCC36534 obtains, ketoglurate dehydrogenase inhibitor is added, 25
~30 DEG C, conversion culture is carried out under 200~250r/min oscillating condition, after conversion culture, conversion culture solution is pure through separating
Change and obtains the α-ketoglutaric acid；Additive amount of the substrate Pidolidone in conversion culture solution is 50g/L；The α-
Ketoglutaric dehydrogenase inhibitor is the mixing of one or both of hydrogen peroxide or methotrexate (MTX), and the hydrogen peroxide is with matter
The form for measuring the aqueous hydrogen peroxide solution of concentration 30% is added, and the additional amount of hydrogen peroxide is in aqueous hydrogen peroxide solution to convert training
Nutrient solution volume is calculated as 40mmol/L；The methotrexate (MTX) is added in the form of 1mmol/L methotrexate (MTX) aqueous solution, the first ammonia butterfly
Methotrexate (MTX) additional amount is calculated as 0.8 μm of ol/L to convert nutrient solution volume in purine aqueous solution；The composition of the conversion culture medium are as follows:
Sucrose 100g/L, yeast extract powder 20g/L, KH2PO45g/L, K2HPO46.5g/L, MgSO41.0g/L, solvent are water, and pH is certainly
So.
2. the method for biotransformation method synthesis α-ketoglutaric acid as described in claim 1, it is characterised in that Marx's Crewe
Tie up yeast ATCC36534 in the form of the conversion culture solution of 6~10g/L of dry mycelium concentration with substrate carry out conversion reaction, described turn
Changing culture solution is kluyveromyces marxianus ATCC36534 inclined-plane thalline to be seeded to conversion culture medium or by dry mycelium concentration 5
~7g/L seed liquor is seeded to what the inverted culture of conversion culture medium obtained with the inoculum concentration of percentage by volume 5-10%.
3. the method for biotransformation method synthesis α-ketoglutaric acid as described in claim 1, it is characterised in that the method is by as follows
Step carries out:
(1) thallus of kluyveromyces marxianus ATCC36534 is inoculated in fresh slant medium, is trained in 25~30 DEG C of constant temperature
It supports and cultivates 24~36h, the kluyveromyces marxianus ATCC36534 strain after being activated in case；The slant medium
Composition are as follows: 10~20g/L of glucose, 5~10g/L of peptone, 3~5g/L of yeast extract powder, 15~20g/L of agar, solvent are
Water, pH are natural；
(2) with 2~3 ring of kluyveromyces marxianus ATCC36534 inclined-plane thalline after oese picking step (1) activation culture,
Be seeded to seed culture medium, the seed culture after inoculation be based on 25~30 DEG C, culture 20 under 200~250r/min oscillating condition~
For 24 hours, the seed liquor that dry mycelium concentration is 5~7g/L is obtained；The seed culture medium composition are as follows: 10~20g/L of glucose, egg
White 5~10g/L of peptone, 3~5g/L of yeast extract powder, solvent are water, and pH is natural；
(3) with 3~5 ring of kluyveromyces marxianus ATCC36534 inclined-plane thalline after oese picking step (1) activation culture,
Or step (2) preparation seed liquor by percentage by volume 5%~10% amount access conversion culture medium, in 25~30 DEG C, 200~
It is cultivated under 250r/min oscillating condition, obtains the conversion culture solution of 6~10g/L of dry mycelium concentration, α-ketoglutaric acid dehydrogenation is added
Enzyme inhibitor adds Pidolidone, carried out under 25~30 DEG C, 200~250r/min oscillating condition conversion culture 20~
For 24 hours, after conversion culture, conversion culture solution is through isolating and purifying to obtain the α-ketoglutaric acid.
4. the method for biotransformation method synthesis α-ketoglutaric acid as claimed in claim 1 or 3, it is characterised in that the conversion culture
The method that liquid isolates and purifies are as follows: after conversion, conversion culture solution is centrifuged off thallus through 3000~4000g, 5~10min
Afterwards, supernatant a is obtained, active carbon is added, filters out active carbon after stirring 30~60min, filtrate decompression is concentrated into the 1/2 of original volume
~1/4, it is centrifuged off sediment again, obtains supernatant b, α-ketoglutaric acid crystal seed is added, be slowly stirred and be cooled to 4 DEG C, it is quiet
8~12h is set, collected by suction α-ketoglutaric acid crystal is dried in vacuo in 45 DEG C, obtains lenticular α-ketoglutaric acid；The activity
Charcoal additional amount is calculated as 5~10g/L with supernatant a volume, and the α-ketoglutaric acid Seed charge is calculated as 10 with supernatant b volume
~20g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510579133.3A CN105177065B (en) | 2015-09-11 | 2015-09-11 | A kind of method of biotransformation method synthesis α-ketoglutaric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510579133.3A CN105177065B (en) | 2015-09-11 | 2015-09-11 | A kind of method of biotransformation method synthesis α-ketoglutaric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105177065A CN105177065A (en) | 2015-12-23 |
| CN105177065B true CN105177065B (en) | 2019-07-23 |
Family
ID=54899491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510579133.3A Expired - Fee Related CN105177065B (en) | 2015-09-11 | 2015-09-11 | A kind of method of biotransformation method synthesis α-ketoglutaric acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105177065B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121316A (en) | 2016-03-02 | 2023-05-16 | 巴斯夫欧洲公司 | Process for the manufacture of L-glufosinate |
| CN113106046B (en) * | 2021-03-31 | 2022-11-25 | 大自然生物集团有限公司 | Genetic engineering bacteria for efficiently synthesizing alpha-ketoglutaric acid and application thereof in synthesizing alpha-ketoglutaric acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250563A (en) * | 2008-03-20 | 2008-08-27 | 江南大学 | Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor |
| CN103911400A (en) * | 2014-04-02 | 2014-07-09 | 江南大学 | Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation |
| CN104195184A (en) * | 2014-09-06 | 2014-12-10 | 青岛科技大学 | Process for producing 2-oxoglutaric acid by virtue of fermentation production |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT1183385E (en) * | 1999-05-21 | 2006-11-30 | Cargill Dow Llc | Methods and materials for the synthesis of organic products |
| DE10055512A1 (en) * | 2000-11-09 | 2002-05-23 | Degussa | L-amino acid oxidase from Rhodococcus species |
| CN101717735B (en) * | 2009-12-11 | 2011-09-07 | 广东环西生物科技股份有限公司 | Structure of recombinant bacterium producing alpha-ketoglutaric acid and method for realizing excessive accumulation of alpha-ketoglutaric acid |
| CN102676438B (en) * | 2012-06-08 | 2013-06-12 | 江南大学 | Alpha-ketoglutarate producing strain and method for producing alpha-ketoglutarate by fermentation with same |
| CN104109698A (en) * | 2013-04-17 | 2014-10-22 | 上海工业生物技术研发中心 | Enzymic method for producing [alpha]-ketoglutaric acid |
| CN103352058B (en) * | 2013-07-23 | 2019-04-23 | 江南大学 | A kind of Biocatalysis method from L-sodium preparation α-ketoglutaric acid |
| CN103484391B (en) * | 2013-10-15 | 2015-03-25 | 江南大学 | Yarrowia lipolytica gene engineering bacterium capable of increasing yield of extracellular alpha-ketoglutarate |
| CN104131041A (en) * | 2014-08-01 | 2014-11-05 | 洛阳华荣生物技术有限公司 | Production method for alpha-ketoglutaric acid |
-
2015
- 2015-09-11 CN CN201510579133.3A patent/CN105177065B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250563A (en) * | 2008-03-20 | 2008-08-27 | 江南大学 | Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor |
| CN103911400A (en) * | 2014-04-02 | 2014-07-09 | 江南大学 | Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation |
| CN104195184A (en) * | 2014-09-06 | 2014-12-10 | 青岛科技大学 | Process for producing 2-oxoglutaric acid by virtue of fermentation production |
Non-Patent Citations (2)
| Title |
|---|
| Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source;Gustavo Graciano Fonseca等;《FEMS YEAST RESEARCH》;20070531;第7卷(第3期);第428页右栏第3段，第432页右栏第5段 |
| 调控光滑球拟酵母碳代谢流促进α-酮戊二酸过量积累;张旦旦;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》;20100515(第（2010）5期);第6页第1.3.1节，图1-4 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105177065A (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100485027C (en) | Method for producing D-lactic acid and spore lactobacillus special for the same | |
| CN103865855A (en) | Bacillus subtilis strain and applications thereof | |
| CN108034599B (en) | One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid from brewed spirit system | |
| CN112239728B (en) | Synthetic medium containing reduced glutathione and suitable for cordyceps militaris culture, preparation method and application | |
| CN101555501B (en) | Method for producing Gamma- propalanine by transformation of Lactococcus lactis cells | |
| CN101255454B (en) | Method for biosynthesis of glutathione by using yeast | |
| CN105505801A (en) | Saccharomyces cerevisiae for high yield of glutathione and application of saccharomyces cerevisiae | |
| CN107058139A (en) | The yeast strain of one plant of high-yield glutathione | |
| CN111100800B (en) | Saccharomyces cerevisiae and application thereof | |
| CN105177065B (en) | A kind of method of biotransformation method synthesis α-ketoglutaric acid | |
| CN101538595B (en) | Method for producing gamma-aminobutyric acid by separated fermentation of enterococcus faecium | |
| CN102994430A (en) | Bacterial cellulose production strain and application thereof | |
| CN112063532B (en) | Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol | |
| CN103451124A (en) | Rhodococcus baikonurensis and application thereof in preparing optically pure (R)-6-hydroxy-8-chlorocaprylate and other optically active chiral alcohol | |
| CN101134943B (en) | Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid | |
| CN105567584B (en) | A kind of (+/-) gamma-lactam that can split obtains bacillus and its screening and application of (+) gamma-lactam | |
| CN102115765B (en) | Method for producing heptadecanedioic acid by fermenting and converting n-heptadecane | |
| CN101538594A (en) | Method for producing gamma-aminobutyric acid by enterococcus faecium | |
| CN105175275B (en) | A kind of isolation and purification method of L ornithine | |
| CN101824438B (en) | Method for preparing (S)-3-hydroxy butyric acid ethyl ester through ethyl acetoacetate microbial conversion | |
| CN105586289B (en) | A kind of (+/-) gamma-lactam that can split obtains pseudomonad and its screening and application of (-) gamma-lactam | |
| CN111424005B (en) | Strain for producing tyrosine ammonia lyase and application thereof | |
| CN102071231B (en) | Method for preparing S-(+)-3-hydroxy tetrahydrofuran through microbial conversion | |
| CN103881922A (en) | Method for harvesting microalgae by using microorganism flocculant | |
| CN106399181A (en) | Lactobacillus buchneri 8-2N and application thereof in preparation of phenyllactic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 312028 Jiangxia Road, Yang Xun Qiao Town, Keqiao District, Shaoxing City, Zhejiang Province, No. 2016 Applicant after: Zhejiang Shuren University Address before: No. 8, Gongshu District tree Man Street, Gongshu District, Hangzhou, Zhejiang Applicant before: Zhejiang Shuren University |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190723 |