CN109321494B - Kluyveromyces intermedia ZJB-17004 and application thereof - Google Patents
Kluyveromyces intermedia ZJB-17004 and application thereof Download PDFInfo
- Publication number
- CN109321494B CN109321494B CN201811153827.0A CN201811153827A CN109321494B CN 109321494 B CN109321494 B CN 109321494B CN 201811153827 A CN201811153827 A CN 201811153827A CN 109321494 B CN109321494 B CN 109321494B
- Authority
- CN
- China
- Prior art keywords
- phenylacetyl
- solution
- reaction
- phosphoryl
- methyl
- 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.)
- Active
Links
- 241000235649 Kluyveromyces Species 0.000 title claims description 15
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 52
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 52
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-UHFFFAOYSA-N 0.000 claims abstract description 51
- 241000881812 Kluyvera intermedia Species 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims description 130
- 239000000243 solution Substances 0.000 claims description 105
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 90
- 239000000758 substrate Substances 0.000 claims description 68
- 238000000034 method Methods 0.000 claims description 47
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 41
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 41
- 239000008367 deionised water Substances 0.000 claims description 26
- 229910021641 deionized water Inorganic materials 0.000 claims description 26
- 229940024606 amino acid Drugs 0.000 claims description 24
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 12
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 12
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 12
- 239000008101 lactose Substances 0.000 claims description 12
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 12
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 12
- 239000011686 zinc sulphate Substances 0.000 claims description 12
- 239000003054 catalyst Substances 0.000 claims description 11
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 8
- 241001052560 Thallis Species 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- PTSRBZOZSRJCKX-UHFFFAOYSA-N 2-[(2-phenylacetyl)amino]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)NC(=O)CC1=CC=CC=C1 PTSRBZOZSRJCKX-UHFFFAOYSA-N 0.000 claims description 5
- FDWFFCURSPACFQ-UHFFFAOYSA-N 2-[(2-phenylacetyl)amino]propanoic acid Chemical compound OC(=O)C(C)NC(=O)CC1=CC=CC=C1 FDWFFCURSPACFQ-UHFFFAOYSA-N 0.000 claims description 5
- KJACRUBFUHSJLG-UHFFFAOYSA-N 3-(4-hydroxyphenyl)-2-[(2-phenylacetyl)amino]propanoic acid Chemical compound C=1C=CC=CC=1CC(=O)NC(C(=O)O)CC1=CC=C(O)C=C1 KJACRUBFUHSJLG-UHFFFAOYSA-N 0.000 claims description 5
- ZTAVORUYXADUPD-UHFFFAOYSA-N 3-hydroxy-2-[(2-phenylacetyl)amino]butanoic acid Chemical compound CC(O)C(C(O)=O)NC(=O)CC1=CC=CC=C1 ZTAVORUYXADUPD-UHFFFAOYSA-N 0.000 claims description 5
- FNHVSHKKUKMXJJ-UHFFFAOYSA-N 3-hydroxy-2-[(2-phenylacetyl)amino]propanoic acid Chemical compound OCC(C(O)=O)NC(=O)CC1=CC=CC=C1 FNHVSHKKUKMXJJ-UHFFFAOYSA-N 0.000 claims description 5
- FJGOZOLLBLTSIJ-UHFFFAOYSA-N 3-methyl-2-[(2-phenylacetyl)amino]butanoic acid Chemical compound CC(C)C(C(O)=O)NC(=O)CC1=CC=CC=C1 FJGOZOLLBLTSIJ-UHFFFAOYSA-N 0.000 claims description 5
- ZULOUGGNZOVTOV-UHFFFAOYSA-N 3-methyl-2-[(2-phenylacetyl)amino]pentanoic acid Chemical compound CCC(C)C(C(O)=O)NC(=O)CC1=CC=CC=C1 ZULOUGGNZOVTOV-UHFFFAOYSA-N 0.000 claims description 5
- LIIPHJDKZNTNII-UHFFFAOYSA-N 3-phenyl-2-[(2-phenylacetyl)amino]propanoic acid Chemical compound C=1C=CC=CC=1CC(=O)NC(C(=O)O)CC1=CC=CC=C1 LIIPHJDKZNTNII-UHFFFAOYSA-N 0.000 claims description 5
- IZQISDWUSBXSGT-UHFFFAOYSA-N 4-methyl-2-[(2-phenylacetyl)amino]pentanoic acid Chemical compound CC(C)CC(C(O)=O)NC(=O)CC1=CC=CC=C1 IZQISDWUSBXSGT-UHFFFAOYSA-N 0.000 claims description 5
- OZQCZEAFOVHVSC-UHFFFAOYSA-N 4-methylsulfanyl-2-[(2-phenylacetyl)amino]butanoic acid Chemical compound CSCCC(C(O)=O)NC(=O)CC1=CC=CC=C1 OZQCZEAFOVHVSC-UHFFFAOYSA-N 0.000 claims description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 239000012429 reaction media Substances 0.000 claims description 4
- 239000013078 crystal Substances 0.000 claims description 3
- 238000004255 ion exchange chromatography Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- SVFKZPQPMMZHLZ-UHFFFAOYSA-N N-Phenylacetylaspartic acid Chemical compound OC(=O)CC(C(O)=O)NC(=O)CC1=CC=CC=C1 SVFKZPQPMMZHLZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 150000008575 L-amino acids Chemical class 0.000 abstract description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- 239000000047 product Substances 0.000 description 30
- 239000007787 solid Substances 0.000 description 24
- 238000004128 high performance liquid chromatography Methods 0.000 description 22
- 238000003756 stirring Methods 0.000 description 21
- 239000006228 supernatant Substances 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 19
- 230000003287 optical effect Effects 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 15
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 15
- 230000009466 transformation Effects 0.000 description 15
- 230000036983 biotransformation Effects 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 13
- 238000001514 detection method Methods 0.000 description 13
- VMZCDNSFRSVYKQ-UHFFFAOYSA-N 2-phenylacetyl chloride Chemical compound ClC(=O)CC1=CC=CC=C1 VMZCDNSFRSVYKQ-UHFFFAOYSA-N 0.000 description 12
- -1 N-phenylacetyl amino Chemical group 0.000 description 12
- 239000012153 distilled water Substances 0.000 description 12
- 238000000967 suction filtration Methods 0.000 description 11
- 239000013076 target substance Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 229910021529 ammonia Inorganic materials 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 229910021642 ultra pure water Inorganic materials 0.000 description 8
- 239000012498 ultrapure water Substances 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 102000004092 Amidohydrolases Human genes 0.000 description 5
- 108090000531 Amidohydrolases Proteins 0.000 description 5
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 229960002989 glutamic acid Drugs 0.000 description 4
- 238000013537 high throughput screening Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000011345 viscous material Substances 0.000 description 4
- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- 229960003767 alanine Drugs 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- 229960003136 leucine Drugs 0.000 description 3
- 229960004452 methionine Drugs 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 229940054441 o-phthalaldehyde Drugs 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 229960002898 threonine Drugs 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 229960004295 valine Drugs 0.000 description 3
- 108700023418 Amidases Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000005561 Glufosinate Substances 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 102000005922 amidase Human genes 0.000 description 2
- 150000003862 amino acid derivatives Chemical class 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000011914 asymmetric synthesis Methods 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 102000005396 glutamine synthetase Human genes 0.000 description 2
- 108020002326 glutamine synthetase Proteins 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 235000006109 methionine Nutrition 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 238000011170 pharmaceutical development Methods 0.000 description 2
- 229960005190 phenylalanine Drugs 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 229960004441 tyrosine Drugs 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- TXUICONDJPYNPY-UHFFFAOYSA-N (1,10,13-trimethyl-3-oxo-4,5,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-17-yl) heptanoate Chemical compound C1CC2CC(=O)C=C(C)C2(C)C2C1C1CCC(OC(=O)CCCCCC)C1(C)CC2 TXUICONDJPYNPY-UHFFFAOYSA-N 0.000 description 1
- LHHFKXFFKGRYJJ-UHFFFAOYSA-N (3,4-diamino-4-oxobutyl)-(hydroxymethoxy)phosphinic acid Chemical compound NC(C(=O)N)CCP(=O)(OCO)O LHHFKXFFKGRYJJ-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- WLJVXDMOQOGPHL-PPJXEINESA-N 2-phenylacetic acid Chemical compound O[14C](=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-PPJXEINESA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- FBWXUNCARHZYFO-UHFFFAOYSA-N C(=O)=C(C(=O)O)CCP(=O)(OCO)O Chemical compound C(=O)=C(C(=O)O)CCP(=O)(OCO)O FBWXUNCARHZYFO-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 102000009127 Glutaminase Human genes 0.000 description 1
- 108010073324 Glutaminase Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 150000008539 L-glutamic acids Chemical class 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- VZVQOWUYAAWBCP-UHFFFAOYSA-N N-acetylphosphinothricin Chemical compound CC(=O)NC(C(O)=O)CCP(C)(O)=O VZVQOWUYAAWBCP-UHFFFAOYSA-N 0.000 description 1
- 108010024026 Nitrile hydratase Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 244000253911 Saccharomyces fragilis Species 0.000 description 1
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 1
- 229910021626 Tin(II) chloride Inorganic materials 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940093740 amino acid and derivative Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- GINJFDRNADDBIN-FXQIFTODSA-N bilanafos Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCP(C)(O)=O GINJFDRNADDBIN-FXQIFTODSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000005282 brightening Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000035572 chemosensitivity Effects 0.000 description 1
- 238000012602 chemosensitivity assay Methods 0.000 description 1
- 239000012069 chiral reagent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000005188 flotation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- CEMZBWPSKYISTN-RXMQYKEDSA-N methyl (2r)-2-amino-3-methylbutanoate Chemical compound COC(=O)[C@H](N)C(C)C CEMZBWPSKYISTN-RXMQYKEDSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 125000005499 phosphonyl group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000001119 stannous chloride Substances 0.000 description 1
- 235000011150 stannous chloride Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- LEIMLDGFXIOXMT-UHFFFAOYSA-N trimethylsilyl cyanide Chemical compound C[Si](C)(C)C#N LEIMLDGFXIOXMT-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/20—Aspartic acid; Asparagine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/225—Tyrosine; 3,4-Dihydroxyphenylalanine
-
- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to Kluyvero intermedia (Kluyvera intermedia) ZJB-17004 and application thereof, wherein the selectivity value of L-amino acid enantiomer produced by catalyzing N-phenylacetyl-DL-amino acid reaches 98%, and the selectivity value of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid enantiomer prepared by catalyzing 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid reaches 99%.
Description
(I) technical field
The invention relates to a bacterial strain for producing amidohydrolase, namely Kluyverainermidium ZJB-17004, and application thereof in catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing N-phenylacetyl substitutes of the amino acid derivatives to prepare chiral amino acid derivatives.
(II) background of the invention
The chemical name of the L-glufosinate-ammonium is as follows: 4- [ hydroxyl (methyl) phosphonyl ] -L-homoalanine is a structural analogue of L-glutamic acid, can inhibit the activity of Glutamine Synthetase (GS), enables ammonia accumulation in plants, blocks the light respiration of the plants due to high-concentration ammonia accumulation, destroys chloroplast structures and cysts of matrixes, and simultaneously inhibits amino acid synthesis to damage cell membranes and kill cells, thereby killing weeds. It has broad spectrum and is herbicide tolerant to the second major transgenic crop in the world.
The existing L-glufosinate-ammonium synthesis method comprises a chemical synthesis method and a biological synthesis method. The chemical method mainly constructs the chiral center of L-glufosinate-ammonium by the following 4 ways: 1) constructing a chiral center by chiral induction by using a chiral auxiliary reagent; 2) taking natural amino acid as a chiral source, and converting to obtain L-glufosinate-ammonium; 3) asymmetric synthesis reaction catalyzed by chiral catalyst; 4) chiral resolution of the racemate. However, the chemical method for synthesizing L-glufosinate-ammonium has the disadvantages of multiple working procedures, low yield, high chiral reagent cost, large amount of three wastes and difficult treatment. The biological method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easy separation and purification and the like, so that the technology for producing the L-glufosinate-ammonium by the biological method has very important industrial development value.
Biological production of L-glufosinate-ammonium according to the substrate used, there are protease hydrolysis bialaphos, hydrolysis of L-3-acetamido-4- (hydroxymethylphosphono) butanamide by phosphodiesterase I, amidase I and glutaminase stepwise synergy maintaining optical activity, resolving bialaphos ethyl ester by α -chymotrypsin, deacetylase resolution of N-acetyl-glufosinate, amidase resolution of 2-amino-4- (hydroxymethylphosphono) -butanamide, ester hydrolase hydrolysis of L-glufosinate-N-carboxylic anhydride, nitrile hydratase hydrolysis of glufosinate nitrile-containing substrate, enzyme catalysis of 2-carbonyl-4- (hydroxymethylphosphono) butyric acid and the like.
The existing chemical method for synthesizing the L-glufosinate-ammonium mainly comprises the following steps: 1) the chiral auxiliary inducing method is characterized in that (S) -2-hydroxy-3-pinone is used as the chiral auxiliary for preparation, the yield is 51 percent, and the e.e. value is 79 percent; the D-valine methyl ester is used as a chiral auxiliary agent, the reaction is carried out at a low temperature of-78 ℃, the yield is 51 percent, and the e.e. value is 93.5 percent. 2) The natural amino acid chiral source method takes L glutamic acid as a chiral source, and the e.e. value is 99.4 percent; l-methionine is chiral source, total yield is 42.3%, e.e. value is 93.5%, and virulent iodomethane is needed. 3) Asymmetric synthesis method, asymmetric catalytic hydrogenation-large catalyst dosage and expensive trimethyl silicon cyanide price; asymmetric Strecker reaction-use highly toxic cyanide; asymmetric Michael addition-the catalyst consumption is large, and the product yield and the e.e. value are low. 4) The racemate resolution method has the highest yield of 86 percent and the highest e.e. value of 99 percent, but the D-glufosinate-ammonium is not converted after the resolution, so that the raw materials are wasted.
The important effective component 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid of the L-glufosinate-ammonium is prepared by selectively catalyzing and resolving 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid by using amidohydrolase, and a basis is provided for realizing the synthesis of the L-glufosinate-ammonium in an industrial route.
Chiral amino acids and derivatives thereof have an increasing role in pharmaceutical development, and the current pharmaceutical development has an increasing demand for chiral purity. The chiral amino acid production process includes chemical resolution, enzyme resolution and other processes. The enzymatic resolution has the advantages of mild condition, strong specificity, less pollution and obvious advantages compared with the chemical resolution. The amide hydrolase selectively catalyzes and resolves the N-phenylacetyl amino acid to produce the chiral amino acid, so that the chiral amino acid has wide application prospect.
Disclosure of the invention
The invention aims to provide a bacterial strain capable of producing amidohydrolase, namely Kluyvera intermedia ZJB-17004, and application thereof in catalyzing N-phenylacetyl-DL-amino acid derivatives (2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid) to prepare L-glufosinate-ammonium important effective components, namely L-amino acid derivatives (2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid) or N-phenylacetyl-DL-amino acid to prepare L-amino acid, so that a new enzyme source is provided for producing L-glufosinate-ammonium effective components and L-amino acid by a chemical-enzymatic method, and the development of green chemical catalysis is promoted.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an amidohydrolase-producing strain, namely Kluyverainermidium sp (Kluyverainermedia) ZJB-17004, which is deposited in China center for type culture Collection with the preservation number of CCTCC No: m2018033, date of deposit 2018, 1 month, 15 days, address: wuhan university, 430072. The strain is separated from soil by the institute of bioengineering of Zhejiang industrial university, and has the capability of catalyzing and synthesizing L-glufosinate-ammonium through detection.
In a second aspect, the present invention provides an application of said Kluyveromyces intermedia ZJB-17004 in the preparation of L-amino acid derivatives (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid) which are important effective ingredients of L-glufosinate-ammonium by microbial catalytic resolution of N-phenylacetyl-DL-amino acid derivatives (preferably 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid), wherein said application comprises: taking wet thalli of Kluyveromyces intermedius ZJB-17004 after fermentation culture as a catalyst, taking N-phenylacetyl-DL-amino acid derivatives (preferably 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid) as a substrate, taking a buffer solution (preferably ammonia water) as a reaction medium to form a reaction system with the pH value of 8.5, carrying out conversion reaction at the temperature of 25-55 ℃, the speed of 100 plus of 200rpm (preferably 30-40 ℃, 150rpm), and after the reaction is finished, separating and purifying the reaction solution to obtain the L-amino acid derivatives (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid). In the reaction system, the dosage of the catalyst is 10-200g/L, preferably 50g/L, and the final concentration of the substrate added initially is 10-500mM, preferably 100mM, calculated by the weight of wet bacteria. The reaction system can also be composed of fermentation liquor and a substrate which are obtained by fermenting and culturing the intermediate Kluyveromyces marxianus ZJB-17004, and the pH value is 8.5; wherein the final concentration of wet thallus in the fermentation liquor in the whole reaction system is 10-200g/L, preferably 50g/L, and the final concentration of the substrate in the reaction system is 10-500mM, preferably 100 mM.
The method for separating and purifying the reaction liquid comprises the following steps: after completion of the reaction, the reaction mixture is extracted with methylene chloride, the pH of the aqueous layer is adjusted to 1.0 to 5.0 (preferably 2.5), loading the sample at a rate of 1-6.0Bv/h (preferably 4Bv/h) for ion exchange chromatography, washing with deionized water, eluting with 0.5-3.0Bv/h (preferably 2Bv/h) with 0.2-4.5M (preferably 1M) ammonia water, detecting the eluate with 0.2% ninhydrin solution-impregnated filter paper, collecting eluate containing target components, distilling under reduced pressure to paste, dissolving in methanol for recrystallization, and drying to obtain L-amino acid derivative (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid).
In a third aspect, the invention also provides an application of the Kluyveromyces intermedia ZJB-17004 in catalyzing N-phenylacetyl-DL-amino acid to prepare L-amino acid, wherein wet thalli obtained by fermentation culture of the Kluyveromyces intermedia ZJB-17004 is used as a catalyst, N-phenylacetyl-DL-amino acid is used as a substrate, a buffer solution (preferably ammonia water) is used as a reaction medium to form a conversion system with the pH of 8.5, the conversion reaction is carried out under the conditions of 25-55 ℃, 100-phase and 200rpm (preferably 30-40 ℃ and 150rpm), and after the reaction is finished, the reaction solution is separated and purified to obtain the L-amino acid. In the transformation system, the dosage of the catalyst is 20-300g/L (preferably 20-60g/L) based on the weight of wet bacteria, and the final concentration of the substrate added is 50-500mM (preferably 100 mM). The N-phenylacetyl-DL-amino acid is one of the following: N-phenylacetyl-DL-alanine, N-phenylacetyl-DL-serine, N-phenylacetyl-DL-glutamic acid, N-phenylacetyl-DL-tyrosine, N-phenylacetyl-DL-threonine, N-phenylacetyl-DL-valine, N-phenylacetyl-DL-aspartic acid, N-phenylacetyl-DL-methionine, N-phenylacetyl-DL-leucine, N-phenylacetyl-DL-isoleucine, N-phenylacetyl-DL-phenylalanine. The method for separating and purifying the reaction liquid comprises the following steps: after the reaction is finished, extracting the reaction solution by using dichloromethane, adjusting the pH of an aqueous layer to 1.0-5.0 (preferably 2.5), loading the aqueous layer to perform ion exchange chromatography at the speed of 1-6.0Bv/h (preferably 4Bv/h), firstly washing by using deionized water, then eluting by using 0.2-4.5M (preferably 1M) of ammonia water at the speed of 0.5-3.0Bv/h (preferably 2Bv/h), detecting the eluent by using filter paper impregnated with 0.2% ninhydrin solution, changing the filter paper to be purple black to indicate that the eluent contains L-amino acid, collecting the eluent containing the target component, distilling under reduced pressure to be pasty, dissolving and recrystallizing by using methanol, taking crystals and drying to obtain the L-amino acid.
The N-phenylacetyl-DL-amino acid is prepared by the following method: adding amino acid and NaOH into distilled water, fully stirring under an ice bath condition of 4 ℃ until the solution is colorless and transparent, dropwise adding phenylacetyl chloride, continuing to react for 2 hours under the ice bath condition of 4 ℃ after dropwise adding is finished, stirring at normal temperature (25-30 ℃) for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to 1.5-5.5 (preferably 2-4), separating out a solid, and performing suction filtration and drying to obtain N-phenylacetyl-DL-amino acid; the molar ratio of the amino acid to NaOH is 1:1 to 7 (preferably 1: 1.5-2.5); the molar ratio of the amino acid to the phenylacetyl chloride is 1: 0.1-2 (preferably 1:0.5-1.0), wherein the volume consumption of the distilled water is 3-10ml/g in terms of the mass of the amino acid; the amino acid is one of the following: alanine, serine, glutamic acid, tyrosine, threonine, valine, aspartic acid, methionine, leucine, isoleucine, phenylalanine.
The wet thallus obtained by fermenting the Kluyveromyces intermedius ZJB-17004 is prepared by the following method:
(1) slant culture:
inoculating Kluyvero intermedia (Kluyvera intermedia) ZJB-17004 to a slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the final concentration of the slant culture medium is as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L，K2HPO40.5-6.0g/L，Na2EDTA 0.01-0.3g/L，MnSO4
0.0001-0.001g/L，MgSO40.001-0.002g/L，ZnSO40.001-0.01g/L，FeSO40.001-0.03g/L of agar, 20g/L of agar, deionized water as a solvent, and 6.5-7.5 of pH value; preferably, the final concentration composition of the slant culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, 20g/L agar, deionized water as solvent and pH 6.8.
(2) Seed culture
Selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution; the final concentration of the seed culture medium is as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L，K2HPO40.5-6.0g/L，Na2EDTA 0.01-0.3g/L，MnSO40.0001-0.001g/L，MgSO40.001-0.002g/L，ZnSO40.001-0.01g/L，FeSO40.001-0.03g/L, deionized water as solvent, and 6.5-7.5 of pH value; preferably, the final concentration composition of the seed culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as solvent, and pH 6.8.
(3) Fermentation culture
Inoculating the seed solution into a fermentation culture medium in an inoculation amount of 1-10% (preferably 1%) by volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L，K2HPO40.5-6.0g/L，Na2EDTA 0.01-0.3g/L，MnSO40.0001-0.001g/L，MgSO40.001-0.002g/L，ZnSO40.001-0.01g/L，FeSO40.001-0.03g/L, deionized water as solvent, and 6.5-7.5 of pH value; preferably, the final concentration of the fermentation medium consists of: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as solvent, and pH 6.8.
The buffer solution with the pH value of 8.5 comprises the following components: a citric acid buffer (100mM, pH 4.0-6.0), a phosphoric acid buffer (100mM, pH 6.0-8.0), Tris-HCl (100mM, pH 7.0-9.0), a boric acid buffer (100mM, pH 9.0-10.5), an aqueous ammonia buffer (pH 7.0-10.0 of the substrate solution adjusted with aqueous ammonia), a Roche buffer (100mM, pH 10.5-12.0); preferably the buffer composition is: the pH of the substrate solution was adjusted to 8.5 with ammonia.
The invention has the following beneficial effects: the invention provides a bacterial strain capable of producing amidohydrolase, namely Kluyvera intermedia ZJB-17004, and also provides a method for preparing 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid which is an important effective component of L-glufosinate-ammonium by using wet thalli obtained by fermentation culture of Kluyvera intermedia ZJB-17004 as a biocatalyst to catalyze 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid, wherein the conversion rate of 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] butyric acid reaches 49.5%, and the product L-glufosinate-ammonium reaches e.e% 99.9% The method for preparing the L-amino acid by the acid has the advantages that the conversion rate can reach 49%, the chiral amino acid of the product can reach 99-99.9%, the resolution preparation process is green and efficient, the strain is easy to culture, collect and apply, the catalytic reaction condition is mild, and the method has important application prospects.
(IV) description of the drawings:
FIG. 1 is a standard curve of OPA/NAC-glufosinate concentration under a high throughput screening method.
FIG. 2 is a liquid phase results diagram of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl ] -D-butyric acid under pre-column derivatization reversed phase high performance liquid chromatography.
FIG. 3 shows the electron microscopic observation result of Kluyvera intermedia (Kluyvera intermedia) ZJB-17004 as the strain obtained by screening.
(V) specific embodiment:
the invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: the ultrapure water is UP water, and is water with the resistivity of 18M omega cm (25 ℃). The ammonia water is an aqueous solution containing 25-28% of ammonia. The normal temperature of the invention is 25-30 ℃. The ice bath was 4 ℃ in the examples of the invention. The preparation method of the 2% ninhydrin solution comprises the following steps: dissolving 2g of ninhydrin and 0.08g of stannous chloride in 100mL of ultrapure water, stirring and filtering, and taking the filtrate and storing away from light.
Example 1: screening of Kluyvera intermedia (Kluyvera intermedia) ZJB-17004
1. Preliminary screening
The invention takes soil samples from all parts of the country, and takes 80 parts of soil samples. The screening method comprises the following specific steps: weighing 1g of soil sample, placing the soil sample in 10 mL0.85% of physiological saline, shaking, standing, taking supernatant into an enrichment medium, and carrying out shaking culture at 30 ℃ and 150r/min for 2-3 days. Adding 1mL of enrichment solution into 50mL of fresh enrichment medium, repeating the process for 3 times, and then carrying out separation and purification.
Bromothymol blue filter paper is selected as indicating filter paper to detect colonies capable of degrading substrates. The principle is as follows: the colonies containing the enzyme of interest, after degradation of the substrate, produce acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) which cause a change in the pH locally on the indicator filter. The indicator bromothymol blue has the color change range of 5.8-7.6 (yellow-blue), so that the color of the periphery of the colony utilizing chlorine-containing organic matters is yellow, and the color of the plate around the colony which cannot be utilized is green.
Gradually diluting the enriched solution with sterile 0.85% physiological saline for 10 gradients, and selecting 10-4、10-5、10-6、10-7、10-8、10-9、10-100.1mL of each of the five dilutions was spread on the plate screening medium and incubated at 30 ℃ for 48 h. Spreading sterile filter paper (indicator filter paper) soaked with 10% bromothymol blue on the plate, picking out corresponding colony showing yellow on the indicator filter paper, inoculating into 96-well plate containing growth medium, placing into shaker at 30 deg.C and 180rpm for constant temperature culture, during which OD is detected600And drawing a growth curve, transferring 300 mu l of fresh fermentation medium when the strain grows to the logarithmic phase, inoculating the residual strain liquid after a 96-well plate is inoculated, sealing and storing in a refrigerator at 4 ℃ for later use. And culturing the transferred 96-well plate containing the fermentation medium for 48 hours at 30 ℃ and 180rpm by a shaking table at constant temperature to ensure that the plate fully grows and produces enzyme to obtain bacterial suspension.
2. OPA/NAC high throughput screening
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid in ammonia water, adding 1mL of the bacterial suspension prepared in the step 1, adjusting the pH value of the solution to 8.5 by using the ammonia water to form a 10mL reaction system, enabling the final concentration of the substrate to be 50mM, and carrying out constant temperature reaction on a shaker at 30 ℃ and 180rpm for 24 hours. The obtained conversion solution is correspondingly diluted by 10 times by ultrapure water respectively, is subjected to ice bath at 4 ℃, is detected by an OPA/NAC high-throughput screening method, and strains with high relative fluorescence values are screened out.
OPA/NAC high throughput screening method: absorbing 40 mul of diluted ice-bath reaction liquid to a 96-hole fluorescence detection plate by using a discharge gun, adding the liquid A after ice-bath, oscillating for 30s in an enzyme labeling instrument,then 100. mu.l of ultrapure water was added thereto, and the mixture was shaken again for 30 seconds at lambdaex＝340nm；λem(455 nm) and the fluorescence intensity value obtained was combined with 2-amino-4- [ hydroxy (methyl) phosphoryl group]Comparing the standard curve of the (E) -L-butyric acid to obtain the 2-amino-4- [ hydroxyl (methyl) phosphoryl group contained in the sample]The concentration of L-butyric acid, from which 2-amino-4- [ hydroxy (methyl) phosphoryl can be calculated]The yield of the (E) -L-butyric acid is obtained, and the obtained strain catalyzes and splits the 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl]2-amino-4- [ hydroxy (methyl) phosphoryl group as important effective component for preparing L-glufosinate-ammonium from-DL-butyric acid]-conversion of L-butyric acid.
The solution A comprises: dissolving N-acetyl-L-cysteine (0.448g) and o-phthalaldehyde (0.185g) in 10mL of absolute ethyl alcohol, adding a boric acid buffer solution (140mM, pH 9.5) under an ice bath condition to a constant volume of 50mL, and storing for 3 days under the ice bath condition, wherein the final concentration of the N-acetyl-L-cysteine in the solution A is 0.313mol/L, and the final concentration of the o-phthalaldehyde is 0.138 mol/L.
The 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid standard curve preparation method: 5g/L of 2-amino-4- [ hydroxy (methyl) phosphoryl group was prepared with ultrapure water]-standard solution of L-butyric acid, diluted in 3 concentration gradients: 0.01-0.1 g/L (0.01, 0.02, 0.04, 0.06, 0.08, 0.1g/L), 0.1-1.0 g/L (0.1, 0.2, 0.4, 0.6, 0.8, 1.0g/L), 1.0-5.0 g/L (1.0, 2.0, 3.0, 4.0, 5.0g/L), respectively sucking 40 μ L of standard solution with different concentration gradients to 96-hole microporous fluorescent detection plate with a row gun, respectively adding 40 μ L of prepared A solution stored in ice bath, oscillating in an enzyme labeling instrument for 30s, then respectively adding 100 μ L of ultrapure water, oscillating for 30s, and then oscillating at λex＝340nm；λemThe fluorescence intensity value was measured at 455nm, and the obtained fluorescence intensity value was plotted on the ordinate and the concentration of the standard on the abscissa to prepare a calibration curve. The standard curves of 3 concentration ranges are prepared, and the good linearity (shown in figure 1) can be known when the sample concentration is 0.01-0.1 g/L, R20.9978, showing that the method has high accuracy and applicability.
3. High performance liquid chromatography rescreening
And (3) inoculating the strains screened in the step (2) to a slant culture medium, culturing at 30 ℃ for 48h, and storing in a refrigerator at 4 ℃.
The strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. Inoculating the seed liquid into a fermentation medium at an inoculation amount of 1% by volume concentration, and performing shaking culture at 30 ℃ and 150rpm for 60 h. The cells were centrifuged at 12000g for 5min to collect wet cells. Taking wet thalli, adding a substrate of 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid and ammonia water, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10mL reaction system, wherein the final concentration of the wet thalli is 50g/L, the final concentration of the substrate is 50mM, and placing the reaction system in a water bath shaker at 30 ℃ for conversion for 24 h. Taking 1mL of conversion solution, centrifuging, taking supernatant fluid for HPLC analysis, and finally obtaining a wild strain with high catalytic activity, and marking the strain as a strain ZJB-17004.
The High Performance Liquid Chromatography (HPLC) re-screening method comprises the following steps: and centrifuging the conversion solution, taking supernatant, diluting the supernatant by 10 times with ultrapure water, taking a clean 1.5mL EP tube with 200. mu.l of derivatization reagent, reacting at 30 ℃ for 5min, adding 600. mu.l of ultrapure water, mixing uniformly, filtering with a syringe filter membrane (0.22 μm), and detecting by HPLC (high performance liquid chromatography) to obtain the concentration of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -butyric acid in the reaction solution.
The derivatization reagent is: 0.1g of o-phthalaldehyde and 0.12g of 0.12g N-phenylacetyl-L-cysteine were dissolved in 10mL of absolute ethanol, and the resulting solution was diluted to 50mL with a boric acid buffer (100mM, pH 9.8).
The HPLC conditions are as follows: a U3000 high performance liquid chromatograph of Daian American and equipped with a fluorescence detector; daian C18A silicon hydroxyl packed column (250mM × 4.6.6 mM), a mobile phase of ammonium acetate (50mM pH 4.7) solution containing 10% pure methanol, a column temperature controlled at 35 deg.C, and a fluorescence excitation wavelength lambdaex350nm, emission wavelength λemThe sample size was 10. mu.l at 450 nm. Under these conditions, 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl]The peak time of the-D-butyric acid is 7.800min and 9.290min (as shown in figure 2).
The enrichment medium comprises the following components: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]1g/L of DL-butyric acid, 5g/L of glucose, 1g/L of sodium chloride, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H2O 0.2g/L，The solvent was deionized water and the pH was 7.
Plate screening medium composition: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]1g/L of DL-butyric acid, 5g/L of glucose, 1g/L of sodium chloride, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H20.2g/L of O, 20g/L of agar, deionized water as a solvent and pH 7.
Growth medium composition: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder and deionized water as a solvent. The composition of the slant medium, seed medium and fermentation medium was the same as in example 3. Example 2: identification of Strain ZJB-17004
1. Morphological identification:
the strain ZJB-17004 obtained by screening in the embodiment 1 of the invention forms a round or nearly round, soft texture, smooth and flat surface, regular edge, glossy and opaque milky colony with the diameter of 2-4mm after being cultured for 24 hours at 37 ℃ on a solid culture medium. And (3) gram staining observation: pink short rod shape, no spores. Solid medium composition: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, 20g/L of agar and deionized water as a solvent.
2. Physiological and biochemical identification:
94 phenotypic tests were performed on strain ZJB-17004 using the Biolog (GEN III) automated microorganism identification system, including 71 carbon source utilization assays and 23 chemosensitivity assays: inoculating the strain to BUG plate medium (BIOLOG UNIVERSAL GROWTH AGAR), culturing at 33 deg.C for 2 days, washing off thallus on the plate with sterile cotton swab, mixing with inoculating liquid (IF-A), making into bacterial suspension, and adjusting to 91% T/IF-A with turbidimeter. The bacterial suspensions were added to each well of the BiologGEN iii microwell assay plate using an 8-well electric applicator, 100 μ L per well. The plate was placed in a 33 ℃ incubator and read on a Biolog reader after 12h, 24h, 36h, 48h incubation, respectively.
The strain ZJB-17004 analyzes the metabolic fingerprint through a Biolog reader, the strain ZJB-17004 can strongly utilize 65 carbon sources, and cannot utilize other 2 carbon sources or has weak utilization capacity; the strain ZJB-17004 is sensitive to 23 chemical substances. The 48h identification results given by the Biolog system are shown in tables 1 and 2.
TABLE 1 ability of the Strain ZJB-17004 to utilize 71 carbon sources on the BiologGEN III plate
TABLE 2 chemosensitivity of Strain ZJB-17004 to 23 chemicals on BiologGEN III plates
3. Molecular biological identification:
the 16S rDNA gene of the strain is amplified by using the total DNA of the strain ZJB-17004 as a template and using primers P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3', after the gene product is connected with a T vector, Shanghai workers are entrusted to amplify and sequence the 16S rDNA of the strain to obtain a 16S rDNA sequence (shown in SEQ ID NO. 1) of the strain, the 16S rDNA gene sequences of related strains in GenBank are searched by BLAST on an NCBI website, and homology comparison is carried out. The strain ZJB-17004 has the highest homology (homology, 99%, basedon 16S ribosomal RNA gene) with Kluyvera intermedia strain 256, and the identified strain basically belongs to a control strain based on the 16S rDNA homology higher than 95% according to the principle of microbial genetics identification. Therefore, the strain ZJB-17004 identified by the experiment is Kluyvero intermedia (Kluyvera intermedia), is planned to be named as Kluyvero intermedia (Kluyvera intermedia) ZJB-17004, is deposited in the China center for type culture Collection, and has the preservation number of CCTCC No: m2018033, date of deposit 2018, 1 month, 15 days, address: wuhan university, 430072.
Any nucleotide sequence obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 in the nucleotide sequence table is within the protection scope of the present invention as long as the nucleotide sequence has homology of more than 90%.
Example 3: preparation of Wet cells
(1) Slant culture:
inoculating Kluyveromyces intermedius ZJB-17004 to a slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus;
the final concentration of the slant culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, 20g/L agar, deionized water as solvent, and pH 6.8.
(2) Seed culture
Selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution;
the final concentration of the seed culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as solvent, and pH 6.8.
(3) Fermentation culture
Inoculating the seed solution into a fermentation culture medium in an inoculation amount of 1% of volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet thalli;
the final concentration of the fermentation medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as solvent, and pH 6.8.
Example 4 bioconversion reactions Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
(1) The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.3g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, and the resulting solution was placed in a 30 ℃ water bath shaker at 150rpm for 24 hours to convert the substrate to a final concentration of 50 mM. 1mL of the transformation solution was added to an ep tube and centrifuged before taking the supernatant for HPLC analysis as described in example 1. The results show that ZJB-17004 can convert the substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid into the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the conversion rate is 49.9%, and the optical purity of the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid is 99.9%.
(2) Separating the conversion liquid containing the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid obtained by the enzyme catalytic reaction to extract the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid.
1) And (3) extraction:
the resulting conversion solution was added to a separatory funnel, and simultaneously, dichloromethane of equal volume was added, and after shaking, it was allowed to stand for 3 hours, the organic layer was discharged from the lower end of the separatory funnel, and the aqueous layer was poured out from the upper end. The aqueous layer obtained is an aqueous solution containing the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
2) Product separation using anion exchange resins
① resin pretreatment
Soaking ion exchange resin 201 × 7 in 50 deg.C warm water to fully expand the resin and remove fine particles (inclined or flotation method), soaking in 1.0M NaOH water solution for 3 hr, washing with deionized water to neutral, soaking in 1.0M HCl water solution for 3 hr, washing with deionized water to neutral, soaking in 1.0M NaOH water solution for 3 hr, and converting into OH-And finally washing the mixture to be neutral by deionized water for later use.
② column packing
The method comprises the steps of filling a column (with the inner diameter of 1.5cm and the height of 40cm) by a wet method, firstly adding deionized water with the height of 13cm into an ion exchange column, then filling 30mL of wet resin 201 × 7 into a glass cup, adding 50mL of deionized water, slowly stirring, pouring suspended resin into the ion exchange column, naturally settling, and enabling the resin to be uniformly secreted in the column without obvious boundary lines, bubbles and the like.
③ and eluting
Adjusting the pH of the aqueous solution obtained in the step 1) to 2.5, loading the aqueous solution at the column flow rate of 4.0Bv/h, taking out effluent liquid at intervals for liquid phase detection, when the adsorption reaches the maximum value, washing the effluent liquid with deionized water, then eluting the effluent liquid with 1.0M ammonia water at the elution speed of 2.0Bv/h, detecting the effluent liquid with filter paper impregnated with 0.2% ninhydrin solution, wherein the filter paper changes purple to show that the eluent contains the target substance 2-amino-4- [ hydroxy (methyl) phosphoryl group]L-butyric acid, collecting the eluate of the target fraction, and detecting the 2-amino-4- [ hydroxy (methyl) phosphoryl group contained therein at intervals]-the content of L-butyric acid. Washing the ion exchange column with deionized water after the elution is finished, and converting the resin into OH-For the next separation.
④ purification
Distilling the eluent under reduced pressure to obtain a yellow viscous substance, adding methanol to dissolve the yellow viscous substance, stirring the yellow viscous substance under ice bath to recrystallize the yellow viscous substance to obtain a white solid, and filtering the white solid to obtain the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
After 1L of the conversion solution prepared under the reaction condition of the step (1) is separated and purified by the method, 4.9g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid solid is obtained, the yield of the product is 98.9%, and the optical purity is 99.9%.
EXAMPLE 5 bioconversion reactions with 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.5g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 9.3g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 93.9% and the optical purity was 99.9%.
Example 6: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.6g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 19.2g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 96.4% and the optical purity was 99.9%.
Example 7: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 2g of the wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, the reaction system was placed in a 30 ℃ water bath shaker, and the mixture was transformed at 150rpm for 24 hours, 1mL of the transformed solution was taken out into an EP tube, and after centrifugation, the supernatant was subjected to HPLC analysis as described in example 1 to determine the conversion rate of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 19.4g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 97.4% and the optical purity was 99.9%.
Example 8: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.5g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 50mM, the reaction system was placed in a 40 ℃ water bath shaker, and the resultant was transformed at 150rpm for 24 hours, and 1mL of the transformed solution was taken out into an EP tube, and after centrifugation, the supernatant was subjected to HPLC analysis as described in example 1 to determine the conversion rate to be 49.9%. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4, 4.87g of the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid was obtained, with a yield of 98.4% and an optical purity of 99.9%.
Example 9: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 2g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, and the reaction system was placed in a 50 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 19.4g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 97.4% and the optical purity was 99.9%.
Example 10: biotransformation reaction using N-phenylacetyl DL-alanine as substrate
Adding 6.0g of alanine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after the dropwise adding is completed, the solution is light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain 13g of white solid namely the N-phenylacetyl-DL-alanine.
Substrate N-phenylacetyl-DL-alanine was dissolved in aqueous ammonia, 0.6g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with aqueous ammonia to prepare 10mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain 49.8% conversion. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-alanine was obtained in an amount of 4.25g, with a yield of 95.5% and an optical purity of 99.9%.
Example 11: biotransformation reaction using N-phenylacetyl DL-threonine as substrate
Adding 6.0g of threonine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after dropwise adding is completed, keeping the solution in light yellow, reacting for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain the white solid, namely 10.35g of N-phenylacetyl-DL-threonine.
Substrate N-phenylacetyl-DL-threonine was dissolved in aqueous ammonia, 3g of the wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with aqueous ammonia to prepare 100mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. 1L of the conversion solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out using a filter paper impregnated with a 0.2% ninhydrin solution, and the filter paper became purple-black indicating that the eluate contained the target substance), the product L-threonine was obtained in an amount of 5.54g, in an yield of 93.1% and in an optical purity of 99.9%.
Example 12: biotransformation reaction using N-phenylacetyl DL-tyrosine as substrate
3.619g of DL-tyrosine and 1.8g of NaOH are added into 25ml of distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 2.875g of phenylacetyl chloride is dropwise added, the solution is milky after the dropwise addition, the ice bath condition is continued for reaction for 2h, the mixture is stirred at normal temperature for reaction for 5h, HCl is added for regulating the pH value to about 2.0, a large amount of white solid is separated out, and the white solid is 5.5g of N-phenylacetyl-DL-tyrosine after suction filtration and drying.
Substrate N-phenylacetyl-DL-tyrosine was dissolved in ammonia water, 0.3g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia water to prepare a 10mL reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.0%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was performed with filter paper impregnated with 0.2% ninhydrin solution, and the filter paper turned purple-black indicating that the eluate contained the target substance), the product L-tyrosine 8.72g was obtained, the yield of the product was 96.3%, and the optical purity was 99.9%.
Example 13: biotransformation reaction using N-phenylacetyl DL-phenylalanine as substrate
Adding 3.3g of DL-phenylalanine and 1.8g of NaOH into 25ml of distilled water, fully stirring under an ice bath condition, thoroughly brightening the solution, dropwise adding 2.875g of phenylacetyl chloride, reacting for 2 hours under the ice bath condition after dropwise adding is finished, adding HCl to adjust the pH value to about 4.0 after stirring and reacting for 5 hours at normal temperature, separating out a large amount of white solid, and performing suction filtration and drying to obtain 5.2g of white solid, namely N-phenylacetyl-DL-phenylalanine.
Substrate N-phenylacetyl-DL-phenylalanine was dissolved in ammonia water, 0.4g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia water to prepare 10mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.2%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-phenylalanine 7.82g was obtained, the yield of the product was 94.7%, and the optical purity was 99.9%.
Example 14: biotransformation reaction using N-phenylacetyl DL-leucine as substrate
Adding 3.92g of leucine and 3.2g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is bright, dropwise adding 6.2g of phenylacetyl chloride, reacting for 2 hours under the ice bath condition continuously, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, precipitating a large amount of white solid, and performing suction filtration and drying to obtain 7.43g of white solid, namely N-phenylacetyl-DL-leucine.
Substrate N-phenylacetyl-DL-leucine was dissolved in ammonia water, 0.6g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia water to prepare 10mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.4%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was performed with filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-leucine (5.94 g) was obtained, the yield of the product was 90.6%, and the optical purity was 99.9%.
Example 15: biotransformation reaction using N-phenylacetyl DL-isoleucine as substrate
Adding 7.84g of isoleucine and 6.4g of NaOH into 60ml of distilled water, fully stirring under an ice bath condition, dripping 5.78g of phenylacetyl chloride into the solution to obtain a light yellow solution, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, enabling the solution to become colorless and transparent, separating out a small amount of white solid, and performing suction filtration and drying to obtain 7.28g of white solid, namely N-phenylacetyl-DL-isoleucine.
Substrate N-phenylacetyl-DL-isoleucine was dissolved in ammonia, 0.5g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia to prepare a 10mL reaction system, and the reaction system was stirred in a 30 ℃ water bath with a final concentration of 100mM and then transferred at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-isoleucine was obtained in an amount of 6.12g, and the product yield was 93.4% and the optical purity was 99.9%.
Example 16: biotransformation reaction using N-phenylacetyl DL-serine as substrate
Adding 6.0g of serine and 4.6g of NaOH into 20ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.2g of phenylacetyl chloride, after dropwise adding is completed, keeping the solution in light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain 11g of white solid, namely N-phenylacetyl-DL-serine.
Substrate N-phenylacetyl-DL-serine is dissolved in ammonia water, 0.5g of wet thallus prepared in the method of example 3 is added, the pH of the solution is adjusted to 8.5 by using ammonia water, a 10mL reaction system is formed, wherein the final concentration of the substrate is added to be 100mM, and the reaction system is placed in a water bath shaker at 30 ℃ and is converted for 24 hours at 150 rpm. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.2%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-serine (4.86 g) was obtained, the yield of the product was 92.5%, and the optical purity was 99.9%.
Example 17: biotransformation reaction using N-phenylacetyl DL-methionine as substrate
Adding 8.94g of methionine and 7.2g of NaOH into 120ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dropwise adding 10.3g of phenylacetyl chloride, after dropwise adding is completed, the solution is transparent, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, separating out a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 14.8g of N-phenylacetyl-DL-methionine.
Substrate N-phenylacetyl-DL-methionine was dissolved in ammonia, 0.45g of wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.3%. After 1L of the transformant was prepared under the same reaction conditions and separated and purified by the method described in example 4 (detection was carried out using a filter paper impregnated with a 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-methionine (7.03 g) was obtained, the yield of the product was 94.4%, and the optical purity was 99.9%.
Example 18: biotransformation reaction using N-phenylacetyl DL-valine as substrate
9.36g of valine and 7.2g of NaOH are added into 60ml of distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 14.24g of phenylacetyl chloride is dropwise added, the solution is yellowish and slightly turbid after the dropwise addition, the mixture is continuously reacted for 2 hours under the ice bath condition, the solution is colorless and transparent after being stirred at normal temperature for 5 hours, HCl is added to adjust the pH value to about 2.0, a large amount of white solid is separated out, and the white solid, namely 17.3g of N-phenylacetyl-DL-valine, is obtained after suction filtration and drying.
Substrate N-phenylacetyl-DL-valine was dissolved in aqueous ammonia, 0.2g of the wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to obtain a conversion of 49.2%. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out using a filter paper impregnated with a 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-valine was obtained in an amount of 5.6g, the yield was 95.7%, and the optical purity was 99.9%.
Example 19: biotransformation reaction using N-phenylacetyl DL-glutamic acid as substrate
Adding 5g of glutamic acid and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dripping 5.735g of phenylacetyl chloride, after the dripping is finished, the solution is transparent, continuing to react for 2h under the ice bath condition, stirring at normal temperature for 5h, adding HCl to adjust the pH value to about 2.0, enabling the solution to become turbid, refrigerating overnight at 4 ℃ in a refrigerator, precipitating a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 8.1g of N-phenylacetyl-DL-glutamic acid.
Substrate N-phenylacetyl-DL-glutamic acid was dissolved in ammonia water, 0.4g of wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with ammonia water to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was added to an ep tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.1%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was performed with filter paper impregnated with 0.2% ninhydrin solution, and the filter paper turned purple-black indicating that the eluate contained the target substance), the product L-glutamic acid (7.12 g) was obtained, the yield of the product was 96.8%, and the optical purity was 99.9%.
Sequence listing
<110> Zhejiang industrial university
<120> Kluyveromyces intermedia ZJB-17004 and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1400
<212>DNA
<213> Kluyvera intermedia
<400>1
cgccctcccg aaggttaagc tacctacttc ttttgcaccc actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgtagcattc tgatctacga ttactagcga 120
ttccgacttc atggagtcga gttgcagact ccaatccgga ctacgacata ctttatgagg 180
tccgcttgct ctcgcgaggt cgcttctctt tgtatatgcc attgtagcac gtgtgtagcc 240
ctactcgtaa gggccatgat gacttgacgt catccccacc ttcctccagt ttatcactgg 300
cagtctcctt tgagttcccg gccgaaccgc tggcaacaaa ggataagggt tgcgctcgtt 360
gcgggactta acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctc 420
agagttcccg aaggcaccaa agcatctctg ctaagttctc tggatgtcaa gagtaggtaa 480
ggttcttcgc gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcatttga gttttaacct tgcggccgta ctccccaggc ggtcgactta acgcgttagc 600
tccggaagcc acgcctcaag ggcacaacct ccaagtcgac atcgtttacg gcgtggacta 660
ccagggtatc taatcctgtt tgctccccac gctttcgcac ctgagcgtca gtctttgtcc 720
agggggccgc cttcgccacc ggtattcctc cagatctcta cgcatttcac cgctacacct 780
ggaattctac ccccctctac aagactctag cctgccagtt tcgaatgcag ttcccaggtt 840
gagcccgggg atttcacatc cgacttgaca gaccgcctgc gtgcgcttta cgcccagtaa 900
ttccgattaa cgcttgcacc ctccgtatta ccgcggctgc tggcacggag ttagccggtg 960
cttcttctgc gagtaacgtc aatcgctgca gttattaact acagcgcctt cctcctcgct 1020
gaaagtactt tacaacccga aggccttctt catacacgcg gcatggctgc atcaggcttg 1080
cgcccattgt gcaatattcc ccactgctgc ctcccgtagg agtctggacc gtgtctcagt 1140
tccagtgtgg ctggtcatcc tctcagacca gctagggatc gtcgcctagg tgagccgtta 1200
ccccacctac tagctaatcc catctgggca catccgatgg tgtgaggccc gaaggtcccc 1260
cactttggtc ttgcgacgtt atgcggtatt agctaccgtt tccagtagtt atccccctcc 1320
atcgggcagt ttcccagaca ttactcaccc gtccgccact cgtcacccaa gagcaagctc 1380
tctgtgctac cgttcgactg 1400
Claims (10)
1. Kluyveromyces intermedius (Kluyvera intermedia) ZJB-17004, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2018033, date of deposit 2018, 1 month, 15 days, address: university of Wuhan, China 430072.
2. Use of Kluyveromyces intermedia ZJB-17004 defined in claim 1 in the catalysis of 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid to produce 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
3. The application as claimed in claim 2, wherein the application comprises using wet thallus obtained by fermentation culture of Kluyveromyces intermedia ZJB-17004 as catalyst, using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate, using buffer solution as reaction medium to form a reaction system with pH of 8.5, performing conversion reaction at 25-55 ℃ and 100-200rpm, and separating and purifying the reaction solution after the reaction is finished to obtain 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
4. The use according to claim 3, wherein in the reaction system, the amount of the catalyst is 10 to 200g/L based on the weight of wet cells, and the final concentration of the substrate is 10 to 500mM when initially added.
5. The use of claim 3, wherein the reaction solution is separated and purified by the following steps: after the reaction is finished, extracting the reaction liquid by using dichloromethane, adjusting the pH value of a water layer to 1.0-5.0, loading the water layer to perform ion exchange chromatography at the speed of 1-6.0Bv/h, washing the water layer by using deionized water, eluting the water layer by using 0.2-4.5M ammonia water at the speed of 0.5-3.0Bv/h, collecting eluent containing the target component, distilling the eluent under reduced pressure to paste, dissolving the paste by using methanol for recrystallization, taking crystals and drying the crystals to obtain the 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid.
6. Use of the intermediate Kluyveromyces intermedia ZJB-17004 of claim 1 in the catalysis of N-phenylacetyl-DL-amino acids to produce N-phenylacetyl-L-amino acids.
7. The method as claimed in claim 6, wherein the method comprises using wet cells obtained from fermentation culture of Kluyveromyces intermedia ZJB-17004 as catalyst, using N-phenylacetyl-DL-amino acid as substrate, using buffer solution as reaction medium to form a conversion system with pH8.5, performing conversion reaction at 25-55 deg.C and 200rpm under 100-.
8. The use according to claim 7, wherein in the conversion system, the amount of catalyst is 20-300g/L based on the weight of wet cells, and the final concentration of the substrate is 50-500mM when initially added.
9. Use according to claim 7, characterized in that the N-phenylacetyl-DL-amino acid is one of the following: N-phenylacetyl-DL-alanine, N-phenylacetyl-DL-serine, N-phenylacetyl-DL-glutamic acid, N-phenylacetyl-DL-tyrosine, N-phenylacetyl-DL-threonine, N-phenylacetyl-DL-valine, N-phenylacetyl-DL-aspartic acid, N-phenylacetyl-DL-methionine, N-phenylacetyl-DL-leucine, N-phenylacetyl-DL-isoleucine, N-phenylacetyl-DL-phenylalanine.
10. The use according to claim 7, wherein the wet cell mass is prepared by the following method: (1) slant culture: inoculating Kluyveromyces intermedius ZJB-17004 to a slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the final concentration of the slant culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002 g/L，FeSO40.01g/L, 20g/L of agar, deionized water as a solvent and 6.8 of pH value;
(2) seed culture: selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution; the final concentration of the seed culture medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as a solvent, and 6.8 of pH value;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 1-10%, carrying out shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration composition of the fermentation medium is as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L，K2HPO41.5g/L，Na2EDTA 0.06g/L，MnSO40.002g/L，MgSO40.001g/L，ZnSO40.002g/L，FeSO40.01g/L, deionized water as solvent, and pH 6.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811153827.0A CN109321494B (en) | 2018-09-30 | 2018-09-30 | Kluyveromyces intermedia ZJB-17004 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811153827.0A CN109321494B (en) | 2018-09-30 | 2018-09-30 | Kluyveromyces intermedia ZJB-17004 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109321494A CN109321494A (en) | 2019-02-12 |
| CN109321494B true CN109321494B (en) | 2020-06-23 |
Family
ID=65266562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811153827.0A Active CN109321494B (en) | 2018-09-30 | 2018-09-30 | Kluyveromyces intermedia ZJB-17004 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109321494B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331153B (en) * | 2019-06-24 | 2021-04-30 | 浙江工业大学 | Kluyveromyces tyrosol lyase mutant and application thereof |
| CN113215048B (en) * | 2021-05-21 | 2022-03-18 | 中国农业科学院农业资源与农业区划研究所 | Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100842450B1 (en) * | 2007-02-16 | 2008-07-01 | 대한민국 | -2 Kluyvera sp. CL-2 and Method of solubilizing insoluble-phosphate immobilized in soils by using the same |
| KR101136480B1 (en) * | 2010-10-28 | 2012-04-19 | 주식회사 두산에코비즈넷 | Novel kluyvera intermedia ds-ebn-gbi and microbial agent comprising the same |
| WO2018140778A1 (en) * | 2017-01-26 | 2018-08-02 | Manus Bio, Inc. | Metabolic engineering for microbial production of terpenoid products |
-
2018
- 2018-09-30 CN CN201811153827.0A patent/CN109321494B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100842450B1 (en) * | 2007-02-16 | 2008-07-01 | 대한민국 | -2 Kluyvera sp. CL-2 and Method of solubilizing insoluble-phosphate immobilized in soils by using the same |
| KR101136480B1 (en) * | 2010-10-28 | 2012-04-19 | 주식회사 두산에코비즈넷 | Novel kluyvera intermedia ds-ebn-gbi and microbial agent comprising the same |
| WO2018140778A1 (en) * | 2017-01-26 | 2018-08-02 | Manus Bio, Inc. | Metabolic engineering for microbial production of terpenoid products |
Non-Patent Citations (1)
| Title |
|---|
| 眼镜蛇中间克吕沃菌的分离鉴定及药敏试验;杜乐妍等;《动物医学进展》;20161220(第12期);73-77 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109321494A (en) | 2019-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109321493B (en) | B-lysine-resistant bacillus ZJB-17007 and application thereof | |
| CN109609475A (en) | Glufosinate-ammonium dehydrogenase mutant and its application for synthesizing L-glufosinate-ammonium | |
| CN109762768B (en) | Bacillus B8W22 and application thereof | |
| CN109321494B (en) | Kluyveromyces intermedia ZJB-17004 and application thereof | |
| US20190360011A1 (en) | Methylopila sp. and use thereof in selective resolution preparation of (s)-a-ethyl-2-oxo-1-pyrrolidineacetate | |
| CN101033454A (en) | Erythro micrococcus ZJB-064 and application thereof | |
| CN105112385A (en) | Recombinant esterase, coding gene, carrier, engineering bacterium and application | |
| CN109321490B (en) | Enterobacter aerogenes ZJB-17003 and application thereof | |
| CN109182201B (en) | Enterobacter hesheng biological I type ZJB-17002 and application thereof | |
| CN109321492B (en) | Non-decarboxylation lechler bacterium ZJB-17008 and application thereof | |
| CN109182200B (en) | Lysinibacillus fusiformis ZJB-17006 and application thereof | |
| CN109321491B (en) | Xiangfang enterobacter ZJB-17001 and application thereof | |
| CN109321497B (en) | Lysine-elongated bacillus ZJB-17009 and application thereof | |
| CN101952418A (en) | Produce (2S, 3R, 4S)-method of 4-hydroxy-L-isoleucine | |
| CN109321495B (en) | Clinkerwell ZJB-17005 and application thereof | |
| CN109321496B (en) | Citrobacter freundii ZJB-17010 and application thereof | |
| CN115786315A (en) | Acylase, coding gene, engineering bacterium and application in hydrolyzing and synthesizing N-fatty acyl-glutamic acid type surfactant | |
| CN107142227B (en) | Acid-producing klebsiella and application thereof | |
| CN113621537A (en) | Novel strain BD-1 for degrading phenylacetic acid and application thereof | |
| KR100352192B1 (en) | A New thermophilic bacterium Brevibacillus borstelensis BCS-1 and A thermostable D-stereospecific amino acid amidase produced therefrom | |
| CN111286470A (en) | Bacillus subtilis capable of degrading erythromycin and application thereof | |
| CN116004470B (en) | Helicobacter sulfide SG202 and application thereof | |
| CN108517306A (en) | A kind of method that bioanalysis prepares L-cysteine | |
| CN116064302B (en) | Vibrio bottom desulfurization SG127 and application thereof | |
| CN101063090A (en) | Human pallid bacillus ZJB-061 and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |