CN106906200B - A kind of PA ase for being used to synthesize Amoxicillin isolates and purifies and immobilization coupling process - Google Patents
A kind of PA ase for being used to synthesize Amoxicillin isolates and purifies and immobilization coupling process Download PDFInfo
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- CN106906200B CN106906200B CN201710236749.XA CN201710236749A CN106906200B CN 106906200 B CN106906200 B CN 106906200B CN 201710236749 A CN201710236749 A CN 201710236749A CN 106906200 B CN106906200 B CN 106906200B
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- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 title claims abstract description 29
- 229960003022 amoxicillin Drugs 0.000 title claims abstract description 29
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 238000010168 coupling process Methods 0.000 title claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 189
- 102000004190 Enzymes Human genes 0.000 claims abstract description 189
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims abstract description 123
- 239000000243 solution Substances 0.000 claims abstract description 82
- 238000000034 method Methods 0.000 claims abstract description 68
- 239000007788 liquid Substances 0.000 claims abstract description 62
- 230000000694 effects Effects 0.000 claims abstract description 49
- 150000003839 salts Chemical class 0.000 claims abstract description 47
- 239000012266 salt solution Substances 0.000 claims abstract description 34
- 239000007787 solid Substances 0.000 claims abstract description 23
- 238000009826 distribution Methods 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 44
- 229910019142 PO4 Inorganic materials 0.000 claims description 14
- 239000010452 phosphate Substances 0.000 claims description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 13
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 230000004913 activation Effects 0.000 claims description 9
- 239000012535 impurity Substances 0.000 claims description 9
- 238000004064 recycling Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- 239000004593 Epoxy Substances 0.000 claims description 4
- 239000012071 phase Substances 0.000 description 77
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 48
- 230000000052 comparative effect Effects 0.000 description 19
- 238000000605 extraction Methods 0.000 description 19
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 19
- 238000000855 fermentation Methods 0.000 description 16
- 230000004151 fermentation Effects 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 239000008213 purified water Substances 0.000 description 14
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- 235000021317 phosphate Nutrition 0.000 description 12
- 238000011084 recovery Methods 0.000 description 12
- 238000000926 separation method Methods 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 10
- 229930182555 Penicillin Natural products 0.000 description 9
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 9
- 229940049954 penicillin Drugs 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000007836 KH2PO4 Substances 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000005070 sampling Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
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- 230000014509 gene expression Effects 0.000 description 7
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- 230000008859 change Effects 0.000 description 6
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- 239000012530 fluid Substances 0.000 description 6
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- 238000012805 post-processing Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000004821 distillation Methods 0.000 description 4
- 229910017053 inorganic salt Inorganic materials 0.000 description 4
- 238000011017 operating method Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-aminopenicillanic acid Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 description 3
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- WZJVQUUBEVDURL-UHFFFAOYSA-N pentanedial;phosphoric acid Chemical compound OP(O)(O)=O.O=CCCCC=O WZJVQUUBEVDURL-UHFFFAOYSA-N 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- WLJVXDMOQOGPHL-PPJXEINESA-N 2-phenylacetic acid Chemical compound O[14C](=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-PPJXEINESA-N 0.000 description 2
- 241000194107 Bacillus megaterium Species 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical group CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 108010073038 Penicillin Amidase Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- IYNDLOXRXUOGIU-LQDWTQKMSA-M benzylpenicillin potassium Chemical compound [K+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 IYNDLOXRXUOGIU-LQDWTQKMSA-M 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- DKLZRLRMFNWRFB-UHFFFAOYSA-N methyl 2-(4-hydroxyanilino)acetate Chemical compound COC(=O)CNC1=CC=C(O)C=C1 DKLZRLRMFNWRFB-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 description 2
- 235000011151 potassium sulphates Nutrition 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- VWWOGKHWOXFHAE-RGMNGODLSA-N C(CC)N[C@@H](CCO)C(=O)O.C1(=CC=CC=C1)O Chemical compound C(CC)N[C@@H](CCO)C(=O)O.C1(=CC=CC=C1)O VWWOGKHWOXFHAE-RGMNGODLSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000165940 Houjia Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000001838 alkalimetric titration Methods 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010959 commercial synthesis reaction Methods 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 230000035614 depigmentation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 150000002366 halogen compounds Chemical class 0.000 description 1
- 230000026030 halogenation Effects 0.000 description 1
- 238000005658 halogenation reaction Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- SZJUWKPNWWCOPG-UHFFFAOYSA-N methyl 2-anilinoacetate Chemical compound COC(=O)CNC1=CC=CC=C1 SZJUWKPNWWCOPG-UHFFFAOYSA-N 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 235000014786 phosphorus Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229960003975 potassium Drugs 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000011833 salt mixture Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- -1 tert-butyl alcohols Chemical class 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000010977 unit operation Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
- C12N9/84—Penicillin amidase (3.5.1.11)
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
-
- 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
- C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
- C12P37/02—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin in presence of phenylacetic acid or phenylacetamide or their derivatives not to be used
-
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Abstract
The present invention relates to bioengineering fields, and in particular to a kind of PA ase for being used to synthesize Amoxicillin isolates and purifies and immobilization coupling process.This method includes the following steps：(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, a concentration of 0.05 0.3g/mL of inorganic salts in the inorganic mixed salt solution；(2) mixed salt solution inorganic obtained by step (1) with the tert-butyl alcohol is mixed, then carries out split-phase, obtain upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and PA ase rich in the tert-butyl alcohol；(3) immobilization that carrier carries out PA ase is added in into phase solution lower obtained by step (2).The method of the present invention is easy to operate, activity of gained PA ase and yield are higher.
Description
Technical field
The present invention relates to bioengineering fields, and in particular to a kind of point for the PA ase for being used to synthesize Amoxicillin
From purifying and immobilization coupling process.
Background technology
PA ase is the key enzyme for preparing beta-lactam antibiotic, successfully by the enzyme application in industrial production
In synthesis Amoxicillin, mostly using immobilised enzymes form.Meet the immobilised enzymes of production requirement to prepare, first by penicillin
Acylase isolates and purifies out from zymotic fluid, then carries out enzyme immobilizatio.The expression vector of PA ase mainly wraps
Include Escherichia coli, bacillus subtilis, bacillus megaterium and Pichia pastoris etc..
Although expression vector is different, PA ase is isolated and purified from zymotic fluid all suffers from some general character and ask
Topic：(1) unconsumed medium component in zymotic fluid is removed；(2) the endogenous expression of PA ase needs to carry out thalline thin
Born of the same parents, which crush, comes out enzyme r e lease；(3) grease, polysaccharide, pigment, amino acid and cell fragment after removal clasmatosis in feed liquid etc.
Impurity；(4) foreign protein after removal clasmatosis in feed liquid improves the purity of PA ase.
PA ase for high-purity is prepared is needed to carry out separation of solid and liquid, concentration and removal and be isolated and purified
Many more manipulations.As industrial enzyme, the fine isolation technics such as chromatographic technique, generally use sheet frame mistake can not be used by being limited by cost
The separation means such as filter, centrifugation, ammonium sulfate precipitation, isoelectric precipitation and ultrafiltration, and be combined to be formed accordingly on this basis
Technique.PA ase known in the art is isolated and purified and is summarized as follows with immobilization technology：(1) fermentation liquor pretreatment：
First with bioanalysis such as chemical methods, the lysozymes such as Mechanical Methods, organic solvent or the surfactants such as high-pressure homogenization or bead mill to bacterium
Body carries out clasmatosis, then adds in flocculant, filter aid etc., and carrying out separation of solid and liquid with centrifugation or plate-frame filtering obtains without bacterium
The crude enzyme liquid of body；(2) the first separation of PA ase：The method of isoelectric precipitation is adjusted to by the miscellaneous egg in part with acid or alkali first
White removal, then with centrifuging or plate-frame filtering obtains PA ase crude enzyme liquid, finally with membrane filtration by PA ase
Crude enzyme liquid is concentrated to suitable concentration；(3) the essence separation of PA ase：Sulfuric acid is carried out to PA ase crude enzyme liquid first
Ammonium salt is analysed, and centrifugation or plate-frame filtering are precipitated；Then it is dissolved and precipitated with buffer salt solution, centrifugation or plate-frame filtering removal are insoluble
Property solid；Finally with impurity such as membrane filtration removal small molecule foreign protein, amino acid, inorganic salts；(4) fixation of PA ase
Change：Buffer salt is added into the isolated PA ase liquid of essence first to suitable concentration, centrifugation or plate-frame filtering removal
Solid impurity, then adds in epoxy base carrier or the amino carrier of activation immobilizes.
Main problem existing for above-mentioned technique is as follows：(1) processing step is excessively cumbersome, and the operating time is long, and production is caused to be imitated
Rate is low；(2) step, which adds upper part enzyme, to be lost due to operating time length, and the total recovery for leading to enzyme is low；(3) main impurity-removing method
For flocculation sediment, ammonium sulfate precipitation, ultrafiltration and plate-frame filtering (or centrifugation), separation means are more traditional, and impurity-eliminating effect is not notable；
(4) not from process integration angle consider separation of solid and liquid, concentration and removal etc. isolate and purify and immobilization operation between mutually interconnection
System, causes step excessive.
Therefore it provides one kind can effectively simplify operating procedure, shorten the operating time, and it is pure to effectively improve enzyme separation
The isolation and purification method of the new PA ase of the total recovery of change is very necessary.
Invention content
The purpose of the invention is to overcome the above problem of the existing technology, one kind is provided for synthesizing Amoxicillin
PA ase isolate and purify and immobilization coupling process.Method of the invention is easy to operate, gained penicillin is acylated
The activity and yield of enzyme are higher.
It was found by the inventors of the present invention that by using the system of organic solvent-inorganic salts, by strictly controlling inorganic salts
Concentration can form three-phase distribution system, and can be enriched to PA ase in lower phase solution (if into above mixing
In liquid, enzyme easily on the one hand is inactivated, on the other hand can not directly be immobilized；If it is enriched to middle phase (solid phase), nothing
Method directly immobilizes, and needs additionally to increase operating procedure)；It is thus possible to directly carry out consolidating for enzyme in the lower phase solution
Fixedization；Method of the invention can complete each step of conventional method in a unit operation as a result, greatly simplifie
Technique, so as to as a kind of process integrated technology of great prospect.Present inventor has further discovered that it uses compared with prior art
Other kinds of alcohol, the tert-butyl alcohol can keep the activity of PA ase to the maximum extent.
It is coupled the present invention provides a kind of for synthesizing the isolating and purifying for PA ase of Amoxicillin with immobilization
Method, wherein, this method includes the following steps：
(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts, the inorganic salts mixing are prepared
A concentration of 0.05-0.3g/mL of inorganic salts in solution；
(2) mixed salt solution inorganic obtained by step (1) with the tert-butyl alcohol is mixed, then carries out split-phase, obtained rich in tertiary fourth
Upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and PA ase of alcohol；
(3) immobilization that carrier carries out PA ase is added in into phase solution lower obtained by step (2).
The method of the present invention compared with prior art existing advantage at least that：
(1) PA ase is isolated and purified the separation of solid and liquid being related to, just separation and essence separation by method of the invention
In multiple operating units completed in an operating unit, and isolating and purifying for enzyme is coupled with immobilization, enormously simplify
Operating procedure, the total recovery for shortening the operating time, improving enzyme extraction；
(2) method of the invention eliminates multistep plate-frame filtering or centrifugation, 1-2 step membrane filtration operations, reduces sewage row
It puts, reduces production cost, improve production efficiency；
(3) method of the invention isolates and purifies the total recovery of PA ase and is significantly higher than the prior art；
(4) enzyme activity of immobilised enzymes that method of the invention is prepared significantly improves；
(5) the recyclable rear reuse of the tert-butyl alcohol used in the present invention, further reduced production cost；
(6) enlarge-effect of method of the invention is small, there is very much industrial applications prospect.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
It is coupled the present invention provides a kind of for synthesizing the isolating and purifying for PA ase of Amoxicillin with immobilization
Method, wherein, this method includes the following steps：
(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts, the inorganic salts mixing are prepared
A concentration of 0.05-0.3g/mL of inorganic salts in solution；
(2) mixed salt solution inorganic obtained by step (1) with the tert-butyl alcohol is mixed, then carries out split-phase, obtained rich in tertiary fourth
Upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and PA ase of alcohol；
(3) immobilization that carrier carries out PA ase is added in into phase solution lower obtained by step (2).
It is the PA ase for synthesizing Amoxicillin, the enzyme that the present invention, which is isolated and purified with the object of immobilization,
Belong to one kind of PA ase, and be the PA ase for being particularly used for synthesis Amoxicillin, is i.e. term " is used for
Synthesize the PA ase of Amoxicillin " this specific PA ase is referred in the present invention.In the present invention
For convenience, " PA ase " will be referred to as " for synthesizing the PA ase of Amoxicillin ", i.e., herein
" PA ase " described in this refers to " for synthesizing the PA ase of Amoxicillin ".
It was found by the inventors of the present invention that the concentration of inorganic salts can be shown in the inorganic mixed salt solution of gained in step (1)
Influence whether three-phase distribution system can be obtained in (2) step with writing, in general, institute in the inorganic mixed salt solution
State inorganic salts concentration reach above-mentioned 0.05-0.3g/mL can so that obtain three-phase distribution system in step (2),
In the case of preferred, a concentration of 0.05-0.2g/mL of inorganic salts, more preferably 0.1- described in the inorganic mixed salt solution
0.2g/mL。
In step (1), the pH of the inorganic mixed salt solution is 5-9, is conducive to maintain the enzyme activity of PA ase
It does not suffer a loss.In order to preferably form three-phase distribution system and PA ase is made to fully enter lower phase as much as possible
In solution, the pH of the inorganic mixed salt solution is preferably 6.5-9, most preferably 7.5-8.5.
In step (1), the inorganic salts can be water-soluble phosphate, sulfate, carbonate, citrate and halogenation
It is one or more in object.Wherein, the water-soluble phosphate is, for example, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, phosphoric acid
Hydrogen two is received, biphosphate is received, phosphoric acid is received；The sulfate is, for example, ammonium sulfate, sodium sulphate, potassium sulfate；The carbonate is for example
For sodium bicarbonate, sodium carbonate, saleratus, potassium carbonate；The citrate is, for example, sodium citrate, potassium citrate；The halogen
Compound is, for example, sodium chloride, potassium chloride, is preferably used cooperatively when the inorganic salts are halide with water-soluble phosphate.
In step (1), in situations where it is preferred, the inorganic salts are water-soluble phosphate and/or sulfate, i.e., preferably
Selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid hydrogen two is received, biphosphate is received, one kind in ammonium sulfate, sodium sulphate and potassium sulfate
It is or a variety of；It is highly preferred that the inorganic salts are water-soluble phosphate, that is, it is most preferably selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, phosphorus
It is one or more in sour disodium hydrogen and sodium dihydrogen phosphate.
In step (1), in order to by the pH of inorganic mixed salt solution controls within the above range, can be by by institute
It states inorganic salts to be combined, and is added step-wise in mixed solution according to acid-base property.For example, a kind of preferred feelings according to the present invention
Condition,, can be in order to adjust pH to 7.5-8.5 when mixture of the inorganic salts for dipotassium hydrogen phosphate and potassium dihydrogen phosphate
It first adds in potassium dihydrogen phosphate and adjusts pH to 6-7, then dipotassium hydrogen phosphate is added to adjust pH to 8-9, be eventually adding potassium dihydrogen phosphate adjusting
PH to 7.5-8.5.
In step (1), term " PA ase crude enzyme liquid " meets the usual definition of this field.The penicillin acyl
Changing enzyme crude enzyme liquid can obtain by using expression vector progress fermented and cultured is then preprocessed；The expression of PA ase
Carrier can be Escherichia coli, bacillus subtilis, bacillus megaterium or Pichia pastoris；The condition of the fermented and cultured can be with
It is carried out using fermentation culture conditions known in the art.The purpose of the pretreatment is fermentation of the fermentation gained containing thalline
The solids such as removal thalline are miscellaneous after the zymotic fluid progress clasmatosis of liquid progress clasmatosis or the gained that will ferment containing thalline
Matter, to be released out the crude enzyme liquid of PA ase.Specific preprocess method is the method for this field routine, herein not
It repeats again.The inorganic salts such as phosphate may be used during pretreatment, the dosage of these inorganic salts is seldom or can be
React removing during pretreatment, therefore the inorganic salt concentration containing salt mixture described in step (1) can't be produced
Life significantly affects.
In step (1), the PA ase crude enzyme liquid can be the untreated penicillin acyl after clasmatosis
Change enzyme crude enzyme liquid, or the PA ase crude enzyme liquid after the solid impurities such as thalline is removed after clasmatosis；It is described
The method for removing the solid impurities such as thalline can be plate-frame filtering or centrifugation.When the PA ase crude enzyme liquid is through cell
Removal thalline, when preparing inorganic mixed salt solution, has when PA ase crude enzyme liquid after solid impurities after broken
A small amount of precipitation generation, it is preferred that carrying out primary removal solid impurity to gained mixture.
In step (1), the inorganic mixed salt solution of the preparation containing PA ase crude enzyme liquid and inorganic salts
Method can include：Inorganic salts are dissolved in the PA ase crude enzyme liquid.
In step (1), the enzyme activity of the crude enzyme liquid is 5-50IU/mL, preferably 20-50IU/mL.
In step (2), the volume ratio of the inorganic mixed salt solution and the tert-butyl alcohol can be 1：0.2-2, preferably 1：
0.2-1, more preferably 1：0.4-0.6.
In step (2), tert-butyl alcohol liquid that the tert-butyl alcohol can be commercially available can also be with the tert-butyl alcohol and water
The form of mixed liquor is mixed with the inorganic mixed salt solution.In order to which the method for the present invention is made to have more economy, it is preferable that
The process of the step (2) further includes：From the upper phase solution recycle the tert-butyl alcohol and recycle for inorganic mixed salt solution phase
During mixing.The method of the recycling is, for example, the way of distillation, and the tert-butyl alcohol of gained is usually the mixed liquor of the tert-butyl alcohol and water,
In the mixed liquor of the tert-butyl alcohol and water the content of the tert-butyl alcohol can be 70-100 weight %, preferably 80-100 weight %,
More preferably 85-95 weight %.
In step (2), there is no particular limitation for the mode of the mixing, for example, vibrates or stirs.
In step (2), there is no particular limitation for the mode of the split-phase, for example, centrifuges or stands.
In step (2), when the mode of the split-phase is centrifugation split-phase, which can be 0-40 DEG C, preferably
It is 4-37 DEG C, more preferably 15-30 DEG C；Centrifugal force can be 500-5000g, more preferably preferably 1000-4000g, 2000-
3500g；Time can be 2-60min, be preferably 2-40min, more preferably 5-20min.
In step (2), when the mode of the split-phase is stands split-phase, which can be 4-30 DEG C, preferably
It is 10-25 DEG C, more preferably 12-17 DEG C；Time is 0.1-4h, preferably 0.3-3h, more preferably 0.5-2h.
It can be obtained rich in the upper phase solution of the tert-butyl alcohol, middle phase solid and rich in salt and enzyme by the method for the present invention
The three-phase distribution system of lower phase solution composition, in the three-phase distribution system, PA ase is entered the nothing of lower phase by enrichment
In machine salting liquid, the activity of enzyme can be kept well, and can lower phase solution directly be carried out enzyme immobilizatio, real
Integrated operation is showed, has been effectively simplified operating procedure, shortens the operating time, so as to reduce industrial cost.On described
Phase solution mainly contains the tert-butyl alcohol and water.One layer of solid layer can be closely accompanied between phase solution and lower phase solution on described,
As described middle phase solid, phase solid mainly includes impurity protein, polysaccharide and bacterial chip that may be present etc. in this.
In step (3), the enzyme activity of the lower phase solution is 5-20IU/mL, if enzyme activity is diluted with water when being higher than 20IU/mL
To above range.
In step (3), the addition of the carrier can according to the addition of this field routine, for example, total enzyme activity with
The ratio between vehicle weight can be 30-60IU/g, preferably 40-50IU/g.
In step (3), the carrier can be known in the art various commercial carriers, such as amino carrier, epoxy
Base carrier etc..It can directly be used without activating when the carrier is epoxy base carrier.When the carrier is amino carrier
When need first to be activated, the activation method of amino carrier carries out in a manner that this field is conventional, for example,：1) it prepares
Glutaraldehyde phosphate buffer：It is weighed dipotassium hydrogen phosphate by 2-7g/L and is put into the beaker equipped with certain volume purified water and dissolved,
Then the glutaraldehyde solution that mass fraction is 40-60% is added in by 30-50mL/L, finally adjusts pH to 7.8- with potassium dihydrogen phosphate
8.2, it adds purified water and is settled to the final concentration of 1-3% (w/v) that scale causes glutaraldehyde；2) a certain amount of amino carrier is weighed
The above-mentioned glutaraldehyde phosphate buffer that 3-5 times of volume (mL/g) is added according to vehicle weight is activated, temperature 20-30
DEG C, rotating speed 100-200rpm, soak time 1.5-4h drain solution after activation；3) 3-5 is added according to vehicle weight
The purified water stirring and washing of times volume (mL/g) 2-4 times, stirs 15-40min every time, drains solution, collects the carrier activated
It is spare.
In step (3), the condition of the enzyme immobilizatio can be the operating condition of this field routine, such as can wrap
It includes：Temperature is 10-40 DEG C, preferably 20-30 DEG C；PH is 5-9, preferably 7-8.5；Rotating speed is 50-300rpm, preferably 100-
200rpm；Time is 4-48h, preferably 16-40h.
The method of the immobilization of step (3) of the present invention can also include immobilised enzymes post processing process, it is post-treated can
To obtain can be directly used for the immobilized penicillin acylated enzyme finished product of commercial synthesis Amoxicillin, the method for the post processing can be with
For the post-processing approach of this field routine, such as including：After enzyme immobilization operates, solution is drained, then according to immobilization
Enzyme weight adds in 2-6 times of volume (mL/g) purified water and cleans 2-4 times, drains solution, collects immobilised enzymes and places in 2-6 DEG C of storehouse
It preserves.
The immobilized penicillin acylated enzyme that the method for the present invention is prepared is applied to the specific side in synthesis Amoxicillin
Method is carried out according to the method for this field routine, and details are not described herein.
The present invention will be described in detail by way of examples below.First, to being made in following embodiment and comparative example
Substance, test method and computational methods are introduced as follows：
1st, carrier used in enzyme immobilizatio
Carrier used in enzyme immobilizatio is the amino carrier (LX- that Xi'an Lanxiao Sci-Tech Co., Ltd. provides
1000HA)。
The carrier is activated in advance before the use, activation method is as follows：1) 2% (w/v) glutaraldehyde phosphoric acid is prepared
Salt buffer：It is weighed dipotassium hydrogen phosphate by 4.76g/L and is put into the beaker equipped with certain volume purified water and dissolved, then pressed
40mL/L adds in the glutaraldehyde solution that mass fraction is 50%, finally adjusts pH to about 8 with potassium dihydrogen phosphate, adds purified water
It is settled to scale；2) 2% (w/v) penta 2 that a certain amount of amino carrier adds in 4 times of volumes (mL/g) according to vehicle weight is weighed
Aldehyde phosphate buffer is activated, and temperature is drained molten for 25 DEG C, rotating speed 150rpm, soak time 2h after activation
Liquid；3) according to the purified water stirring and washing of vehicle weight 4 times of volumes (mL/g) of addition three times, 20min is stirred every time, is drained molten
It is spare to collect the carrier activated for liquid.
2nd, the measure of enzyme activity
Enzyme activity determination uses alkalimetric titration method.The definition of enzyme activity is：Under the conditions of 28 DEG C, pH 8.0, the penicillin of unit
The enzyme activity that acylase 1min consumes 1 μm of ol potassium penicillin G is a unit (IU).Principle is：In 28 DEG C, 8.0 conditions of pH
Under, PA ase hydrolyzing penicillin G sylvite generates equimolar 6-APA and phenylacetic acid, is demarcated with the NaOH of 0.1mol/L
Liquid, which accurately titrates phenylacetic acid, can calculate the consumption of potassium penicillin G.
It is as follows：1) preparation of buffer solution：Weigh 4.76g dipotassium hydrogen phosphates and the dissolving of 0.55g potassium dihydrogen phosphates
In 900mL distilled water, with potassium dihydrogen phosphate tune pH to 7.8, then constant volume to 1L.2) 0.1mol/L NaOH solutions are prepared：First
Saturation NaOH solution is prepared, supernatant 5mL is taken to be added in water of the 1000mL without carbon dioxide after clarifying, is shaken up.It reuses
Substance is demarcated on the basis of Potassium Hydrogen Phthalate, calculates its concentration.3) 3mol/L NaOH solutions are prepared：It is quick to use totally small
After beaker weighs 30g NaOH, with being poured into the volumetric flask of 250mL after purifying water dissolution, rinse beaker 3 times with purified water and fall
Enter in volumetric flask, constant volume shake up after be 3mol/L NaOH solutions.4) preparation of substrate：2g potassium penicillin Gs are weighed, are dissolved in
In 40mL enzyme activity buffer solutions, pH is adjusted to 8.0 with 3mol/L NaOH, it is now with the current.5) enzyme activity determination：The certain body of accurate measuring
Long-pending liquid enzymes or the immobilised enzymes of certain mass is taken to keep temperature simultaneously to being preheating in advance in 28 DEG C of 40mL substrate solutions
Quick stirring starts timing with 0.1 or 3mol/L NaOH solution tune pH to 8.0, and the NaOH calibration liquid of 0.1mol/L is added dropwise in interruption
It is 8.0 to maintain pH, and reaction 5min or so, record adds NaOH titration liquid measures and reaction time.6) liquid enzymes work is calculated：Enzyme activity=
v1*c*1000/t*v2.Wherein, enzyme activity unit IU/mL；v1Represent the NaOH titrating solution volumes consumed in minute
(mL)；C represents NaOH titrating solutions (0.1mol/L) concentration (mol/L)；1000 expressions mM and micromolar conversion scale；t
Represent the reaction time (min)；v2Represent liquid enzymes volume (mL).7) immobilised enzymes work is calculated：Enzyme activity=v1*c*1000/t*w。
Wherein, enzyme activity unit IU/g；v1Represent the NaOH titrating solutions volume (mL) consumed in minute；C represents NaOH titration
Liquid (0.1mol/L) concentration (mol/L)；1000 expressions mM and micromolar conversion scale；T represents the reaction time (min)；w
Represent immobilised enzymes weight (g).
3rd, PA ase is than calculating living
PA ase is than calculation formula living：Than work (IU/mg)=enzyme activity (IU/mL)/protein concentration (mg/
ML), wherein the measure of protein concentration uses Coomassie Brilliant Blue.
4th, the calculating of enzyme yield
(1) often the calculation formula of step processing PA ase yield (Y, %) is：Y=c*v/ (c0*v0) * 100%,
In, after c expressions processing in feed liquid PA ase concentration (IU/mL)；Material liquid volume (mL) after v expressions processing；c0It represents
The concentration (IU/mL) of PA ase in before processing feed liquid；v0Represent before processing material liquid volume (mL)；
(2) the purification procedures yield of enzyme extraction total recovery (%)=fermentation liquor pretreatment step yield (%) × enzyme
(%).
5th, the immobilised enzymes product of gained is used to synthesize Amoxicillin
(1) synthetic method
It weighs 91.69g immobilised enzymes to be placed into reactor, adds in 500mL purified water stirring and washings, add after draining solution
Enter the purified water 800mL being pre-chilled in advance；Then 86.5g 6-amino-penicillanic acids (6-APA) and 74.65g are weighed respectively to hydroxyl
In Phenylglycine methyl ester input reactor, it is stirred.Controlled between 9.5-10.5 DEG C in reaction process, reaction terminates
Time is judged that (pH value can constantly rise during the reaction, answer the moment when rising to peak according to the variation of pH
Observation judges that reaction terminates when pH stable declines 0.01).
(2) (chemical equation of synthesis Amoxicillin can be expressed as the measure of 6-amino-penicillanic acid (6-APA) content
6-amino-penicillanic acid+p-hydroxyphenylglycine methyl ester → Amoxicillin+D-pHPG, the present invention is by 6-APA
The degree of the measure characterization reaction of residual quantity)
6-APA is detected with liquid chromatography, specific as follows：1. mobile phase is prepared：6.80g potassium dihydrogen phosphates are weighed,
1000mL purifying water dissolutions are added in, is dissolved with 1mol/l potassium hydroxide and adjusts pH value 5.8, it is fully mixed to add 40mL acetonitriles
It is even, after 0.45 μm of filtering with microporous membrane, ultrasound degassing 20min；2. reference substance is configured：It is sweet to weigh Amoxicillin, para hydroxybenzene
Propylhomoserin, 6-amino-penicillanic acid and each 20mg of p-hydroxyphenylglycine methyl ester are added in the volumetric flask of 100mL clean drieds, are added
Flowing phased soln is simultaneously diluted to scale, through 0.45 μm of membrane filtration after shaking up；3. sample treatment：Measure 100 μ L reaction solutions use
Mobile phase is diluted to 10mL, through 0.45 μm of membrane filtration after shaking up；4. liquid-phase condition：Pillar is C18 (150mm*4.6mm),
Sample size is 20 μ L, flow velocity 1mL/min, Detection wavelength 225nm.
Preparation example
(1) fermented and cultured of PA ase
Using the large intestine bacteria strain of recombinant expression, using existing literature, (Lu Jianmei, synthesis Amoxicillin are acylated with penicillin
Expression [D] of the enzyme in Escherichia coli, Beijing University of Chemical Technology's master thesis, 2013) method described in carries out fermentation training
It supports, until the enzyme activity of PA ase is 35.7IU/mL in zymotic fluid.
(2) fermentation liquor pretreatment：
(i) zymotic fluid 4500g is centrifuged 20min, collects thalline by fermentation ends；Then phosphate-buffered is pressed into thalline
Phosphate buffer of the volume mass of liquid/thalline than adding in 0.1mol/L pH 8.0 for 2.5mL/g is high under 900bar pressure
Broken cell homogenate is pressed, time 80min, homogenate is secondary, and interval sampling and measuring enzyme activity substantially completely, obtains enzyme to clasmatosis
PA ase crude enzyme liquid I living for 32.2IU/mL；
(ii) after clasmatosis, feed liquid is warming up to 47 DEG C, maintains temperature as 47 DEG C of processing 60min, then quickly
Cool the temperature to less than 40 DEG C；It is then slowly added into 0.4g/mL calcium chloride solutions and adjusts pH to 5.1；Finally by feed liquid 4500g from
Heart 20min, collects supernatant, which is the PA ase crude enzyme liquid II that enzyme activity is 27.6IU/mL.
Respectively according to the method described in following embodiment and comparative example to the PA ase crude enzyme liquid I obtained by preparation example
Or II carry out enzyme isolate and purify and immobilization.
Embodiment 1
(1) under conditions of 14 DEG C, according to the dosage of a concentration of 0.15g/mL by phosphate (by K2HPO4.3H2O and
KH2PO4Composition) it is dissolved in 90min in the PA ase crude enzyme liquid II that preparation example obtains and is adjusted during addition
The pH value of solution is saved as 8.0 (first plus KH2PO4PH is adjusted to 6.5, then adds K2HPO4.3H2O adjusts pH to 8.3, is eventually adding
KH2PO4PH is adjusted to 8.0), adding while salt stirring that salt is made fully to dissolve, speed of agitator 250rpm；Then 4500g is centrifuged
20min collects supernatant, obtains inorganic mixed salt solution；
(2) mixed salt solution inorganic obtained by step (1) and the tert-butyl alcohol of step (3) recycling gained and the mixed liquor of water are pressed
According to 1：0.6 volume ratio mixes, and vortex oscillation 2min is to being sufficiently mixed, and then 3500g centrifuges 10min, is formed rich in tertiary fourth
Upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and PA ase of alcohol, will
Three-phase solution is detached；
(3) the upper phase solution obtained by step (2) is taken, distillation obtains the tert-butyl alcohol and water of a concentration of 85 weight % of the tert-butyl alcohol
Mixed liquor, and be recycled in step (2)；
(4) the lower phase solution obtained by step (2) is diluted with water to enzyme activity as 15IU/mL, then by total enzyme activity and vehicle weight
The ratio between the amino carrier of activation added in for 45IU/g carry out enzyme immobilizatio, operating condition is：Temperature is 27 DEG C, pH 8, rotating speed
For 150rpm, time 30h；
(5) immobilised enzymes post-processes：After enzyme immobilizatio operates, solution is drained, then according to immobilised enzymes weight
It adds in 4 times of volume (mL/g) purified water cleanings three times, drains solution, collect and preserved in immobilised enzymes 4 DEG C of storehouses of placement.
Fermentation liquor pretreatment yield note is calculated in table 1.The enzyme activity of the lower phase solution obtained by above-mentioned steps (2) is measured by sampling
And protein concentration, ratio work and the yield of enzyme are calculated, result is remembered in table 1.The enzyme of immobilised enzymes obtained by step (5) is measured by sampling
Living and water content, by acquired results note in table 1.
Embodiment 2
(1) under conditions of 12 DEG C, according to the dosage of a concentration of 0.2g/mL in 90min by phosphate (by
K2HPO4.3H2O and KH2PO4Composition) it is dissolved in the PA ase crude enzyme liquid I that preparation example obtains and during addition
The pH value of solution is adjusted as 8.5 (first plus KH2PO4PH is adjusted to 7, then adds K2HPO4.3H2O adjusts pH to 9, is eventually adding KH2PO4
PH is adjusted to 8.5), adding stirring (speed of agitator 250rpm) while salt that salt is made fully to dissolve, obtains inorganic mixed salt solution；
(2) mixed salt solution inorganic obtained by step (1) and the tert-butyl alcohol of step (3) recycling gained and the mixed liquor of water are pressed
According to 1：0.4 volume ratio mixes, and vortex oscillation 2min is to being sufficiently mixed, and then 2000g centrifuges 20min, is formed rich in tertiary fourth
Upper phase solution, middle phase solid and the three-phase distribution system of the lower phase solution composition rich in salt and enzyme of alcohol, by three-phase solution into
Row separation；
(3) the upper phase solution obtained by step (2) is taken, distillation obtains the tert-butyl alcohol and water of a concentration of 95 weight % of the tert-butyl alcohol
Mixed liquor, and be recycled in step (2)；
(4) the lower phase solution obtained by step (2) is diluted with water to enzyme activity as 5IU/mL, then is in the ratio of enzyme/carrier
The amino carrier that 50IU/g adds in activation immobilizes, and the operating condition of immobilization is：Immobilization temperature is 20 DEG C, and pH is
8.5, rotating speed 100rpm, time 40h；
(5) immobilised enzymes post-processes：After enzyme immobilization operates, solution is drained, is then added according to immobilised enzymes weight
Enter 4 times of volume (mL/g) purified water cleanings three times, drain solution, collect and preserved in immobilised enzymes 4 DEG C of storehouses of placement.
Fermentation liquor pretreatment yield note is calculated in table 1.The enzyme activity of the lower phase solution obtained by above-mentioned steps (2) is measured by sampling
And protein concentration, ratio work and the yield of enzyme are calculated, result is remembered in table 1.The enzyme of immobilised enzymes obtained by step (5) is measured by sampling
Living and water content, by acquired results note in table 1.
Embodiment 3
(1) the PA ase crude enzyme liquid II that preparation example obtains is taken, is added in tank body, 13 DEG C of pot temperature is controlled, stirs
Speed 270rpm is mixed, then add in phosphate according to the dosage of a concentration of 0.2g/mL in 90min and is controlled during the addition process
The pH value of solution is about 7.8 (first plus KH2PO4PH is adjusted to 6.5, then adds K2HPO4.3H2O adjusts pH to 8.0, is eventually adding
KH2PO4PH is adjusted to 7.8)；Then a small amount of diatomite is added in, plate-frame filtering collects filtrate to get to inorganic mixed salt solution；
(2) mixed salt solution inorganic obtained by step (1) and the tert-butyl alcohol of step (3) recycling gained and the mixed liquor of water are pressed
According to 1：0.5 volume ratio mixes, and 350rpm stirrings 30min is sufficiently mixed, and pot temperature is then controlled to stop stirring at 15 DEG C
It mixes, stands split-phase 1 hour, form the upper phase solution rich in the tert-butyl alcohol, middle phase solid and rich in salt and PA ase
The three-phase distribution system of lower phase solution composition, three-phase solution is detached；
(3) the upper phase solution obtained by step (2) is taken, distillation obtains the tert-butyl alcohol and water of a concentration of 88 weight % of the tert-butyl alcohol
Mixed liquor, and be recycled in step (2)；
(4) the lower phase solution obtained by step (2) is diluted with water to enzyme activity as 20IU/mL, then by total enzyme activity and vehicle weight
The ratio between the amino carrier of activation added in for 40IU/g carry out enzyme immobilizatio, operating condition is：Temperature is 30 DEG C, pH 7, rotating speed
For 200rpm, time 40h；
(5) immobilised enzymes post-processes：After enzyme immobilization operates, solution is drained, is then added according to immobilised enzymes weight
Enter 4 times of volume (mL/g) purified water cleanings three times, drain solution, collect and preserved in immobilised enzymes 4 DEG C of storehouses of placement.
Fermentation liquor pretreatment yield note is calculated in table 1.The enzyme activity of the lower phase solution obtained by above-mentioned steps (2) is measured by sampling
And protein concentration, ratio work and the yield of enzyme are calculated, result is remembered in table 1.The enzyme of immobilised enzymes obtained by step (5) is measured by sampling
Living and water content, by acquired results note in table 1.
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Fermentation liquor pretreatment yield (%) | 80 | 93 | 80 |
| Isolate and purify yield (%) | 94 | 79 | 95 |
| Enzyme extraction total recovery (%) | 75 | 74 | 76 |
| Enzyme is detached than (IU/mg) living | 5.3 | 4.2 | 5.4 |
| Immobilization enzyme activity (IU/g) | 41.0 | 37.5 | 41.8 |
| Cure enzyme water content (%) | 51 | 53 | 50 |
Embodiment 4 and comparative example 1
Embodiment 4 (including embodiment 4-1~4-4) and comparative example 1 (including comparative example 1-1~1-2) are for illustrating difference
The influence that is isolated and purified to PA ase of extraction system.
Embodiment 4 is the procedure of Example 1 was followed except that the phosphate in step (1) is replaced with identical
Other inorganic salts of quality, it is specific as shown in table 2.
Comparative example 1 is the procedure of Example 1 was followed except that the tert-butyl alcohol in step (2) is replaced with identical
Other organic solvents of quality, it is specific as shown in table 2.
The separately sampled enzyme activity and protein concentration for measuring the lower phase solution obtained by above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note in table 2.
Table 2
From table 2 it can be seen that it can cause the ratio for the enzyme being obtained by extraction is lived to reach by using the extraction system of the present invention
More than 4.0IU/mg, yield reach more than 83%, are especially obtained by extraction when the tert-butyl alcohol-phosphate system for using embodiment 1
The ratio work of enzyme can reach 5.3IU/mg, yield can reach 94%.And when the tert-butyl alcohol is replaced with ethyl alcohol by comparative example, it is green
Mycin acylase is inactivated, and when the tert-butyl alcohol is replaced with isopropanol, the yield of PA ase is only 5%.
Embodiment 5 and comparative example 2
Embodiment 5 (including embodiment 5-1~5-3) and comparative example 2 (including comparative example 2-1~2-2) are for illustrating step
(1) influence that inorganic salt concentration isolates and purifies PA ase in inorganic mixed salt solution in.
Embodiment 5 the procedure of Example 1 was followed except that the phosphatic weight that changes cause it is phosphatic
Concentration changes, but the pH value for being to maintain solution is constant, specific as shown in table 3.
Comparative example 2 the procedure of Example 1 was followed except that the phosphatic weight that changes cause it is phosphatic
Concentration changes, but the pH value for being to maintain solution is constant, specific as shown in table 3.
The separately sampled enzyme activity and protein concentration for measuring the lower phase solution obtained by above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note in table 3.
Table 3
From table 3 it can be seen that when the concentration of inorganic salts is in the preferred scope of the present invention, the ratio for the enzyme being obtained by extraction is lived
Reach more than 4.6IU/mg, yield can reach more than 91%.And it can not be formed as inorganic salt concentration too low (comparative example 2-1)
Three-phase distribution system；As inorganic salt concentration excessively high (comparative example 2-2), most of PA ase is assigned to middle phase, lower phase
The ratio for the enzyme being obtained by extraction is lived and yield has significant decline.
Embodiment 6
Embodiment 6 (including embodiment 6-1~6-5) is for illustrating the pH value of inorganic mixed salt solution in step (1) to blueness
Mycin is acylated the influence of enzyme purification.
The procedure of Example 1 was followed except that on the basis of keeping phosphate weight constant, change phosphorus
The proportioning of sour hydrogen dipotassium and potassium dihydrogen phosphate makes the pH value of the inorganic mixed salt solution of gained reach as shown in table 4, in the feelings of needs
It can be finely adjusted under condition with the NaOH of phosphoric acid or 3mol/L.
The separately sampled enzyme activity and protein concentration for measuring the lower phase solution obtained by above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note in table 4.
Table 4
From table 4, it can be seen that when the pH value of inorganic mixed salt solution is in the wide range of the present invention, it is obtained by extraction
The ratio work of enzyme can reach more than 5.2IU/mg, and yield can reach more than 66%；It is extracted when in most preferred range of the present invention
The yield that isolates and purifies of obtained enzyme improves significantly to more than 87%.
Embodiment 7
Embodiment 7 (including embodiment 7-1~7-5) is for illustrating that the dosage of the tert-butyl alcohol in step (2) is acylated penicillin
The influence of enzyme purification.
The procedure of Example 1 was followed except that changing the step in (2), inorganic salts mixing is molten obtained by step (1)
The volume ratio of liquid and the tert-butyl alcohol of step (3) recycling gained and the mixed liquor of water,
It is specific as shown in table 5.
The separately sampled enzyme activity and protein concentration for measuring the lower phase solution obtained by above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note in table 5.
Table 5
As can be seen from Table 5, obtained by the step (1) tert-butyl alcohol of inorganic mixed salt solution and step (3) recycling gained and
When the volume ratio of the mixed liquor of water is in the preferred scope of the present invention, the ratio work for the enzyme being obtained by extraction reaches more than 5.1IU/mg,
Yield can reach more than 73%；When the present invention most preferred range in when be obtained by extraction enzyme ratio work reach 5.3IU/mg
More than, yield can reach more than 86%.
Embodiment 8
Time of repose when embodiment 8 (including embodiment 8-1~8-2) is for illustrating in step (2) using standing split-phase
The influence isolated and purified to PA ase.
It is carried out according to the method for embodiment 3, the difference is that the different time points that split-phase is stood in step (2) carry out
Sampling measures the enzyme activity and protein concentration of the lower phase solution obtained by above-mentioned steps (2), ratio work and the yield of enzyme is calculated, by result
Remember in table 6, specific time point setting is as shown in table 6.
Table 6
As can be seen from Table 6, with the growth of time of repose, the ratio for the enzyme being obtained by extraction is lived and yield is increased,
But when between upon standing more than 2 hours, the ratio for the enzyme being obtained by extraction is lived and slight decline occurs in yield, and production efficiency is dropped
It is low.
Embodiment 9
The embodiment (including embodiment 9-1~9-5) is sharp for illustrating the tert-butyl alcohol is carried out repeatedly to recycle in step (2)
The influence of Amoxicillin is synthesized with immobilization and immobilised enzymes with to isolating and purifying for PA ase.
Carry out test of many times according to the method for embodiment 3, the difference is that the tert-butyl alcohol used in step (2) and
The tert-butyl alcohol that the mixture of water is obtained for last experiment recycling, that is to say, that the used tert-butyl alcohol of often carrying out a test
Cycle-index increase it is primary, it is specific as shown in table 7.It measures and calculates the fermentation liquor pretreatment yield (%) of gained, point of enzyme
From purifying yield (%), enzyme extraction total recovery (%) (i.e. the total recovery for isolating and purifying two steps of fermentation liquor pretreatment and enzyme), consolidate
Surely change enzyme activity (IU/g) and immobilised enzymes water content (%) is reported in Table 7 below.
Table 7
As can be seen from Table 7, the data redundancy tested respectively using 5 recycling gained tert-butyl alcohols is good, fermentation
The data base for isolating and purifying yield, enzyme extraction total recovery, immobilization enzyme activity and immobilised enzymes water content of liquid pretreatment yield, enzyme
Originally it is maintained in error range；Purification procedures average yield is 96 ± 1%, and the average total recovery of enzyme extraction is 77 ± 1%, is obtained
The immobilised enzymes finished product arrived is averaged enzyme activity as 41.5 ± 1.4IU/g, and average water content is 51 ± 1%.
In addition, the immobilised enzymes obtained by Example 9-1, embodiment 3 and embodiment 9-2 synthesizes Amoxicillin, weight respectively
React 15 batches again, average reaction time is respectively 69,64,66min, average 6-APA residuals are respectively 2.80,2.60,
2.60mg/mL, it was demonstrated that gained immobilised enzymes it is functional.
Comparative example 3
This comparative example for illustrate when using state of the art isolate and purify with immobilized penicillin acylated enzyme and
Immobilised enzymes synthesizes the situation of Amoxicillin.
Using the PA ase crude enzyme liquid II obtained by preparation example, isolate and purify as follows and immobilized penicillin
Acylase：
(1) PA ase crude enzyme liquid II 1-3mol/L NaOH solution tune pH to 8.4-8.6, add a small amount of diatomite,
Plate-frame filtering after mixing is stirred, collects filtrate；Then it is 15-30IU/mL to concentrate the filtrate to enzyme activity with hollow-fibre membrane,
Collect concentrate；
(2) 25-30% ammonium sulfate is slowly added in concentrate, while Yanbian is added to stir, 30min is stirred for after adding salt,
Then plus a small amount of diatomite plate-frame filtering, collection precipitate；
(3) precipitation, Ran Houjia are dissolved with close 8.0 phosphate buffers of 0.05mol/L pH of crude enzyme liquid II volumes
A small amount of diatomite plate-frame filtering, collects filtrate；
(4) filtrate adds purified water to rinse repeatedly with hollow cellulose film, removes depigmentation, inorganic salts and small molecular protein, makes
Conductance is less than 1000 μ S/mL, and the enzyme activity that enzyme solution is finally concentrated into PA ase is 15-25IU/mL, collects concentrate；
(5) enzyme immobilizatio and post processing：Filtrate adds dipotassium hydrogen phosphate and potassium dihydrogen phosphate to inorganic by a certain percentage
The final concentration of 0.1mol/L of salt, other modes of operation are identical with the mode of operation of immobilization and the post processing of embodiment 1, consolidate
Surely change enzyme.
Test case
The immobilised enzymes product of 3 gained of embodiment 3 and comparative example is respectively used to synthesis Amoxicillin, repeats reaction 15 batches
Secondary, results are averaged by general, as shown in table 8.
Table 8
| Number | Embodiment 3 | Comparative example 3 |
| Fermentation liquor pretreatment yield (%) | 80 | 80 |
| Enzyme isolates and purifies yield (%) | 95 | 70 |
| Enzyme extraction total recovery (%) | 76 | 56 |
| Immobilization enzyme activity (IU/g) | 41.8 | 34 |
| Immobilised enzymes water content (%) | 50 | 52 |
| Synthesising reacting time (min) | 64 | 86 |
| 6-APA residual quantities (mg/mL) | 2.60 | 2.68 |
As can be seen from Table 8, compared with the method for comparative example, the yield isolated and purified of enzyme has significantly method of the invention
Increase, so as to enzyme extraction total recovery dramatically increase.And the enzyme activity of the immobilised enzymes of gained of the invention is apparently higher than comparison
Example, so as to which the reaction time for making synthesis Amoxicillin is obviously shortened than comparative example, the efficiency of synthesis Amoxicillin in the unit interval
It significantly improves.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In the skill of the present invention
In art conception range, a variety of simple variants can be carried out to technical scheme of the present invention, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, belongs to
Protection scope of the present invention.
Claims (9)
1. a kind of exist for synthesizing the isolating and purifying for PA ase of Amoxicillin with immobilization coupling process, feature
In this method includes the following steps：
(1) the inorganic mixed salt solution being made of PA ase crude enzyme liquid and inorganic salts is prepared, the inorganic salts mixing is molten
A concentration of 0.1-0.2g/mL of inorganic salts in liquid；
(2) mixed salt solution inorganic obtained by step (1) with the tert-butyl alcohol is mixed, then carries out split-phase, obtained rich in the tert-butyl alcohol
The three-phase distribution system of upper phase solution, middle phase solid and the lower phase solution composition rich in salt and PA ase；
(3) immobilization that carrier carries out PA ase is added in into phase solution lower obtained by step (2)；
Wherein, the inorganic salts are water-soluble phosphate；
Wherein, the pH of the inorganic mixed salt solution is 7.5-8.5；
Wherein, the volume ratio of the inorganic mixed salt solution and the tert-butyl alcohol is 1：0.4-0.6.
2. according to the method described in claim 1, wherein, in step (1), the PA ase crude enzyme liquid is through cell
The broken rear untreated PA ase crude enzyme liquid for being used to synthesize Amoxicillin is that thalline is removed after clasmatosis
Wait the PA ase crude enzyme liquid for being used to synthesize Amoxicillin after solid impurities.
3. according to the method described in claim 2, wherein, the enzyme activity of the PA ase crude enzyme liquid is 5-50IU/mL.
4. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is centrifuges split-phase, this point
Phase temperature is 0-40 DEG C, centrifugal force 500-5000g, time 2-60min.
5. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is stands split-phase, this point
Phase temperature is 4-30 DEG C, time 0.1-4h.
6. according to the method described in claim 1, wherein, the process of the step (2) further includes：It is returned from the upper phase solution
During receiving the tert-butyl alcohol and recycling for being mixed with inorganic mixed salt solution.
7. according to the method described in claim 1, wherein, in step (3), the enzyme activity of the lower phase solution is 5-20IU/mL,
If it is diluted with water to above range higher than 20IU/mL.
8. the method according to claim 1 or 7, wherein, in step (3), the addition of the carrier for total enzyme activity with
The ratio between vehicle weight is 30-60IU/g.
9. according to the method described in claim 8, wherein, the carrier is epoxy base carrier or the amino carrier of activation.
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| CN101693735A (en) * | 2009-10-30 | 2010-04-14 | 大连理工大学 | Method for extracting protein and enzyme by aqueous two-phase extraction technology |
| CN103695405A (en) * | 2013-11-11 | 2014-04-02 | 华北制药河北华民药业有限责任公司 | Novel beta-lactam antibiotic synthetase production method |
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| CN101693735A (en) * | 2009-10-30 | 2010-04-14 | 大连理工大学 | Method for extracting protein and enzyme by aqueous two-phase extraction technology |
| CN103695405A (en) * | 2013-11-11 | 2014-04-02 | 华北制药河北华民药业有限责任公司 | Novel beta-lactam antibiotic synthetase production method |
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| Title |
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| Tuning permeabilization of microbial cells by three-phase partitioning;Smita Raghava等;《Analytical Biochemistry》;20081017;第385卷;20-25 * |
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