CN110438113A - The process for fixation of D-Psicose 3- epimerase - Google Patents
The process for fixation of D-Psicose 3- epimerase Download PDFInfo
- Publication number
- CN110438113A CN110438113A CN201910668643.6A CN201910668643A CN110438113A CN 110438113 A CN110438113 A CN 110438113A CN 201910668643 A CN201910668643 A CN 201910668643A CN 110438113 A CN110438113 A CN 110438113A
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- China
- Prior art keywords
- psicose
- weight
- parts
- epimerase
- enzyme
- Prior art date
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- Granted
Links
- 108030002106 D-psicose 3-epimerases Proteins 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 50
- 230000008569 process Effects 0.000 title claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 175
- 102000004190 Enzymes Human genes 0.000 claims abstract description 174
- 239000007788 liquid Substances 0.000 claims abstract description 87
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims abstract description 71
- 239000011347 resin Substances 0.000 claims abstract description 49
- 229920005989 resin Polymers 0.000 claims abstract description 49
- 150000003839 salts Chemical class 0.000 claims abstract description 41
- 238000000926 separation method Methods 0.000 claims abstract description 23
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 239000000872 buffer Substances 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 235000002639 sodium chloride Nutrition 0.000 claims description 53
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 24
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- 229910052799 carbon Inorganic materials 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 16
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 15
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 14
- 238000009938 salting Methods 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 12
- 239000001103 potassium chloride Substances 0.000 claims description 12
- 235000011164 potassium chloride Nutrition 0.000 claims description 12
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 11
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 11
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 11
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000012452 mother liquor Substances 0.000 claims description 5
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 5
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 5
- 235000011151 potassium sulphates Nutrition 0.000 claims description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 229920001661 Chitosan Polymers 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 2
- 159000000013 aluminium salts Chemical class 0.000 claims description 2
- 229910000329 aluminium sulfate Inorganic materials 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000006911 enzymatic reaction Methods 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract 1
- 230000003000 nontoxic effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 63
- 238000006243 chemical reaction Methods 0.000 description 28
- 230000000694 effects Effects 0.000 description 26
- 238000002360 preparation method Methods 0.000 description 20
- 238000000855 fermentation Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 12
- 229930091371 Fructose Natural products 0.000 description 10
- 239000005715 Fructose Substances 0.000 description 10
- 238000006555 catalytic reaction Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 230000004151 fermentation Effects 0.000 description 9
- 239000007787 solid Substances 0.000 description 8
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 238000005189 flocculation Methods 0.000 description 7
- 230000016615 flocculation Effects 0.000 description 7
- 239000000758 substrate Substances 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 239000004744 fabric Substances 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 230000001376 precipitating effect Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 102000004195 Isomerases Human genes 0.000 description 4
- 108090000769 Isomerases Proteins 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 208000008589 Obesity Diseases 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 235000003599 food sweetener Nutrition 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000020824 obesity Nutrition 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- YRWWOAFMPXPHEJ-OFBPEYICSA-K sodium L-ascorbic acid 2-phosphate Chemical compound [Na+].[Na+].[Na+].OC[C@H](O)[C@H]1OC(=O)C(OP([O-])([O-])=O)=C1[O-] YRWWOAFMPXPHEJ-OFBPEYICSA-K 0.000 description 3
- 229940048058 sodium ascorbyl phosphate Drugs 0.000 description 3
- 239000008174 sterile solution Substances 0.000 description 3
- 239000003765 sweetening agent Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 2
- 101710109941 D-tagatose 3-epimerase Proteins 0.000 description 2
- 101710141886 Ketose 3-epimerase Proteins 0.000 description 2
- 208000007976 Ketosis Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 2
- 108090001066 Racemases and epimerases Proteins 0.000 description 2
- 102000004879 Racemases and epimerases Human genes 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 150000002584 ketoses Chemical class 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 210000002784 stomach Anatomy 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
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000364825 Mesoaciditoga lauensis Species 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000001994 activation Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000021433 fructose syrup Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical group O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
-
- 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
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention relates to D-Psicose 3- epimerism enzyme immobilization technology fields, a kind of process for fixation of D-Psicose 3- epimerase is disclosed, described method includes following steps: macroreticular resin buffer being rinsed and clean macroreticular resin is obtained by filtration；Obtained clean macroreticular resin and D-Psicose 3- epimerism enzyme solution are mixed into row enzyme immobilizatio, mixed liquor is obtained and is separated by solid-liquid separation, obtain the immobilization D-Psicose 3- epimerase using macroreticular resin as carrier；Wherein, inorganic salts -2 are 5-25 parts by weight in D-Psicose 3- epimerism enzyme solution described in 100 parts by weight.The D-Psicose 3- epimerase that can be only intended for single use realization is used repeatedly in the present invention, so that enzymatic reaction cost substantially reduces in industrial chain, and later separation cost is also declined, and resin itself is nontoxic, securely and reliably, there is good industrialized production and the prospect of marketing.
Description
Technical field
The present invention relates to Biochemical Engineering technical fields, and in particular to a kind of D-Psicose 3- epimerism enzyme immobilizatio
Method.
Background technique
As obesity, diabetes, " three high " chronic diseases are stretched in the world." the health intake " of sugar in diet
As new healthy project, wherein the development and application of low calorie sweetener is maximally efficient one of approach.
Psicose (D-psicose, Psi) is the epimer of D-Fructose (D-fructose, Fru) C-3, sugariness phase
When in the 70% of sucrose, heat is equivalent to sucrose 0.3%, and volume characteristics and mouthfeel are close with sucrose, can be used as in food medicine industry
The substitute of sucrose.There is the risk for causing fat and diabetes relative to high fructose syrup, psicose have obesity controlling and
The effect of diabetes, can obviously inhibit the increase of weight and the accumulation of stomach fat, can be exported by the core of glucose production kinases,
The tolerance of glucose and the sensibility of insulin are maintained, weight and stomach fat are controlled, reaches obesity controlling and diabetes
Effect.
CJ First Sugar Co., Ltd., South Korea applied for D- to U.S. Food and Drug Administration (FDA) in 2011
" it is generally acknowledged that safety " (GRAS) of psicose assert, obtains the answer (GRAS of " there is no problem " in June, 2012
Notice No.GRN000400)。
Song Gu group obtains in the GRAS identification to its product D-Psicose of FDA application in 2013 in June, 2014
The reply (GRAS Notice No.GRN000498) of " there is no problem ".This report is summarized, the daily ingestion of 31-33g of human experimentation
D-Psicose is no any side effect.Therefore, D-Psicose is classified as a kind of conventional carbohydrate substitution
Product, and any safety problem will not be constituted.FDA in 2011 approves that sweetener D-Psicose can be used as food additives and make
With.D-Psicose is rapidly developed since then, occurs a variety of products containing D-Psicose in the market, as patient of diabetes
Person can choose such highly-safe sweetener low in calories, to equally enjoy happy grow under the premise of maintaining health diet
Taste.Seen by sugar and the service condition of sweetener in the Asia new product development activity of 2012-2016, the use ratio of sucrose by
Gradually reduce, and the ratio of other natural sweeteners gradually rises, and reaches 17% within 2016 years.China market about the country about D- Ah
The research work of Lip river ketose is started late, and has been substantially carried out bacterial screening, enzyme gene clone and enzyme immobilizatio research etc., there is no
Industrialization report.Therefore, the sugar market space is wide.
But D-Psicose 3- isomery enzymatic is fructose converting for during psicose, since enzyme can not reuse,
So that the high process cost, and after reaction, D-Psicose 3- isomerase itself is used as foreign protein, needs from reactant
It is removed in system, increases separation costs, therefore, D-Psicose 3- isomery fixation techniques for enzyme is developed, so that D- A Luo ketone
Sugared 3- isomerase can reuse, and not only can substantially compress fermentation costs, later separation cost can also be reduced, to A Luo
The industry development of ketose has a very important significance.
Summary of the invention
Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a kind of D-Psicose 3- differences to different
Structure enzyme immobilizatio method, the method realize D-Psicose 3- epimerism enzyme immobilization, keep D-Psicose 3- poor
It can be used repeatedly to isomerase, reduce enzymic catalytic reaction cost.
To achieve the goals above, the present invention provides a kind of process for fixation of D-Psicose 3- epimerase,
Described method includes following steps:
Macroreticular resin buffer is rinsed and clean macroreticular resin is obtained by filtration；
Obtained clean macroreticular resin and D-Psicose 3- epimerism enzyme solution are mixed into row enzyme immobilizatio, obtained
It to mixed liquor and filters, obtains the immobilization D-Psicose 3- epimerase using macroreticular resin as carrier；
It wherein, is 5-25 weight containing inorganic salts -2 in D-Psicose 3- epimerism enzyme solution described in 100 parts by weight
Part.
Preferably, the present invention provides a kind of D-Psicose 3- epimerism enzyme solutions is prepared by following steps
:
(1) the production bacterium of D-Psicose 3- epimerase activated, fermented, microorganism collection, bacterial cell disruption and
Separation of solid and liquid obtains crude enzyme liquid；
(2) crude enzyme liquid is heated, inorganic salts -1 is then added, obtained mother liquor is filtered to obtain D- A Luo
Ketose 3- epimerase salting liquid；
(3) active carbon is added in Xiang Suoshu D-Psicose 3- epimerase salting liquid, after mixing is sufficiently stirred, solid-liquid
It is separated off active carbon, obtains D-Psicose 3- epimerism enzyme solution.
Through the above technical solutions, compared with the prior art, the invention has the following beneficial effects:
Firstly, the method for the invention can prepare the D-Psicose 3- epimerase of immobilization, substantially reduce
The cost of psicose enzymatic reaction, in addition, avoiding zymoprotein since enzyme is fixed on carrier and being directly dissolved in band in reaction solution
The later separation problem come, reduces later separation cost.Moreover, having during the present invention prepares product without using any
The harmful reagent of poison, therefore product safety.
Secondly, it is high using the stability of the immobilised enzymes of method of the present invention preparation, it can be repeated several times use, 55
Under the conditions of DEG C, the enzymatic activity of immobilised enzymes is still without obvious decaying after 18 batch of successive reaction.Under the conditions of 70 DEG C, reaction 10
Enzymatic activity is without obvious decaying after batch.
In addition, the immobilization D-Psicose 3- difference obtained using preferred technical solution purifying of the present invention is to different
Structure enzyme accesses relatively high enzyme activity yield and enzyme purity multiple.
Detailed description of the invention
Fig. 1 be in the present invention one be preferably carried out D-Psicose 3- epimerism enzyme immobilizatio side described in mode
The flow chart of method；
Fig. 2 is the immobilization D-Psicose 3- epimerase of method preparation described in embodiment 1 in 55 DEG C and 70 DEG C
Under the conditions of successive reaction attenuation curve.
Specific embodiment
Below in conjunction with attached drawing, detailed description of the preferred embodiments.It should be understood that this place is retouched
The specific embodiment stated is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
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 as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In the present invention, in the absence of explanation to the contrary, term " enzyme activity (Enzyme Activity) " is also referred to as enzyme
Activity, 1min is interior to be defined as 1 enzyme-activity unit (U) for the enzyme amount that 1mg substrate is converted into product.D-Psicose 3- difference is to different
The measuring method of structure enzyme enzyme activity are as follows: under the excessive reaction condition of substrate fructose, measure product in the initial 5min reaction time
The generation weight of D-Psicose, the then vigor of enzyme=D- psicose weight/5min.
Term " enzyme activity yield " refers to the enzyme activity after each step process operation and preoperative enzyme activity ratio multiplied by 100%.
Term " enzyme purity multiple " refers to that the ratio of enzyme is lived, i.e. the purity multiple of enzyme=(each Rate activity)/(first time
Rate activity).
Term " ratio of enzyme is living " is the measurement of enzyme purity, refers to the vigor of enzyme possessed in the protein of Unit Weight
Units is generally indicated with IU/mg protein.Specifically, measuring initial 5min under the excessive reaction condition of substrate fructose
The generation weight of product D-Psicose in reaction time, then ratio work=(weight/5min of D-Psicose)/total protein of enzyme
Weight.
The attenuation curve method for drafting of immobilised enzymes: with a batch enzyme, the continuous catalysis different batches within the same reaction time
Substrate, the attenuation trend of the conversion ratio of enzymic catalytic reaction is the attenuation curve of enzyme.In the present invention, multiple continuous catalysis
Afterwards, when the conversion ratio of enzymic catalytic reaction decays to 50% or less conversion ratio when being catalyzed for the first time, which is denoted as immobilization
Most numbers that enzyme uses.
In the present invention, the source of the D-Psicose 3- epimerase can not be particularly limited, such as can
D-Psicose 3- epimerase disclosed in patent application 201711458026.0 is thought, specifically, D-Psicose 3- is poor
To isomerase from the thermoacidophile Mesoaciditogalauensis of deep-sea hot spring, gene order is as follows:
MNFGVYLYLWEDRILEEEKALKIFKTIAELGYDGIEIPLNNPNLIDPFL ARKLAKEFELNITTSVAL
PQNINFMSDDESERDKAKEFLTNCVDLCNTMG SAVLGGVLYAPWGRTDVDKSEKKIGFLVEGLREISKYAEERGI
NLYLEPV NRFETNVLNTVKEGIDLIEKINSNNVSLLLDTFHMNIEEKDLSTAITEAGN LVGHFHTCENDRGIPG
TGHIPWKDIVQSLKKINYDGFLVFEAFSVKKEEIL NSANIWRSQELIPNPDKAAYESISFFKSIIY。
The invention discloses a kind of process for fixation of D-Psicose 3- epimerase, and the method includes walking as follows
It is rapid:
Macroreticular resin buffer is rinsed and clean macroreticular resin is obtained by filtration；
Obtained clean macroreticular resin and D-Psicose 3- epimerism enzyme solution are mixed into row enzyme immobilizatio, obtained
It to mixed liquor and filters, obtains the immobilization D-Psicose 3- epimerase using macroreticular resin as carrier；
Wherein, inorganic salts -2 are 5-25 parts by weight in D-Psicose 3- epimerism enzyme solution described in 100 parts by weight.
In the present invention, D-Psicose 3- epimerism enzyme solution refers to comprising the molten of D-Psicose 3- epimerase
Liquid, the inorganic salts -2 containing 5-25 parts by weight in the D-Psicose 3- epimerism enzyme solution, such as can for 5,10,15,
20, the amount ranges formed between 25 parts by weight and any two value, preferably 10-20 parts by weight.
The D-Psicose 3- epimerism enzyme solution can be D-Psicose 3- epimerase is dissolved in it is inorganic
Obtained in salting liquid, be also possible to enzyme solution that self-control obtains (can be as needed, such as pH or ionic strength, selection add or
Do not add inorganic salts), prepare D-Psicose 3- epimerism enzyme solution preferably by following in the present invention, add or
Inorganic salts acquisition is not added.The D-Psicose 3- epimerase, which voluntarily can be processed or be prepared, to be made, also commercially available
It buys.
The inorganic salts -2 in the D-Psicose 3- epimerism enzyme solution can be disodium hydrogen phosphate, biphosphate
At least one in sodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium sulphate and potassium sulfate
Kind, the combination preferably at least containing sodium dihydrogen phosphate and disodium hydrogen phosphate or the group containing potassium dihydrogen phosphate and dipotassium hydrogen phosphate
It closes, can be the combination of potassium dihydrogen phosphate, dipotassium hydrogen phosphate for example, can be the combination of sodium dihydrogen phosphate, disodium hydrogen phosphate,
It can be the combination of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, can be potassium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium chloride
Combination, can be sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium sulphate combination, can be potassium dihydrogen phosphate, dipotassium hydrogen phosphate
With the combination of potassium sulfate.The pH of the D-Psicose 3- epimerism enzyme solution can be 7.5-8.5, preferably 7.8-8.2.
Macroreticular resin commonly used in the art is used equally for the present invention, can be used for macroreticular resin of the invention and preferably wraps
At least one of (but being not limited to) alkalescent macroreticular resin is included, it is highly preferred that heretofore described macroreticular resin is epoxy
Base macroreticular resin or amino macroreticular resin.The macroreticular resin can it is self-produced or it is commercially available obtain, for example can be purchased from Xi'an indigo plant
Know at least one of LX-1000EP, LX-1000HA and LX-1000EA of new material Science and Technology Ltd..
It will be understood by those skilled in the art that the alkalescent macroreticular resin that can be used in the present invention in addition to using trade name institute above
Other than the specific product of restriction, further includes any commercial resins product with other trade names or voluntarily prepare or process and obtain
Resin, as long as they have and the same or similar structure of the specific product and absorption or switching performance.
In the present invention, relative to the D-Psicose 3- epimerism enzyme solution of 100 parts by weight, the macroreticular resin
It can be 5-60 parts by weight, such as can be 5,10,15,20,25,30,35,40,45,50,55,60 parts by weight and any
Any range formed between two values, preferably 20-40 parts by weight.
In rinsing, the buffer that the buffer can be commonly used in the art, preferably phosphate buffer.The phosphorus
The concentration of acid buffer is 0.05%-1%, preferably 0.1%-0.8%.The pH of the buffer can be 7.5-8.5, into one
Step is preferably 7.8-8.2.
In rinsing, buffer can select in a wider scope with macroreticular resin weight ratio, it is preferable that relative to
The macroreticular resin of 100 parts by weight, the buffer are 200-500 parts by weight.
Conventional technical means in the art can be used and carry out enzyme immobilizatio.Preferably, in the present invention, the fixation of the enzyme
The condition of change may include: that immobilization temperature is 10-40 DEG C, such as can be 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35
DEG C, 40 DEG C, further preferably 15-35 DEG C；The immobilization time is 24-48h, further preferably 30-40h；Immobilization is stirred
Mixing revolving speed is 90-150rpm.
The mixed liquor after immobilization can be separated by solid-liquid separation using conventional technical means in the art.Preferably, immobilization D- Ah
The separate mode of Lip river ketose 3- epimerase and residual liquid can be filtering, and filter pore size can be 40-500 mesh, such as
It can be 40 mesh, 60 mesh, 100 mesh, 200 mesh, 300 mesh, 500 mesh, preferably 60-200 mesh.
Preferably, in the present invention, the D-Psicose 3- epimerism enzyme solution is prepared by following steps:
(1) the production bacterium of D-Psicose 3- epimerase activated, fermented, microorganism collection, bacterial cell disruption and
Separation of solid and liquid obtains crude enzyme liquid；
(2) crude enzyme liquid is heated, inorganic salts -1, the filtrate that obtained mother liquor is filtered then is added
For D-Psicose 3- epimerase salting liquid；
(3) active carbon is added in Xiang Suoshu D-Psicose 3- epimerase salting liquid, after mixing is sufficiently stirred, solid-liquid
It is separated off active carbon, obtains D-Psicose 3- epimerism enzyme solution.
In the present invention, described " the production bacterium of D-Psicose 3- epimerase ", which can be, passes through genetic engineering means
The carrier bacterium of the D-Psicose 3- epimerase of building is also possible to be also possible to two kinds or more through taming bacterium naturally
Combination.For example, can be the Escherichia coli (Escherichia for carrying D-Psicose 3- epimerase gene order
Coli), bacillus subtilis (Bacillus subtilis) or clostridium (Clostridium.sp).The D-Psicose 3-
Epimerase gene order can be obtained from patent application 201711458026.0, can also use conventional technical means,
As Mutation induction carries out codon optimization to its sequence.
Conventional technical means in the art can be used to activate the production bacterium of D-Psicose 3- epimerase, send out
Ferment, microorganism collection, broken and separation of solid and liquid.
Wherein, thallus activation process can will be stored in carrying D-Psicose 3- epimerase base in ultra low temperature freezer
Because the production bacterium of sequence saves pipe natural thaw, seed liquor then is obtained after shaking flask and at least one level seeding tank spread cultivation.
Wherein, fermentation process, which can be, is inoculated into seed liquor in the fermentor equipped with fluid nutrient medium commonly used in the art
Fermented and cultured is carried out, the fluid nutrient medium can be LB liquid medium.Preferably, the condition of the fermented and cultured is inoculation
Amount is 0.1-5%, and speed of agitator 60-200rpm, temperature is 30-38 DEG C, ventilating ratio 1:(0.1-0.4), cultivate 12-36h
Afterwards, stop fermentation, obtain fermentation liquid.
Wherein, microorganism collection mode can be centrifuged for disc-stack centrifuge or tubular type；Centrifugal rotational speed can be in biggish model
It can be 5000rpm-20000rpm, preferably 8000rpm-15000rpm in enclosing.The fermentation liquid that centrifugal treating obtains collects
Obtain thallus.
Wherein, after obtaining thallus, sterile solution can be used and thallus is carried out dilution processing is resuspended；The sterile solution can
To be water, physiological saline or buffer etc., it is preferable that using the phosphate buffer that concentration is 0.1%, pH is 8.2, the nothing
The parts by weight of bacterium solution are optional, and preferably relative to 100 parts by weight thallus weight, the sterile solution weight is 100-200 weight
Measure part.
Wherein, the bacterial cell disruption mode can be homogenizer crush method or ultrasonic fragmentation；It is broken using homogenizer
When the processing of broken method, pressure can be 5MPa-60MPa, preferably 10MPa-40MPa；When being handled using ultrasonic fragmentation, ultrasonic wave
Power density is 100W/L-1000W/L, preferably 300W/L-700W/L.
In the present invention, flocculant can be added before or after the separation of solid and liquid in step (1), it is broken to remove thallus
Piece and other impurities.It was found by the inventors of the present invention that adding flocculant before separation of solid and liquid more can be improved separation of solid and liquid
Effect improves the purity and the rate of recovery of finally obtained enzyme, additionally it is possible to reduce the energy consumption of separation of solid and liquid, such as by the way that flocculation is added
Agent reduces revolving speed when centrifuge separation.
Therefore, in the preferred embodiment of the application, described in step (1) be separated by solid-liquid separation the step of before also
Including adding flocculant；Preferably, the flocculant can for aluminium salt, molysite, calcium chloride, acrylamide, in chitosan at least
It is a kind of；Relative to thallus described in 100 parts by weight, the dosage of the flocculant can be 0.05-1.5 parts by weight, for example, can be
0.05, any model formed between 0.1,0.2,0.5,0.7,0.9,1.0,1.2,1.5 parts by weight and any two numerical value
It encloses, preferably 0.2-0.8 parts by weight.
In the present invention, can be using conventional solid-liquid separation means separating thallus fragment and crude enzyme liquid, for example can be
Centrifugation or filtering.The condition of the centrifugation can be 2000-8000rpm；The filtering mesh number can be 60-500 mesh, such as can
Think one of 60 mesh, 80 mesh, 100 mesh, 200 mesh, 300 mesh, 400 mesh and 500 mesh；Those skilled in the art can basis
Actual needs is selected.
It was found by the inventors of the present invention that heating the egg that can promote thermal stability difference in crude enzyme liquid to crude enzyme liquid
The denaturation of white or other impurities, sedimentation, if after inorganic salt solution (preferably phosphatic inorganic salts) processing heating is used in combination
Crude enzyme liquid can further increase the rate of recovery and purity of enzyme.In step (2), the heating temperature can be 40-60 DEG C,
It such as can be 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, preferably 45-55 DEG C；The heating time can be 30-300min,
Such as can be formed between 30min, 60min, 120min, 180min, 240min, 300min and any two numerical value
Any range, preferably 60min-180min.
In step (2), the inorganic salts -1 can be disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid hydrogen
At least one of dipotassium, sodium chloride, potassium chloride, sodium sulphate and potassium sulfate, preferably include phosphate.Relative to 100 weight
The crude enzyme liquid of part, the inorganic salts -1 can be 1-25 parts by weight, preferably 10-20 parts by weight.By the way that the nothing is added
Machine salt -1, so that the pH of the crude enzyme liquid after the heating is 7.5-8.5, preferably 7.8-8.2.
In the present invention, be added active carbon purpose be in order to adsorb the pigment and partial impurities in enzyme solution, active carbon
Additive amount can be in the larger context.Relative to the crude enzyme liquid of 100 parts by weight, the dosage of the active carbon can be
0.05-1 parts by weight, for example, can between 0.05,0.1,0.2,0.5,0.7,0.9,1 parts by weight and any two numerical value group
At any range, preferably 0.1-0.5 parts by weight.
In the present invention, the enzyme solution body of the enzyme solution volume of the crude enzyme liquid and the D-Psicose 3- epimerism enzyme solution
Difference between product is ignored.
It such as Fig. 1, is preferably carried out in mode at of the invention one, a kind of fixation of D-Psicose 3- epimerase
Change method includes the following steps:
(1) the production bacterium of D-Psicose 3- epimerase activated, fermented, microorganism collection, bacterial cell disruption and
Separation of solid and liquid obtains crude enzyme liquid；
(2) crude enzyme liquid is heated, inorganic salts -1, the filtrate that obtained mother liquor is filtered then is added
For D-Psicose 3- epimerase salting liquid；
(3) active carbon is added in Xiang Suoshu D-Psicose 3- epimerase salting liquid to decolourize, is sufficiently stirred mixed
After even, it is separated by solid-liquid separation and removes active carbon, obtain D-Psicose 3- epimerism enzyme solution；
(4) macroreticular resin buffer is rinsed and clean macroreticular resin is obtained by filtration；
(5) obtained clean macroreticular resin and D-Psicose 3- epimerism enzyme solution are mixed into the fixation of row enzyme
Change, obtains mixed liquor and be separated by solid-liquid separation, obtain the immobilization D- psicose 3- epimerase using macroreticular resin as carrier.
The present invention will be described in detail by way of examples below.
Following embodiment and comparative examples:
Used D-Psicose 3- epimerase gene order is shown in patent application 201711458026.0.It is used
Bacterium is produced by the way that the D-Psicose 3- epimerase gene order is imported Escherichia coli (Escherichia coli)
It obtains.
The LX-1000EP of the model Xi'an Lan Xiao new material Science and Technology Ltd. of the macroreticular resin.
When phosphate buffer is prepared to obtain by disodium hydrogen phosphate and sodium dihydrogen phosphate, referred to as sodium phosphate buffer；When
When phosphate buffer is prepared to obtain by dipotassium hydrogen phosphate and potassium dihydrogen phosphate, referred to as potassium phosphate buffer.Wherein, phosphoric acid hydrogen two
Sodium is seven water disodium hydrogen phosphates；Sodium dihydrogen phosphate is sodium dihydrogen phosphate-water；Dipotassium hydrogen phosphate is three water dipotassium hydrogen phosphates；Phosphoric acid
Potassium dihydrogen is anhydrous potassium dihydrogenphosphate.
Not specifically specified substance and material are routine experiment articles, can pass through commercially available acquisition.
Thallus spreads cultivation and fermentation medium: LB liquid medium, 1% peptone, 0.5% yeast powder, 1% sodium chloride, pH
=6.8-7.0.
Enzyme activity determination method: taking mass concentration is the fructose soln 10ml of 45%-60%, and enzyme 0.5g to be determined is added
(or ml).5min is reacted under the conditions of 55-70 DEG C, is measured the generation weight of product D-Psicose, is calculated the vigor of enzyme.Enzyme
Vigor=D-Psicose quality (unit mg)/5min.
The attenuation curve method for drafting of immobilised enzymes: with a batch enzyme, the continuous catalysis different batches within the same reaction time
Substrate, the attenuation trend of the conversion ratio of enzymic catalytic reaction is the attenuation curve of enzyme.In the following embodiments, with embodiment 1
The immobilised enzymes of acquisition draws the attenuation curve of the immobilised enzymes, as shown in Figure 2 as catalyst.Specifically, with 500g/L
Fructose soln be substrate, using embodiment 1 obtain immobilised enzymes as catalyst, by every 100mL fructose soln be added 5g urge
The ratio catalysis fructose of agent generates D-Psicose, and reaction filters out immobilised enzymes after completing once from reaction solution, carries out
Second of catalysis reaction, successively carries out, until detecting that the enzyme activity of immobilised enzymes is reduced to the 50% of protoenzyme.
The measuring method of the conversion ratio of enzymic catalytic reaction is the content of fructose before and after measuring catalyzed conversion, conversion ratio=(turn
The content of fructose before content/conversion of fructose after change) × 100%.
Preparation example 1
This preparation example is used to illustrate the acquisition of the production bacterium thallus of D-Psicose 3- epimerase of the invention
The Escherichia coli for carrying D-Psicose 3- epimerase gene order in ultra low temperature freezer will be stored in save
Pipe shifts to an earlier date 0.5h natural thaw, obtains seed liquor after shaking flask, first class seed pot spread cultivation.
Primary seed solution is inoculated into the 100L fermentor equipped with 50L LB liquid medium, inoculum concentration 1%, is stirred
Revolving speed 150rpm, temperature is 35 DEG C, ventilating ratio 1:0.4, after cultivating 16h, stops fermentation, obtains fermentation liquid.
The fermentation liquid being centrifuged using tube centrifuge 10000rpm collects thallus.
Preparation example 2
This preparation example is used to illustrate the preparation of macroreticular resin clean in the present invention
1kg macroreticular resin LX-1000EP is taken, is that the phosphate buffer that 0.1%, pH is 7.8-8.2 rinses 2h with 3L concentration,
Clean macroreticular resin is obtained by filtration.
Wherein, the cation in the phosphate buffer (sodium ion or potassium ion) selects as needed, i.e., in preparation D-
Sodium ion and potassium ion select a use during psicose 3- epimerase immobilised enzymes.
Embodiment 1
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
1) acquisition of crude enzyme liquid
The 500g thallus that preparation example 1 obtains is resuspended using 750ml sodium phosphate buffer, wherein the sodium ascorbyl phosphate is slow
Fliud flushing concentration is 0.1%, pH 8.2.Bacterium after being crushed the resuspension under conditions of surge pressure 25MPa using homogenizer
Body.Then the flocculation of 3g calcium chloride is added into the solution after break process, is centrifugated precipitating under the revolving speed of 5000rpm, obtains
1000ml supernatant, as crude enzyme liquid.
2) acquisition of D-Psicose 3- epimerism enzyme solution
Inorganic salts -1 150g is added after being down to room temperature, so that slightly in 50 DEG C of heating 120min of crude enzyme liquid that step (1) is obtained
The pH of enzyme solution is 8.2.The inorganic salts -1 are the mixture of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride, wherein sodium chloride
Weight be 80g.By D-Psicose 3- epimerase salting liquid is obtained by filtration；2g active carbon is added, is filtered to remove
Active carbon obtains D-Psicose 3- epimerism enzyme solution.
3) acquisition of immobilization D-Psicose 3- epimerase
The clean macroreticular resin of the 300g that preparation example 2 is prepared is added in enzyme solution, 20 DEG C, 120rpm stirring 35h,
100 mesh filter-cloth filterings obtain immobilization D-Psicose 3- epimerase.
4) data determination and calculating
The D-Psicose 3- epimerism enzyme solution that the homogeneous post-fermentation liquid and step (2) for taking step (1) to obtain obtain, is surveyed
Its fixed enzyme activity and ratio are lived, and the purity multiple of enzyme is calculated.
The ratio for the immobilization D-Psicose 3- epimerase that determination step (3) obtains is living and at 55 DEG C and 70 DEG C
Catalyzed conversion produces the conversion ratio of D-Psicose, obtains at most repeating batch.
It the results are shown in Table 1.
Embodiment 2
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
1) acquisition of crude enzyme liquid
The 500g thallus that preparation example 1 obtains is resuspended using 500ml potassium phosphate buffer, wherein the potassium phosphate is slow
Fliud flushing concentration is 0.1%, pH 8.Thallus after being crushed the resuspension under conditions of surge pressure 40MPa using homogenizer.
Then the flocculation of 4g iron chloride is added into the solution after break process, is centrifugated precipitating under the revolving speed of 2000rpm, obtains 750ml
Supernatant, as crude enzyme liquid.
2) acquisition of D-Psicose 3- epimerism enzyme solution
Inorganic salts -1 150g is added after being down to room temperature, so that slightly in 45 DEG C of heating 180min of crude enzyme liquid that step (1) is obtained
The pH of enzyme solution is 8.The inorganic salts -1 are the mixture of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium chloride, wherein potassium chloride
Weight is 75g.By D-Psicose 3- epimerase salting liquid is obtained by filtration；0.75g active carbon is added, crosses and filters out
Deactivation charcoal obtains D-Psicose 3- epimerism enzyme solution.
3) acquisition of immobilization D-Psicose 3- epimerase
The clean macroreticular resin of the 150g that preparation example 2 is prepared is added in enzyme solution, 15 DEG C, 90rpm stirring 40h,
300 mesh filter-cloth filterings obtain immobilization D-Psicose 3- epimerase.
4) data determination and calculating
The D-Psicose 3- epimerism enzyme solution that the homogeneous post-fermentation liquid and step (2) for taking step (1) to obtain obtain, is surveyed
Its fixed enzyme activity and ratio are lived, and the purity multiple of enzyme is calculated.
The ratio for the immobilization D-Psicose 3- epimerase that determination step (3) obtains is living and at 55 DEG C and 70 DEG C
Catalyzed conversion produces the conversion ratio of D-Psicose, obtains at most repeating batch.
It the results are shown in Table 1.
Embodiment 3
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
1) acquisition of crude enzyme liquid
The 500g thallus that preparation example 1 obtains is resuspended using 1000ml potassium phosphate buffer, wherein the potassium phosphate is slow
Fliud flushing concentration is 0.1%, pH 7.8.Bacterium after being crushed the resuspension under conditions of surge pressure 10MPa using homogenizer
Body.Then 1g flocculate with chitosan is added into the solution after break process, is centrifugated precipitating under the revolving speed of 8000rpm, obtains
1250ml supernatant, as crude enzyme liquid.
2) acquisition of D-Psicose 3- epimerism enzyme solution
Inorganic salts -1 125g is added after being down to room temperature, so that slightly in 55 DEG C of heating 60min of crude enzyme liquid that step (1) is obtained
The pH of enzyme solution is 7.8, wherein the inorganic salts -1 are the mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.By being obtained by filtration
D-Psicose 3- epimerase salting liquid；6.25g active carbon is added, active carbon is filtered to remove, obtains D-Psicose 3-
Epimerism enzyme solution.
3) acquisition of immobilization D-Psicose 3- epimerase
It weighs 25g potassium chloride to be added in enzyme solution after mixing, it is clean to add 500g that preparation example 2 is prepared
Macroreticular resin stirs 30h in 35 DEG C, 150rpm, and 60 mesh filter-cloth filterings obtain immobilization D-Psicose 3- epimerase.
4) data determination and calculating
The D-Psicose 3- epimerism enzyme solution that the homogeneous post-fermentation liquid and step (2) for taking step (1) to obtain obtain, is surveyed
Its fixed enzyme activity and ratio are lived, and the purity multiple of enzyme is calculated.
The ratio for the immobilization D-Psicose 3- epimerase that determination step (3) obtains is living and at 55 DEG C and 70 DEG C
Catalyzed conversion produces the conversion ratio of D-Psicose, obtains at most repeating batch.
It the results are shown in Table 1.
Embodiment 4
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
1) acquisition of crude enzyme liquid
The 500g thallus that preparation example 1 obtains is resuspended using 750ml sodium phosphate buffer, wherein the sodium ascorbyl phosphate is slow
Fliud flushing concentration is 0.1%, pH 8.2.Bacterium after being crushed the resuspension under conditions of surge pressure 25MPa using homogenizer
Body.Then the flocculation of 0.25g calcium chloride is added into the solution after break process, is centrifugated precipitating under the revolving speed of 5000rpm, obtains
1000ml supernatant, as crude enzyme liquid.
2) acquisition of D-Psicose 3- epimerism enzyme solution
Inorganic salts -1 240g is added after being down to room temperature, so that slightly in 40 DEG C of heating 300min of crude enzyme liquid that step (1) is obtained
The pH of enzyme solution is 8.2.The inorganic salts -1 are the mixture of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride, wherein sodium chloride
Weight be 170g.By D-Psicose 3- epimerase salting liquid is obtained by filtration；0.5g active carbon is added, crosses and filters out
Deactivation charcoal obtains D-Psicose 3- epimerism enzyme solution.
3) acquisition of immobilization D-Psicose 3- epimerase
The clean macroreticular resin of the 600g that preparation example 2 is prepared is added in enzyme solution, 10 DEG C, 120rpm stirring 48h,
100 mesh filter-cloth filterings obtain immobilization D-Psicose 3- epimerase.
4) data determination and calculating
The D-Psicose 3- epimerism enzyme solution that the homogeneous post-fermentation liquid and step (2) for taking step (1) to obtain obtain, is surveyed
Its fixed enzyme activity and ratio are lived, and the purity multiple of enzyme is calculated.
The ratio for the immobilization D-Psicose 3- epimerase that determination step (3) obtains is living and at 55 DEG C and 70 DEG C
Catalyzed conversion produces the conversion ratio of D-Psicose, obtains at most repeating batch.
It the results are shown in Table 1.
Embodiment 5
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
1) acquisition of crude enzyme liquid
The 500g thallus that preparation example 1 obtains is resuspended using 750ml sodium phosphate buffer, wherein the sodium ascorbyl phosphate is slow
Fliud flushing concentration is 0.1%, pH 8.2.Bacterium after being crushed the resuspension under conditions of surge pressure 25MPa using homogenizer
Body.Then the flocculation of 7.5g calcium chloride is added into the solution after break process, is centrifugated precipitating under the revolving speed of 5000rpm, obtains
1000ml supernatant, as crude enzyme liquid.
2) acquisition of D-Psicose 3- epimerism enzyme solution
Inorganic salts -1 50g is added after being down to room temperature, so that thick enzyme in 60 DEG C of heating 30min of crude enzyme liquid that step (1) is obtained
The pH of liquid is 8.2, wherein the inorganic salts -1 are the mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate.By D- is obtained by filtration
Psicose 3- epimerase salting liquid；Add 10g active carbon, be filtered to remove active carbon, obtain D-Psicose 3- difference to
Isomery enzyme solution.
3) acquisition of immobilization D-Psicose 3- epimerase
It weighs 10g sodium chloride to be added in enzyme solution after mixing, adds clean big of 50g that preparation example 2 is prepared
Hole resin, 40 DEG C, 120rpm stirring for 24 hours, 100 mesh filter-cloth filterings obtain immobilization D-Psicose 3- epimerase.
4) data determination and calculating
The D-Psicose 3- epimerism enzyme solution that the homogeneous post-fermentation liquid and step (2) for taking step (1) to obtain obtain, is surveyed
Its fixed enzyme activity and ratio are lived, and the purity multiple of enzyme is calculated.
The ratio for the immobilization D-Psicose 3- epimerase that determination step (3) obtains is living and at 55 DEG C and 70 DEG C
Catalyzed conversion produces the conversion ratio of D-Psicose, obtains at most repeating batch.
It the results are shown in Table 1.
Embodiment 6
The present embodiment is used to illustrate the process for fixation of D-Psicose 3- epimerase of the invention.
Embodiment 6-1, other conditions are same as Example 3, unlike the inorganic salts -1 for 2) adding in step be
25g potassium chloride adds the mixture of 125g dipotassium hydrogen phosphate and potassium dihydrogen phosphate in the 3) step into enzyme solution, then again plus
Enter clean macroreticular resin.
Embodiment 6-2, other conditions are same as Example 3, unlike the 2) do not add inorganic salts -1, In in step
3) the adds the mixture of 150g dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium chloride in step into enzyme solution, then add dry
Net macroreticular resin；Wherein, the weight of the potassium chloride of addition is 25g, and the mixture of addition makes the pH of crude enzyme liquid be 7.8.
Embodiment 6-3, other conditions are same as Example 3, unlike the inorganic salts -1 for 2) adding in step be
150g potassium chloride does not add inorganic salts in 3) step.
It the results are shown in Table 1.
Embodiment 7
The present embodiment is used to illustrate the effect of addition flocculant in step 2).
Embodiment 7-1, other conditions are same as Example 1, unlike do not add flocculant, revolving speed improve to
10000rpm。
Embodiment 7-2, other conditions are same as Example 1, unlike elder generation's 10000rpm centrifugal breaking that treated is molten
Then liquid adds the flocculation of 3g calcium chloride, 5000rpm centrifugal treating after flocculation.
It the results are shown in Table 1.
Comparative example 1
This comparative example 1 is used to illustrate the influence of Inorganic Salts and additive amount
Comparative example 1-1, other conditions are same as Example 1, the difference is that inorganic salts -1 are sodium chloride, weight 38.5g.
Comparative example 1-2, other conditions are same as Example 1, the difference is that inorganic salts -1 are disodium hydrogen phosphate and di(2-ethylhexyl)phosphate
The mixture of hydrogen sodium, total weight 26g, addition inorganic salts -1 make the pH of crude enzyme liquid be 8.
Comparative example 1-3, other conditions are same as Example 1, the difference is that inorganic salts -1 are disodium hydrogen phosphate, biphosphate
The mixture of sodium and sodium chloride, total weight 26g, addition inorganic salts -1 make the pH of crude enzyme liquid be 8.
It the results are shown in Table 1.
Table 1
It can be seen that in immobilised enzymes preparation process from the data of embodiment and comparative example, control -2 mesopodium of inorganic salts
Enough ionic strengths enable to the ratio work of immobilised enzymes relatively high, additionally it is possible to improve immobilised enzymes in 55 DEG C and 70 DEG C of items
The most repetition batches being catalyzed under part.
From the point of view of embodiment 1 to embodiment 5 is compared with embodiment 6, inorganic salts -1 include in the preparation process of enzyme solution
Phosphate can be improved the enzyme activity yield and enzyme purity multiple of the enzyme solution being prepared, in addition, being prepared for subsequent
The enzyme of immobilised enzymes produces positive influence than living and repetition batch.
From the point of view of embodiment 1 is compared with embodiment 7, enzyme solution can be significantly improved by first adding the method that flocculant is centrifuged again
Enzyme activity yield and enzyme purity multiple, achieve beneficial effect.
It is described the prefered embodiments of the present invention in detail above in conjunction with attached drawing, still, the present invention is not limited to above-mentioned realities
The detail in mode is applied, within the scope of the technical concept of the present invention, a variety of letters can be carried out to technical solution of the present invention
Monotropic type, these simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of process for fixation of D-Psicose 3- epimerase, which is characterized in that described method includes following steps:
Macroreticular resin buffer is rinsed and clean macroreticular resin is obtained by filtration；
Obtained clean macroreticular resin and D-Psicose 3- epimerism enzyme solution are mixed into row enzyme immobilizatio, mixed
It closes liquid and is separated by solid-liquid separation, obtain the immobilization D-Psicose 3- epimerase using macroreticular resin as carrier；
Wherein, inorganic salts -2 are 5-25 parts by weight in D-Psicose 3- epimerism enzyme solution described in 100 parts by weight.
2. according to the method described in claim 1, wherein, the buffer is phosphate buffer；The phosphate buffer it is dense
Degree is 0.05-1 weight %, preferably 0.1-0.8 weight %；
The pH of the buffer is 7.5-8.5, preferably 7.8-8.2；It is described relative to the macroreticular resin of 100 parts by weight
Buffer is 200-500 parts by weight.
3. according to the method described in claim 1, wherein, the macroreticular resin is at least one of alkalescent macroreticular resin；
Relative to the D-Psicose 3- epimerism enzyme solution of 100 parts by weight, the macroreticular resin is 5-60 parts by weight, excellent
It is selected as 20-40 parts by weight.
4. the pH of the D-Psicose 3- epimerism enzyme solution is 7.5-8.5 according to the method described in claim 1, wherein,
Preferably 7.8-8.2；
Preferably, the inorganic salts -2 be disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride,
At least one of potassium chloride, sodium sulphate and potassium sulfate.
It include: immobilization temperature in the condition of the enzyme immobilizatio is 10- 5. according to the method described in claim 1, wherein
40 DEG C, preferably 15-35 DEG C；The immobilization time is 24-48h, preferably 30-40h；The speed of agitator of immobilization is 90-
150rpm。
6. process for fixation described in any one of -5 according to claim 1, wherein the D-Psicose 3- epimerism
Enzyme solution is prepared by following steps:
(1) the production bacterium of D-Psicose 3- epimerase activated, fermented, microorganism collection, bacterial cell disruption and solid-liquid
Isolated crude enzyme liquid；
(2) crude enzyme liquid is heated, inorganic salts -1 is then added, the filtrate that obtained mother liquor is filtered is D-
Psicose 3- epimerase salting liquid；
(3) active carbon is added in Xiang Suoshu D-Psicose 3- epimerase salting liquid, after mixing is sufficiently stirred, is separated by solid-liquid separation
Active carbon is removed, D-Psicose 3- epimerism enzyme solution is obtained.
Further include 7. according to the method described in claim 6, wherein, before the step of being separated by solid-liquid separation described in step (1) plus wadding
Solidifying agent；Preferably, the flocculant is at least one of aluminium salt, molysite, calcium chloride, acrylamide, chitosan；Relative to
Thallus described in 100 parts by weight, the dosage of the flocculant are 0.05-1.5 parts by weight, preferably 0.2-0.8 parts by weight.
8. according to the method described in claim 6, wherein, in step (2), the heating temperature is 40-60 DEG C, preferably 45-
55℃；The heating time is 30min-300min, preferably 60min-180min.
9. according to the method described in claim 6, wherein, in step (2), the inorganic salts -1 are disodium hydrogen phosphate, phosphoric acid
At least one of sodium dihydrogen, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride, sodium sulphate and potassium sulfate；Relative to
The crude enzyme liquid of 100 parts by weight, the inorganic salts -1 are 1-25 parts by weight, preferably 10-20 parts by weight；
The pH of the mother liquor is 7.5-8.5, preferably 7.8-8.2.
10. according to the method described in claim 6, wherein, in step (2), relative to the crude enzyme liquid of 100 parts by weight,
The dosage of the active carbon is 0.05-1 parts by weight, preferably 0.1-0.5 parts by weight.
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