CN106967704B - A kind of PA ase for being used to prepare 6 aminopenicillanic acids isolates and purifies and immobilization coupling process - Google Patents
A kind of PA ase for being used to prepare 6 aminopenicillanic acids isolates and purifies and immobilization coupling process Download PDFInfo
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- CN106967704B CN106967704B CN201710236750.2A CN201710236750A CN106967704B CN 106967704 B CN106967704 B CN 106967704B CN 201710236750 A CN201710236750 A CN 201710236750A CN 106967704 B CN106967704 B CN 106967704B
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- 238000010168 coupling process Methods 0.000 title claims abstract description 8
- 239000002253 acid Substances 0.000 title abstract description 5
- 150000007513 acids Chemical class 0.000 title abstract 2
- 108090000790 Enzymes Proteins 0.000 claims abstract description 215
- 102000004190 Enzymes Human genes 0.000 claims abstract description 215
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims abstract description 120
- 239000000243 solution Substances 0.000 claims abstract description 84
- 238000000034 method Methods 0.000 claims abstract description 73
- 239000007788 liquid Substances 0.000 claims abstract description 70
- 230000000694 effects Effects 0.000 claims abstract description 61
- 150000003839 salts Chemical class 0.000 claims abstract description 48
- 239000012266 salt solution Substances 0.000 claims abstract description 36
- 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 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 49
- 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 claims description 25
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 claims description 24
- 229910019142 PO4 Inorganic materials 0.000 claims description 21
- 239000010452 phosphate Substances 0.000 claims description 21
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 20
- 238000004064 recycling Methods 0.000 claims description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 8
- 239000004593 Epoxy Substances 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 150000004820 halides Chemical class 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 239000012071 phase Substances 0.000 description 79
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 54
- 230000000052 comparative effect Effects 0.000 description 21
- 238000000605 extraction Methods 0.000 description 20
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 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 16
- 235000021317 phosphate Nutrition 0.000 description 16
- 238000000855 fermentation Methods 0.000 description 15
- 230000004151 fermentation Effects 0.000 description 15
- 229930182555 Penicillin Natural products 0.000 description 14
- 229940049954 penicillin Drugs 0.000 description 14
- 238000001914 filtration Methods 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- 235000019796 monopotassium phosphate Nutrition 0.000 description 11
- 238000011084 recovery Methods 0.000 description 11
- 238000000926 separation method Methods 0.000 description 11
- 239000007836 KH2PO4 Substances 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
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 239000002585 base Substances 0.000 description 9
- 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 9
- 238000005070 sampling Methods 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 108700023418 Amidases Proteins 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 102000005922 amidase Human genes 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 229910017053 inorganic salt Inorganic materials 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000008213 purified water Substances 0.000 description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000012805 post-processing Methods 0.000 description 5
- 239000001103 potassium chloride Substances 0.000 description 5
- 235000011164 potassium chloride Nutrition 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- 244000025254 Cannabis sativa Species 0.000 description 4
- 108010073038 Penicillin Amidase Proteins 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
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000004821 distillation Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000005374 membrane filtration Methods 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
- 238000012360 testing method Methods 0.000 description 4
- 241000235058 Komagataella pastoris Species 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 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
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-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
- 241000894006 Bacteria Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-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
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 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
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 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
- 239000007791 liquid phase Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229960003975 potassium Drugs 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 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
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- -1 salt salt Chemical class 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
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
- ZENKESXKWBIZCV-UHFFFAOYSA-N 2,2,4,4-tetrafluoro-1,3-benzodioxin-6-amine Chemical group O1C(F)(F)OC(F)(F)C2=CC(N)=CC=C21 ZENKESXKWBIZCV-UHFFFAOYSA-N 0.000 description 1
- 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
- XXZCIYUJYUESMD-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-3-(morpholin-4-ylmethyl)pyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C=1C(=NN(C=1)CC(=O)N1CC2=C(CC1)NN=N2)CN1CCOCC1 XXZCIYUJYUESMD-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
- 239000005711 Benzoic acid Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- ITIONVBQFUNVJV-UHFFFAOYSA-N Etomidoline Chemical compound C12=CC=CC=C2C(=O)N(CC)C1NC(C=C1)=CC=C1OCCN1CCCCC1 ITIONVBQFUNVJV-UHFFFAOYSA-N 0.000 description 1
- 240000005708 Eugenia stipitata Species 0.000 description 1
- 235000006149 Eugenia stipitata Nutrition 0.000 description 1
- 241000165940 Houjia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 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
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid 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
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling 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
- 230000035614 depigmentation Effects 0.000 description 1
- 239000012153 distilled water Substances 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
- 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
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000010354 integration Effects 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
- 239000012982 microporous membrane Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 235000019371 penicillin G benzathine Nutrition 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000014786 phosphorus Nutrition 0.000 description 1
- 239000000049 pigment Substances 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
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 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
- 229940006198 sodium phenylacetate Drugs 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 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
- 238000010977 unit operation 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
- C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position
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- C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase
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Abstract
The present invention relates to bioengineering field, and in particular to a kind of PA ase for being used to prepare 6 aminopenicillanic acids isolates and purifies and immobilization coupling process.This method comprises the following steps：(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, which is 0.05 0.35g/mL；(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, 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 into lower phase solution obtained by step (2).Method of the invention is easy to operate, the activity of gained PA ase and yield are higher.
Description
Technical field
The present invention relates to bioengineering field, and in particular to a kind of penicillin for being used to prepare 6-amino-penicillanic acid is acylated
Enzyme isolates and purifies 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 preparing 6-amino-penicillanic acid, use immobilised enzymes form more.To prepare the immobilised enzymes for meeting production requirement, first will
PA ase isolates and purifies out from zymotic fluid, then carries out enzyme immobilizatio.The expression vector of PA ase
Mainly 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 is, it is necessary 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.
Need separation of solid and liquid, concentration and removal and isolate and purify for the PA ase of high-purity is prepared
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 bioanalysises such as chemical method, the lysozymes such as Mechanical Method, organic solvent or the surfactants such as high-pressure homogenization or bead mill to bacterium
Body carries out clasmatosis, then adds flocculant, filter aid etc., is carried out separation of solid and liquid with centrifugation or plate-frame filtering and is obtained being free of 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
It is white to remove, 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 dissolved and precipitated with buffer salt solution, centrifugation or plate-frame filtering removal are insoluble
Property solid；Finally the impurity such as small molecule foreign protein, amino acid, inorganic salts are removed with membrane filtration；(4) fixation of PA ase
Change：Buffer salt to suitable concentration, centrifugation or plate-frame filtering is added into the isolated PA ase liquid of essence first to remove
Solid impurity, then adds 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 operating time length, causes production to be imitated
Rate is low；(2) step, which adds upper part enzyme, to be lost because the operating time is long, cause the total recovery of enzyme 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.
The content of the invention
The purpose of the invention is to overcome the above problem existing in the prior art, there is provided one kind is used to prepare 6- amino green grass or young crops
The PA ase of mould alkanoic acid isolates and purifies and immobilization coupling process.Method of the invention is easy to operate, gained mould
The activity and yield of plain acylase 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 is directly immobilized, it is necessary to additionally increase operating procedure)；It is thus possible to penicillin is directly carried out in the lower phase solution
Acylated enzyme immobilizatio；Thus, method of the invention can complete each step of conventional method in a unit operation, pole
The earth simplifies technique, so that as a kind of process integrated technology of great prospect.Present inventor has further discovered that compared to existing
There is the other kinds of alcohol that technology uses, the tert-butyl alcohol can keep the activity of PA ase to greatest extent.
The present invention provides a kind of isolating and purifying with fixing for PA ase for being used to prepare 6-amino-penicillanic acid
Change coupling process, wherein, this method comprises the following steps：
(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, this contains mixed salt solution
Described in the concentration of inorganic salts be 0.05-0.35g/mL；
(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, then carries out split-phase, obtain being 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 into lower phase solution 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
Put, reduce 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 for the 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, reduce further 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.
Embodiment
The endpoint of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectively
It can be combined with each other between the endpoint value of a scope and single point value, and individually between point value and obtain one or more
New number range, these number ranges should be considered as specific open herein.
The present invention provides a kind of isolating and purifying with fixing for PA ase for being used to prepare 6-amino-penicillanic acid
Change coupling process, wherein, this method comprises the following steps：
(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, this contains mixed salt solution
Described in the concentration of inorganic salts be 0.05-0.35g/mL；
(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, then carries out split-phase, obtain being 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 into lower phase solution obtained by step (2).
It is that the penicillin for being used to prepare 6-amino-penicillanic acid is acylated that the present invention, which is isolated and purified with the object of immobilization,
Enzyme, the enzyme belong to one kind of PA ase, and are the penicillin acylations for being particularly used for preparing 6-amino-penicillanic acid
Enzyme, i.e. term " PA ase for being used to prepare 6-amino-penicillanic acid " refer to this specific mould in the present invention
Plain acylase.In the present invention for convenience, will " PA ase for being used to prepare 6-amino-penicillanic acid " abbreviation
For " PA ase ", i.e., " PA ase " refers to " be used to prepare 6- aminopenicillanics in this text
The PA ase of acid ".
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 0.05-0.35g/mL can so that obtain three-phase distribution system in step (2),
In the case of preferable, the concentration of inorganic salts described in the inorganic mixed salt solution is 0.15-0.3g/mL, more preferably 0.2-
0.25g/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
Do not suffer a loss.In order to preferably form three-phase distribution system and PA ase is fully entered lower phase as much as possible
In solution, the pH of the inorganic mixed salt solution is preferably 5-8, is most preferably 6-8, more preferably 7-8.
In step (1), the inorganic salts can be water-soluble phosphate, sulfate, carbonate, citrate and halogenation
One or more in thing.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 acid carbonate, 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
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
State inorganic salts to be combined, and be added step-wise to according to acid-base property in mixed solution.A kind of for example, preferred feelings according to the present invention
Condition,, can be with order to adjust pH to 7.5-8.5 when mixture of the inorganic salts for dipotassium hydrogen phosphate and potassium dihydrogen phosphate
First add potassium dihydrogen phosphate and adjust pH to 6-7, then add dipotassium hydrogen phosphate 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 area.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
Carried out using fermentation culture conditions known in the art.The purpose of the pretreatment is thick enzyme of the fermentation gained containing thalline
Liquid carries out clasmatosis, or the solid such as removal thalline is miscellaneous after crude enzyme liquid progress clasmatosis of 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 area routine, herein not
Repeat 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.
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 PA ase is 50-300IU/mL in the PA ase crude enzyme liquid,
Preferably 70-300IU/mL.Preferably, the crude enzyme liquid is the penicillin after the solid impurity such as removal thalline after clasmatosis
Acylase crude enzyme liquid, when using this crude enzyme liquid, the enzyme activity of PA ase is excellent in the PA ase crude enzyme liquid
Elect 150-300IU/mL as.
In step (2), the volume ratio of the inorganic mixed salt solution and the tert-butyl alcohol can be 1：0.1-2, is preferably 1：
0.2-1.5, more preferably 1：0.25-0.6.
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 make the method for the present invention have more economy, it is preferable that
The process of the step (2) further includes：The tert-butyl alcohol is recycled from the upper phase solution and is circulated and is used for and 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,
The content of the tert-butyl alcohol can be 70-100 weight % in the mixed liquor of the tert-butyl alcohol and water, be 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 centrifuges split-phase, which can be 0-40 DEG C, preferably
For 4-37 DEG C, more preferably 15-30 DEG C；Centrifugal force can be 500-5000g, be preferably 1000-4000g, more preferably 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
For 10-25 DEG C, more preferably 12-17 DEG C；Time is 0.1-4h, is preferably 0.3-3h, more preferably 0.5-2h.
Upper phase solution rich in the tert-butyl alcohol, middle phase solid can be obtained and rich in salt and mould by the method for the present invention
The three-phase distribution system of the lower phase solution composition of plain acylase, in the three-phase distribution system, PA ase be enriched with into
In the inorganic salt solution for entering lower phase, the activity of enzyme can be kept well, and lower phase solution directly can be used for green grass or young crops
Mycin is acylated enzyme immobilizatio, realizes integrated operation, is effectively simplified operating procedure, shortens the operating time, so that
Reduce industrial cost.The upper phase solution mainly contains the tert-butyl alcohol and water.The meeting between phase solution and lower phase solution on described
One layer of solid layer is closely accompanied, is the middle phase solid, the solid in this in phase solid mainly includes impurity protein, polysaccharide
And bacterial chip that may be present etc..
In step (3), carrier is added into lower phase solution obtained by step (2) and carries out enzyme immobilizatio, which can be with
Including：The enzyme activity of the lower phase solution is 50-150IU/mL, is preferably 100-150IU/mL, if enzyme activity is higher than 150IU/mL
Above range is diluted with water to, phosphate to inorganic salts is then added and accounts for the concentration of total system and (be preferably for 0.2-0.45g/mL
0.3-0.4g/mL), add carrier and carry out enzyme immobilizatio；It is described add phosphatic process be referred to step (1) into
OK, in preferred control ph during adding in the range of the pH value required by step (1).The addition of the carrier can
According to the addition of this area routine, to be, for example, that the ratio between total enzyme activity and vehicle weight can be 500-1000IU/g, be preferably
700-900IU/g。
In step (3), the carrier can be known in the art various commercial carriers, such as amino carrier, epoxy
Base carrier etc..Can directly it be used without activating when the carrier is epoxy base carrier.When the carrier is amino carrier
Shi Youxuan is first activated, and the activation method of amino carrier carries out in the way of this area is conventional, and details are not described herein.
In step (3), the condition of the enzyme immobilizatio can be the operating condition of this area routine, such as can wrap
Include：Temperature is 10-40 DEG C, is preferably 20-30 DEG C；PH is 5-9, is preferably 7-9；Rotating speed is 50-300rpm, is preferably 100-
200rpm；Time is 4-48h, is preferably 16-40h.
The method of the enzyme immobilizatio of step (3) of the present invention can also include the process of immobilised enzymes post processing, locate after
Reason can obtain can be directly used for the immobilized penicillin acylated enzyme finished product of industrially prepared 6-amino-penicillanic acid, the post processing
Method can be this area routine post-processing approach, such as including：After enzyme immobilizatio operates, solution is drained, so
2-6 times of volume (mL/g) purified water is added according to immobilised enzymes weight afterwards to clean 2-4 times, drain solution, collect immobilised enzymes and put
Put in 2-6 DEG C of storehouse and preserve.
The immobilized penicillin acylated enzyme that the method for the present invention is prepared is applied to prepare in 6-amino-penicillanic acid
Specific method is carried out according to the method for this area 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 embodiments and comparative example
Material, test method and computational methods are introduced as follows：
1st, carrier used in immobilization
The epoxy base carrier (L-11) that carrier used in immobilization far provides for Cangzhou into Chemical Co., Ltd..
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.
Comprise the following steps that：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, takes supernatant 5mL to be added in water of the 1000mL without carbon dioxide after clarifying, shakes up.Reuse
Material 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, poured into after being dissolved with purified water in the volumetric flask of 250mL, 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, the NaOH calibration liquid of 0.1mol/L is added dropwise in interruption with 0.1 or 3mol/L NaOH solution tune pH to 8.0
It is 8.0 to maintain pH, reaction 5min or so, record plus 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 of consumption 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) of consumption 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, immobilized penicillin acylated enzyme is used to prepare 6-amino-penicillanic acid
(1) preparation method
Weigh immobilized penicillin acylated enzyme 50g to be placed into reactor, the scotcil salting liquid after concentration is used
It is 130000U/mL reaction solutions that 0.25% boric acid solution, which is configured to potassium penicillin G potency, matching while using.Used in reaction process
3mol ammonium hydroxide controls pH value 8.0, and temperature control is 30 DEG C, reaction time 40min.During every 5 minutes record primary first-order equations
Between, temperature, pH value and ammonium hydroxide usage amount, the content of sampling liquid chromatography for measuring potassium penicillin G and 6-APA, calculates blue or green
The residual potency of mycin G sylvite and the conversion ratio of 6-APA.
(2) measure of potassium penicillin G
Potassium penicillin G is detected with liquid chromatography.It is specific as follows：1) mobile phase is prepared：Weigh potassium dihydrogen phosphate
14.85g adds water 1400mL, stirring and dissolving, pH value is adjusted to 4.5, Ran Houyu with 1mol/L KOH solutions in 2000mL beakers
600mL trifluoroacetic acid aqueous solutions mix, after 0.45 μm of filtering with microporous membrane, ultrasound degassing 20min；2) reference substance configures：Weigh green grass or young crops
Mycin G sylvite reference substance 0.0315g are diluted with water constant volume in 50mL volumetric flasks, and 5mL is taken from above-mentioned solution to 50mL capacity
In bottle, with water constant volume again；3) sample treatment：Measure 2mL reaction solutions and be diluted with water to 50mL；4) liquid-phase condition：Pillar is C18
(150mm*4.6mm), sample size are 20 μ L, flow velocity 1mL/min, Detection wavelength 215nm.
(3) measure of 6-APA
6-APA is detected with liquid chromatography.It is specific as follows：1) mobile phase is prepared：Weigh disodium hydrogen phosphate 5.0g and
Potassium dihydrogen phosphate 2.7g, is dissolved in 2000mL beakers, adds water 1900mL, stirring and dissolving, with 1mol/L KOH solution tune pH value
To 7.0, then mixed with 100mL acetonitriles, after 0.45 μm of membrane filtration, ultrasound degassing 20min；2) reference substance configures：Weigh
6-APA reference substance 0.1000g, sodium phenylacetate 0.0750g add the buffer solution of pH 7.0 to be settled in same 500mL volumetric flasks
Scale；3) sample treatment：Sample dilutes 250 times using the buffer solution identical with reference substance；4) liquid-phase condition：Pillar is C18
(150mm*4.6mm), sample size are 20 μ L, flow velocity 1mL/min, Detection wavelength 220nm.
4th, 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.
5th, the calculating of enzyme yield
(1) often the calculation formula of step processing PA ase yield (Y, %) is：Y=c*v/ (c0*v0) * 100%, its
In, after c expressions processing in feed liquid PA ase concentration (IU/mL)；Material liquid volume (mL) after v expressions processing；c0Represent
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
(%).
Preparation example
(1) fermented and cultured of PA ase
Using the Pichi strain of recombination expression, using existing literature (Luo Qian, recombinant yeast pichia pastoris production PGA high density
Fermentation and its immobilization [D], Sino-South African Forestry University of Science and Technology's master thesis, 2013) method described in carries out fermented and cultured,
The enzyme activity of PA ase is 83.2IU/mL in test gained zymotic fluid.
(2) fermentation liquor pretreatment：
(i) after fermentation, by material liquid volume plus the ammonium sulfate of 0.01g/mL, stirring and dissolving, is then added by material liquid volume
The cetyl trimethylammonium bromide of 0.008g/mL, stirs 25min；Feed liquid is warming up to 45 DEG C, while with 2mol/L NaOH
Solution tune pH to 7.6；After temperature and pH are transferred to position, it is 45 DEG C to maintain temperature, and interruption adds 3mol/L NaOH solutions and maintains pH to exist
7.6 ± 0.1,80min is soaked, interval sampling and measuring enzyme activity substantially completely, obtains PA ase crude enzyme liquid to clasmatosis
I, the enzyme activity of PA ase crude enzyme liquid I is 79.8IU/mL, protein concentration 11.9mg/mL after measured, is than work
6.7IU/mg；
(ii) after clasmatosis, less than 40 DEG C are cooled the temperature to, by material liquid volume plus 0.06g/mL phosphate (according to
PH adjusts the ratio of dipotassium hydrogen phosphate and dipotassium hydrogen phosphate), pH to 8.0 is adjusted, stirring is completely dissolved salt, is then slowly added into
0.4g/mL calcium chloride solutions adjust pH to 5.1；By material liquid volume plus 0.09g/mL diatomite, sheet frame mistake after mixing is stirred
Filter, collects filtrate.
(iii) filtrate adds a small amount of diatomite with 2mol/L NaOH solution tune pH to 8.0, is stirred sheet frame mistake after mixing
Filter, collects filtrate；Then filtrate is concentrated with hollow-fibre membrane, collects concentrate, be PA ase crude enzyme liquid II, pass through
The enzyme activity for measuring PA ase crude enzyme liquid II is 210.2IU/mL, protein concentration 12.0mg/mL, is than work
17.5IU/mg。
Respectively according to the method described in following embodiments and comparative example to the PA ase crude enzyme liquid I obtained by preparation example
Or II carry out PA ase isolate and purify and immobilization.
Embodiment 1
(1) under conditions of 14 DEG C, according to the dosage that concentration is 0.2g/mL by phosphate (by K2HPO4.3H2O and KH2PO4
Composition) it is dissolved in the PA ase crude enzyme liquid II that preparation example obtains and is adjusted during addition molten in 90min
The pH value of liquid for 8.0 (first plus KH2PO4PH is adjusted to 6.5, then adds K2HPO4.3H2O adjusts pH to 8.3, is eventually adding KH2PO4Adjust
8.0) section pH is to, obtaining inorganic mixed salt solution；
(2) inorganic mixed salt solution 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, and formation is 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 separated；
(3) the upper phase solution obtained by step (2), the tert-butyl alcohol and water that the concentration that distillation obtains the tert-butyl alcohol is 85 weight % are taken
Mixed liquor, and be recycled in step (2)；
(4) it is 100IU/mL that the lower phase solution obtained by step (2), which is diluted with water to enzyme activity, and it is dense to phosphate to add phosphate
Spend for 0.4g/mL, and by adjusting K during adding2HPO4.3H2O and KH2PO4Proportioning pH value is adjusted to 8.0, then
Epoxy base carrier is added by the ratio between total enzyme activity and vehicle weight for 900IU/g to immobilize, operating condition is：Temperature is 27
DEG C, pH 8, rotating speed 150rpm, time 30h；
(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 2
(1) under conditions of 12 DEG C, according to concentration be 0.3g/mL dosage 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 6 (first plus KH2PO4PH is adjusted to 5, then adds K2HPO4.3H2O adjusts pH to 7, is eventually adding KH2PO4Adjust
Section pH obtains inorganic mixed salt solution to 6), adding stirring (speed of agitator 250rpm) while salt salt is fully dissolved；
(2) inorganic mixed salt solution obtained by step (1) and the tert-butyl alcohol of step (3) recycling gained and the mixed liquor of water are pressed
According to 1：0.25 volume ratio mixes, and vortex oscillation 2min is to being sufficiently mixed, and then 2000g centrifuges 20min, and formation is 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 separated；
(3) the upper phase solution obtained by step (2), the tert-butyl alcohol and water that the concentration that distillation obtains the tert-butyl alcohol is 95 weight % are taken
Mixed liquor, and be recycled in step (2)；
(4) it is 50IU/mL that the lower phase solution obtained by step (2), which is diluted with water to enzyme activity, by total enzyme activity and vehicle weight it
Immobilized than adding epoxy base carrier for 700IU/g, operating condition is：Temperature is 20 DEG C, pH 9, rotating speed 100rpm,
Time is 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 phosphate is added according to the dosage that concentration is 0.25g/mL in 90min and is controlled in adition process
The pH value of solution processed 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), that is, obtains inorganic salts mixed solution；
(2) inorganic mixed salt solution 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 then controls pot temperature to stop stirring at 15 DEG C
Mix, stand split-phase 1 it is small when, formed rich in the upper phase solution of 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 separated；
(3) the upper phase solution obtained by step (2), the tert-butyl alcohol and water that the concentration that distillation obtains the tert-butyl alcohol is 88 weight % are taken
Mixed liquor, and be recycled in step (2)；
(4) concentration that the lower phase solution obtained by step (2) is diluted with water to PA ase is 100IU/mL, adds phosphorus
Hydrochlorate to phosphate concn is 0.35g/mL, and by adjusting K during adding2HPO4.3H2O and KH2PO4Proportioning make
PH value is adjusted to 7.8, and adding epoxy base carrier by the ratio between total enzyme activity and vehicle weight for 800IU/g immobilizes, and operates bar
Part is：Temperature is 26.5 DEG C, and pH 8, rotating speed 150rpm, time 32h, finally stands 8h；
(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 (%) | 71 | 92 | 71 |
| Isolate and purify yield (%) | 93 | 68 | 95 |
| Enzyme extraction total recovery (%) | 66 | 63 | 67 |
| Enzyme is separated than (IU/mg) living | 31.3 | 18.2 | 26.1 |
| Immobilization enzyme activity (IU/g) | 404 | 313 | 353 |
| Cure enzyme water content (%) | 55 | 55 | 55 |
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 used to illustrate 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 enzyme activity and protein concentration of lower phase solution obtained by separately sampled measure above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note in table 2.
Table 2
Reach from table 2 it can be seen that the ratio for the enzyme that can be obtained by extraction by using the extraction system of the present invention is lived
More than 22.8IU/mg, yield reach more than 78%, are especially obtained by extraction when using the tert-butyl alcohol-phosphate system of embodiment 1
The ratio work of enzyme can reach 31.3IU/mg, yield can reach 93%.And when the tert-butyl alcohol is replaced with ethanol or different by comparative example
During propyl alcohol, PA ase is inactivated.
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 used to illustrate 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 enzyme activity and protein concentration of lower phase solution obtained by separately sampled measure 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 24.6IU/mg, yield can reach more than 81%；When the concentration of inorganic salts is in the preferred scope of the present invention,
The ratio for the enzyme being obtained by extraction, which is lived, reaches more than 28.4IU/mg, and yield can reach more than 90%.And when inorganic salt concentration is too low
Three-phase distribution system can not be formed when (comparative example 2-1).It is most of blue or green as inorganic salt concentration excessive (comparative example 2-2)
Mycin acylase is assigned to middle phase, and the ratio for the enzyme that lower phase is obtained by extraction is lived and yield has significant decline.
Embodiment 6
The pH value that embodiment 6 (including embodiment 6-1~6-5) is used to illustrate inorganic mixed salt solution in step (1) is to green grass or young crops
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 enzyme activity and protein concentration of lower phase solution obtained by separately sampled measure 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 preferred scope of the present invention, it is obtained by extraction
The ratio work of enzyme can reach more than 27.0IU/mg, and yield can reach more than 73%；Extracted when in most preferred range of the present invention
The ratio work of obtained enzyme can reach more than 27.0IU/mg, and yield improves significantly to more than 89%.
Embodiment 7
Embodiment 7 (including embodiment 7-1~7-5) is used to illustrate that the dosage of the tert-butyl alcohol in step (2) to be 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)
Liquid and the tert-butyl alcohol and the volume ratio of the mixed liquor of water obtained by step (3) recycling, it is specific as shown in table 5.
The enzyme activity and protein concentration of lower phase solution obtained by separately sampled measure 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 for the enzyme being obtained by extraction, which is lived, reaches more than 22.4IU/mg,
Yield can reach more than 83%；The ratio for the enzyme being obtained by extraction when in the most preferred range in the present invention, which is lived, reaches 28.8IU/
More than mg, yield can reach more than 93%.
Embodiment 8
Time of repose when embodiment 8 (including embodiment 8-1~8-2) is used to illustrate in step (2) using standing split-phase
The influence isolated and purified to PA ase.
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 sets as shown in table 6.
The enzyme activity and protein concentration of lower phase solution obtained by separately sampled measure above-mentioned steps (2), calculate enzyme ratio live and
Yield, by result note 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 has 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 has dropped
It is low.
Embodiment 9
Embodiment 9 (including embodiment 9-1~9-4) is used for the enzyme for illustrating the PA ase in step (3) after dilution
Influence of the concentration to immobilized penicillin amidase.
Carry out according to the method for embodiment 3, the difference is that in step (3), will mix respectively under step (2) gained
Liquid is diluted to different enzyme activity, specific as shown in table 7.
The enzyme activity and water content of the separately sampled final gained immobilised enzymes of measure, by result note in table 7.
Table 7
As can be seen from Table 7, larger model of the enzyme concentration of PA ase after dilution in step (3) in the present invention
When enclosing interior, the enzyme activity of gained immobilised enzymes can reach more than 332IU/g, and water content can be in the range of 53-56%；When
The enzyme activity of gained immobilised enzymes can reach more than 351U/mg when in the most preferred range of the present invention, and water content can be in 53-
In the range of 55%.
Embodiment 10
Embodiment 10 (including embodiment 10-1~10-4) is used to illustrating to add in step (3) resulting solution after inorganic salts
Influence of the inorganic salt concentration to immobilized penicillin amidase.
Carried out according to the method for embodiment 3, the difference is that in step (3), add dipotassium hydrogen phosphate and biphosphate
The mass volume ratio difference that potassium (control ph is constant) to inorganic salts account for total system is as shown in table 8.
The enzyme activity and water content of the separately sampled final gained immobilised enzymes of measure, by result note in table 8.
Table 8
As can be seen from Table 8, after inorganic salts are added in step (3) resulting solution inorganic salt concentration the present invention compared with
When in a wide range of, the enzyme activity of gained immobilised enzymes can reach more than 302IU/g, and water content can be in the range of 54-56%；
The enzyme activity of immobilised enzymes can reach more than 332IU/g obtained by when in the most preferred range in the present invention, and water content can be
In the range of 54-56%.
Embodiment 11
Embodiment 11 (including embodiment 11-1~11-4) is used to illustrate the use of PA ase and carrier in step (3)
Amount compares the influence of immobilized penicillin amidase.
Carried out according to the method for embodiment 3, the difference is that in step (3), change the addition of carrier so that is blue or green
The amount ratio of mycin acylase and carrier is as shown in table 9.
The enzyme activity and water content of separately sampled measure immobilization yield, final gained immobilised enzymes, result is remembered in table 9
In.
Table 9
As can be seen from Table 9, when in step (3) amount ratio of PA ase and carrier the present invention in a big way
When interior, immobilization yield can reach more than 39.3%, and the enzyme activity of gained immobilised enzymes can reach more than 311IU/g, and water contains
Amount can be in the range of 54-56%；When the present invention most preferred range in when immobilization yield can reach 42.4% with
On, the enzyme activity of gained immobilised enzymes can reach more than 352IU/g, and water content can be in the range of 55-56%.
Embodiment 12
The embodiment 12 (including embodiment 12-1~12-5) is used to illustrate repeatedly to follow the tert-butyl alcohol in step (2)
Ring utilizes the influence isolated and purified with immobilization to 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 obtains for last experiment recycling, that is to say, that the used tert-butyl alcohol of often carrying out a test
Cycle-index increase once, it is specific as shown in table 10.
Measure and calculate the fermentation liquor pretreatment yield (%) of gained, enzyme isolate and purify yield (%), enzyme extraction is total receives
Rate (%) (i.e. the total recovery for isolating and purifying two steps of fermentation liquor pretreatment and enzyme), immobilization enzyme activity (IU/g) and immobilised enzymes water
Content (%) is reported in Table 10 below.
Table 10
As can be seen from Table 10, 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 68 ± 1%, is obtained
The immobilised enzymes finished product arrived the enzyme activity that is averaged is 353 ± 8IU/g, and average water content is 55 ± 1%.
In addition, the immobilised enzymes finished product obtained by Example 12-1, embodiment 12-2 and embodiment 12-4 prepares 6- respectively
Aminopenicillanic acid repeats 15 batches of reaction, and average potassium penicillin G residual potency is respectively 2737,2652 and 2807U/mL,
Prove that the catalytic performance of gained immobilised enzymes is good.
Comparative example 3
This comparative example be used for illustrate when using state of the art isolate and purify with immobilized penicillin acylated enzyme and
Immobilised enzymes prepares the situation of 6-amino-penicillanic acid.
Using the PA ase crude enzyme liquid II obtained by preparation example, isolate and purify as follows and immobilized penicillin
Acylase：
(1) 36-40% ammonium sulfate is slowly added in PA ase crude enzyme liquid II, while adding Yanbian to stir, adds salt to terminate
After be stirred for 30min, then plus a small amount of diatomite plate-frame filtering, collect precipitation；
(2) 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；
(3) 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 it is 150-250IU/mL that enzyme liquid finally is concentrated into the enzyme activity of PA ase, collects concentration
Liquid；
(4) add the benzoic acid of 0.4-0.45%, adjust pH 6.5-6.7, be warming up to 50-52 DEG C, maintain 10-15min, then
Fast cooling adds a small amount of diatomite, plate-frame filtering, collects filtrate to less than 30 DEG C；
(5) enzyme immobilizatio and post processing：The mode of operation of immobilization and post processing with embodiment 1 is identical, consolidate
Surely enzyme is changed.
Test case
The immobilised enzymes product of embodiment 3 and the gained of comparative example 3 is respectively used to prepare 6-amino-penicillanic acid, is repeated anti-
15 batches are answered, results are averaged by general, as shown in table 11.
Table 11
| Numbering | Embodiment 3 | Comparative example 3 |
| Fermentation liquor pretreatment yield (%) | 71 | 71 |
| Enzyme isolates and purifies yield (%) | 95 | 70 |
| Enzyme extraction total recovery (%) | 67 | 50 |
| Immobilization enzyme activity (IU/g) | 353 | 305 |
| Immobilised enzymes water content (%) | 55 | 55 |
| Reaction time (min) | 35 | 39 |
| Potassium penicillin G residual potency (U/mL) | 2805 | 3025 |
As can be seen from Table 11, method of the invention is compared with the method for comparative example, and the yield isolated and purified of enzyme has aobvious
The increase of work, so that enzyme extraction total recovery dramatically increases.And the enzyme activity of the immobilised enzymes of gained of the invention is apparently higher than right
Ratio so that synthesis 6-amino-penicillanic acid reaction time shortened than comparative example.In addition, the benzyl penicillin of the present invention
Sylvite residual potency ratio comparative example is substantially less, and the immobilised enzymes of this explanation present invention gained is more preferable than comparative example catalytic performance.
The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present invention
In art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, these simple variants and combination should equally be considered as content disclosed in this invention, belong to
Protection scope of the present invention.
Claims (13)
1. isolate and purify and the immobilization coupling process of a kind of PA ase for being used to prepare 6-amino-penicillanic acid, it is special
Sign is that this method comprises the following steps：
(1) the inorganic mixed salt solution containing PA ase crude enzyme liquid and inorganic salts is prepared, this contains institute in mixed salt solution
The concentration for stating inorganic salts is 0.15-0.3g/mL；
Wherein, the pH of the inorganic mixed salt solution is 5-8；
(2) inorganic mixed salt solution obtained by step (1) is mixed with the tert-butyl alcohol, 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；
Wherein, the volume ratio of the inorganic mixed salt solution and the tert-butyl alcohol is 1：0.2-1.5；
(3) immobilization that carrier carries out PA ase is added into lower phase solution obtained by step (2)；
Wherein, the enzyme activity of the lower phase solution is 50-150IU/mL, if being diluted with water to above range higher than 150IU/mL.
2. according to the method described in claim 1, wherein, in step (1), inorganic salts is dense in the inorganic mixed salt solution
Spend for 0.2-0.3g/mL.
3. method according to claim 1 or 2, wherein, in step (1), the inorganic salts are water-soluble phosphate, sulphur
One or more in hydrochlorate, carbonate, citrate and halide.
4. according to the method described in claim 3, wherein, the inorganic salts are water-soluble phosphate and/or sulfate.
5. according to the method described in claim 4, wherein, the inorganic salts are water-soluble phosphate.
6. method according to claim 1 or 2, wherein, in step (1), the PA ase crude enzyme liquid is warp
The untreated PA ase crude enzyme liquid for being used to prepare 6-amino-penicillanic acid after clasmatosis, or for through clasmatosis
The PA ase crude enzyme liquid for being used to prepare 6-amino-penicillanic acid after the solid impurities such as thalline is removed afterwards.
7. method according to claim 1 or 2, wherein, the enzyme activity of the PA ase crude enzyme liquid is 50-300IU/
mL。
8. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is to centrifuge split-phase, this point
Phase temperature is 0-40 DEG C, centrifugal force 500-5000g, time 2-60min.
9. according to the method described in claim 1, wherein, in step (2), the mode of the split-phase is to stand split-phase, this point
Phase temperature is 4-30 DEG C, time 0.1-4h.
10. according to the method described in claim 1, wherein, the process of the step (2) further includes：From the upper phase solution
The recycling tert-butyl alcohol is simultaneously circulated for during being mixed with inorganic mixed salt solution.
11. according to the method described in claim 1, wherein, in step (3), add phosphate to inorganic salts and account for total system
Concentration is 0.2-0.45g/mL.
12. the method according to claim 1 or 11, wherein, in step (3), the addition of the carrier is total enzyme activity
It is 500-1000IU/g with the ratio between vehicle weight.
13. according to the method for claim 12, wherein, the carrier is epoxy base carrier or the amino carrier of activation.
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