CN103451255A - Production method of inosinic acid - Google Patents
Production method of inosinic acid Download PDFInfo
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- CN103451255A CN103451255A CN2013101066995A CN201310106699A CN103451255A CN 103451255 A CN103451255 A CN 103451255A CN 2013101066995 A CN2013101066995 A CN 2013101066995A CN 201310106699 A CN201310106699 A CN 201310106699A CN 103451255 A CN103451255 A CN 103451255A
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- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 239000004245 inosinic acid Substances 0.000 title claims abstract description 43
- 229940028843 inosinic acid Drugs 0.000 title claims abstract description 43
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 title claims abstract description 42
- 235000013902 inosinic acid Nutrition 0.000 title claims abstract description 42
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 32
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims abstract description 112
- 229950006790 adenosine phosphate Drugs 0.000 claims abstract description 112
- 229920005989 resin Polymers 0.000 claims abstract description 85
- 239000011347 resin Substances 0.000 claims abstract description 85
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims abstract description 57
- 150000001450 anions Chemical class 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims description 114
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 239000008367 deionised water Substances 0.000 claims description 31
- 229910021641 deionized water Inorganic materials 0.000 claims description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000002253 acid Substances 0.000 claims description 27
- 238000006555 catalytic reaction Methods 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 238000002203 pretreatment Methods 0.000 claims description 21
- 229920001429 chelating resin Polymers 0.000 claims description 16
- 230000001105 regulatory effect Effects 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 238000000967 suction filtration Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 11
- 239000002585 base Substances 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 9
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 8
- 125000000524 functional group Chemical group 0.000 claims description 8
- 239000012266 salt solution Substances 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 241000370738 Chlorion Species 0.000 claims description 6
- 239000008351 acetate buffer Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 5
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 5
- 230000008929 regeneration Effects 0.000 claims description 5
- 238000011069 regeneration method Methods 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 4
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- 239000003456 ion exchange resin Substances 0.000 claims description 4
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 4
- 125000001302 tertiary amino group Chemical group 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000009413 insulation Methods 0.000 claims description 3
- 229920003180 amino resin Polymers 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- 150000003141 primary amines Chemical group 0.000 claims description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims 1
- 230000009466 transformation Effects 0.000 abstract description 11
- 238000000605 extraction Methods 0.000 abstract description 7
- 238000000926 separation method Methods 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 4
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 abstract 1
- 102000055025 Adenosine deaminases Human genes 0.000 abstract 1
- 238000010924 continuous production Methods 0.000 abstract 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 40
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 33
- 108090000790 Enzymes Proteins 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 22
- 230000035484 reaction time Effects 0.000 description 15
- 239000007788 liquid Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 13
- 230000008569 process Effects 0.000 description 12
- 238000006481 deamination reaction Methods 0.000 description 11
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 10
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 10
- 238000010907 mechanical stirring Methods 0.000 description 10
- 235000019837 monoammonium phosphate Nutrition 0.000 description 10
- 239000012071 phase Substances 0.000 description 10
- 239000007791 liquid phase Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
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- 230000003197 catalytic effect Effects 0.000 description 4
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- 238000002791 soaking Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 3
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- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-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
- 238000009825 accumulation Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 2
- 235000013928 guanylic acid Nutrition 0.000 description 2
- 239000004226 guanylic acid Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002213 purine nucleotide Substances 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- RQFCJASXJCIDSX-UHFFFAOYSA-N 14C-Guanosin-5'-monophosphat Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(O)=O)C(O)C1O RQFCJASXJCIDSX-UHFFFAOYSA-N 0.000 description 1
- CRIZPXKICGBNKG-UHFFFAOYSA-N 3,7-dihydropurin-2-one Chemical class OC1=NC=C2NC=NC2=N1 CRIZPXKICGBNKG-UHFFFAOYSA-N 0.000 description 1
- DCTLYFZHFGENCW-UUOKFMHZSA-N 5'-xanthylic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 DCTLYFZHFGENCW-UUOKFMHZSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
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- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a production method of inosinic acid. The method comprises the step of adopting adenosine deaminase fixed in anion resin to catalyze the generation of the inosinic acid from adenylic acid. According to the method provided by the invention, the adenylic acid is taken as a substrate, the supply of raw materials is sufficient, the requirements for equipment are low, the separation and extraction are convenient, the semi-continuous production of the inosinic acid can be realized, the substrate is convenient and easy to obtain, the transformation is thorough, and the cost is relatively low.
Description
Technical field
The invention belongs to the biosynthesizing field, be specifically related to a kind of production method of inosinic acid.
Background technology
Inosinic acid, have another name called hypoxanthylic acid, t-inosinic acid or oxypurine Nucleotide, abbreviation 5 '-IMP, it is the important mesostate of purine nucleotides internal metabolism, it is the tie of adenylic acid (AMP) and guanylic acid metabolism, can realize the mutual conversion of adenylic acid (AMP) and guanylic acid, with the balance of metabolism of purine nucleotide in holder.Inosinic acid participates in the synthetic of energy i (in vivo) metabolism and nucleoprotein, activation pyruvic oxidase system, improve the activity of coenzyme A, make the histocyte under low energy, anoxic condition can continue successfully to carry out metabolism, and activation liver function, make undermined liver cell accelerate to repair, and stimulate generation antibody in body, be applicable to white corpuscle and thrombocytopenia, myocardial damage, hepatitis and liver cirrhosis.Simultaneously, a kind of in inosinic acid or flavour nucleotide, share the umami effects that can significantly strengthen monosodium glutamate with the sweet acid of bird.
At present, inosinic acid mainly contains the production methods such as chemical synthesis, natural matter extraction method, microbe fermentation method, fermentation-catalyzed combination method and enzyme transforming process.And above several production method has certain defect.Chemical synthesis because productive rate is low, by product is many and separation difficulty, the more high factor of equipment requirements are unfavorable for industrial production; The natural matter extraction method can not scale operation owing to being subject to raw material sources restrictions, is difficult to satisfy social needs; Microbe fermentation method and fermentation-catalyzed combination method realizes the production of hypoxanthylic acid with cheap raw material, yet the amount of the hypoxanthylic acid of microbial accumulation is limited, thereby causes in fermented liquid the concentration of hypoxanthylic acid on the low side, causes difficulty to the separation and Extraction in later stage; Enzyme transforming process is because the intrinsic defect of enzyme is restricted.These defects cause the inosinic acid supply shortage.
Patent application (CN1406273A) discloses a kind of industrial microorganism bacterial strain-product ammonia rod bacillus CJIP009(KCCM-10226 that utilizes) the mutant production technique of producing inosinic acid.Wherein in flask fermentation and 5L fermentor tank, the accumulation volume of hypoxanthylic acid can reach respectively 19.1g/L, 70.3g/L, nonetheless, and separation and Extraction inosinic acid difficult still from 7.03% solution.
Summary of the invention
For the problems referred to above, main purpose of the present invention is to provide a kind of production method of inosinic acid, the method be take adenylic acid (AMP) as substrate, the raw material supply abundance, and low for equipment requirements, separation and Extraction is convenient, can realize the semicontinuous production of hypoxanthylic acid, substrate conveniently is easy to get and transforms thoroughly, and cost is lower.
The invention provides a kind of production method of xanthylic acid, described method comprises and adopts the adenylic deaminase catalysis adenylic acid (AMP) be fixed in resin anion(R.A) to generate inosinic acid.
Further, described resin anion(R.A) is skeleton for take styrene polymer or acrylate copolymer, take the resin that primary amine groups, tertiary amine groups or quaternary amine base be functional group;
Preferably, described resin anion(R.A) is for take styrene polymer as skeleton, take the season amino resin that is functional group; Further preferably, described resin anion(R.A) is for take vinylbenzene-divinylbenzene as skeleton, the resin that the tertiary amino of take is functional group; Perhaps take vinylbenzene-divinylbenzene as skeleton, the resin that the quaternary ammonium group of take is functional group.
Further preferably, described resin anion(R.A) is Amberlite IRA-900, Amberlite IRA-93 or Amberlite IRA-400 resin.
Further, described production method specifically comprises:
Step 1, carry out pre-treatment to resin anion(R.A), and adenylic deaminase is fixed in to resin anion(R.A);
Step 2, be dissolved to the substrate adenylic acid (AMP) in deionized water, then add the immobilization adenylic deaminase obtained in step 1, makes the adenylic acid (AMP) reaction generate inosinic acid.
Further, described step 1 specifically comprises:
Step 1a, adopt alcoholic solution to soak, wash resin anion(R.A), suction filtration, and then through strong acid solution immersion, washing, suction filtration, then through strong base solution immersion, washing, suction filtration; And then adopt the immersion of neutral salt acid salt solution, washing, suction filtration, obtain chlorion type resin anion(R.A);
Preferably, described alcoholic solution is anhydrous methanol or dehydrated alcohol, preferably dehydrated alcohol;
Preferably, described strong acid solution is HCl or H
2sO
4solution; The concentration of described strong acid solution is 1～3M preferably, more preferably 1M; The HCl solution of the preferred 1M of described strong acid solution;
Preferably, described strong base solution is NaOH or KOH solution; The concentration of described strong base solution is 1～3M preferably, more preferably 1M; The NaOH solution of the preferred 1M of described strong base solution;
Preferably, described neutral salt acid salt solution is NaCl or KCl solution; The concentration of described neutral salt acid salt solution is 0.5～3M, preferably 1M; The NaCl solution of the preferred 1M of described neutral salt acid salt solution.
Step 1b, be placed in by the chlorion type resin anion(R.A) and the adenylic deaminase solution that obtain the immobilization of carrying out adenylic deaminase on shaking table, 20～35 ℃ of set temperatures, and washing after taking out, be fixed on resin adenylic deaminase;
Preferably, before step 1b, adenylic deaminase solution is placed in to buffered soln, regulating pH is 5～8, and preferably pH is 5～6; Preferably, described buffered soln is acetate buffer solution, buffered with bicarbonate solution, borate buffer solution or Tris-HCl buffered soln.
Further, described step 2 specifically comprises:
The substrate adenylic acid (AMP) is dissolved in deionized water, regulating pH is 5～8, preferably pH is 5～6,40～55 ℃ of preheating 10-30min, preferred 10min, then add the immobilization adenylic deaminase obtained in step 1, then 40～55 ℃ of insulations, 50～120 rev/mins of stirrings, make the adenylic acid (AMP) reaction generate inosinic acid;
Preferably, the substrate adenylic acid (AMP) is dissolved in deionized water, adopts the HCl solution of 1～3M or the acetic acid solution of 3～6M, preferably the HCl solution of 3M is regulated pH.
Further, the concentration of described substrate adenylic acid (AMP) is 100～300g/L.
Further, described production method also comprises the regeneration step of resin anion(R.A).
Further, described regeneration comprises with alkali cleaning exhaustion of yin ion exchange resin, and then adopts NaCl solution, preferably the NaCl solution of 1M soaked, washing, suction filtration, change the oh type resin anion(R.A) into chlorion type resin anion(R.A).
Be below the detailed description of technical solution of the present invention:
Ultimate principle of the present invention is as follows: the iso-electric point of adenylic deaminase protein molecular, in 5.6 left and right, shows that the electronegative state of protein molecule is the advantage form in neutrality and slightly acidic environment, can form ionic linkage with positively charged ion.Utilize this mechanism, the present invention fixes adenylic deaminase by the resin anion(R.A) of selection, to reach efficiently enzyme effect admittedly, thereby utilizes the immobilization adenylic deaminase to realize the semicontinuous production of inosinic acid.
Wherein, the adenylic deaminase that the present invention adopts is commercial desaminase, and this enzyme can be made by honey aspergillus fermentation purifying, and the unit enzyme is lived as 10U/ml enzyme liquid (generating the required enzyme amount of 1mg inosinic acid under the 1U=experiment condition in per minute).
The present invention carries out alcohol-alkali-acid-alkali to the resin anion(R.A) of selecting and processes, use successively alcohol, highly basic, strong acid, highly basic carries out pre-treatment to resin anion(R.A), remove resin anion(R.A) residual alcohol in preparation process, strong acid and highly basic soluble impurity, finally with hydrochloride solution, resin anion(R.A) is made the transition, then the resin after transition is placed in to the acetate buffer solution of pH5～6, buffered with bicarbonate solution, carry out the immobilization of adenylic deaminase in borate buffer solution or Tris-HCl buffered soln, because the adenylic deaminase protein molecular is electronegative in neutral and slightly acidic solution environmental, therefore can with the Cl on resin anion(R.A) surface
-exchanged, realized the immobilization of zymoprotein.
Concrete immobilization step is as follows:
Steps A, take the resin anion(R.A) of certain mass in container, adds anhydrous alcohol solution, soaked overnight, adopt deionized water wash to residual without alcohol in elutriant, then suction filtration, wherein said absolute alcohol alcoholic solution is anhydrous methanol or dehydrated alcohol, preferably dehydrated alcohol;
Step B, the strong base solution soaked overnight by the resin that obtains in steps A with the preferred 1M of 1～3M, adopt deionized water wash to elutriant to be pH neutrality, suction filtration then, wherein said strong base solution is NaOH or KOH solution, preferably the NaOH solution of 1M;
Step C, the strong acid solution soaked overnight by the resin that obtains in step B with the preferred 1M of 1～3M, adopt deionized water wash to elutriant to be pH neutrality, suction filtration then, wherein said strong acid solution is HCl or H
2sO
4solution, preferably the HCl solution of 1M;
Step D, the strong base solution soaked overnight by the resin that obtains in step C with the preferred 1M of 1～3M, adopt deionized water wash to elutriant to be pH neutrality, suction filtration then, wherein said strong base solution is NaOH or KOH solution, preferably the NaOH solution of 1M;
Step e, neutral salt acid salt solution soaked overnight by the resin that obtains in step D with the preferred 1M of 0.5～3M, adopt deionized water wash to elutriant to be pH neutrality, then suction filtration, obtain chlorion type resin anion(R.A), wherein said hydrochloride solution is NaCl or KCl solution, preferably the NaCl solution of 1M;
Step F, by the chlorion type resin anion(R.A) that obtains in step e and the adenylic deaminase solution of certain volume, be placed in 20～35 ℃, and immobilization 2h on the shaking table of 150～250rpm lives and protein residue without enzyme to elutriant with deionized water wash after taking-up;
Preferably, before step F, adenylic deaminase solution is placed in to buffered soln, regulating pH is 5～8, and preferably pH is 5～6; Preferably, described buffered soln is acetate buffer solution, buffered with bicarbonate solution, borate buffer solution or Tris-HCl buffered soln;
Step G, the immobilized enzyme that step F is obtained is preserved under 4 ℃, being fixed adenylic deaminase.
According to immobilized enzyme method provided by the invention, the enzyme work of resulting immobilized enzyme can reach 10～23U/g wet resin.
The method of the enzyme activity determination in the present invention is as follows:
The immobilized enzyme that step 1g is obtained takes 0.3g after drying at room temperature, join 4ml in the substrate buffer solution (75g/L) of preheating, 55 ℃ of insulation 15min, then add the perchloric acid solution of 4ml7% to stop enzyme reaction, the content of inosinic acid in the Liquid Detection reaction solution.
Wherein, the acetic acid buffer solution system that the damping fluid adopted in the said determination method is 0.2M, pH is 5.6.
The Liquid Detection condition is as follows: moving phase is the 0.02M ammonium dihydrogen phosphate, contains 3.5% methyl alcohol (v/v), and flow velocity is 1ml/min, and chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm.
Further, the concrete preparation process of inosinic acid of the present invention is as follows:
Adenylic acid (AMP) is dissolved in deionized water, with the HCl solution of 1～3M or the acetic acid solution of 3～6M, preferably the HCl solution of 3M adjusting pH is preferably pH5～6, pH5～8, 40～55 ℃ of lower water-bath preheating 10～30min, preferred 10min, then add immobilization adenylic deaminase obtained above, 40～55 ℃ of water bath heat preservations, 50～120rpm mechanical stirring, the content of adenylic acid (AMP) and inosinic acid in timing sampling detection reaction liquid, to determine transforming degree, no longer descend or Liquid Detection goes out peak-to-peak signal less than inosinic acid to OD265nm, now think that adenylic acid (AMP) all is converted into inosinic acid, finish a production cycle.
Wherein, need strictly to control pH in above-mentioned reaction process, pH is stabilized in the preferred pH5 in pH5～8～6 scopes.
Wherein, after a production cycle finishes, can, by immobilization adenylic deaminase deionized water drip washing of the present invention, then put into the next production cycle or prepare against follow-up use in 4 ℃ of preservations.
In addition, when immobilization adenylic deaminase of the present invention, when repeatedly reacting catalytic performance reduces, can the de-mode of alkali cleaning be regenerated by resin anion(R.A), then adopt hydrochloride to be made the transition the resin anion(R.A) after regeneration, to reuse.
Compared with prior art, the production method of inosinic acid provided by the invention at least has the following advantages:
One, the present invention be take enzyme immobilization technology as basis, take anionite-exchange resin as fixation support to being fixed of adenylic deaminase (EC3.5.4.6), produce inosinic acid, can realize the semicontinuous conversion production of adenylic acid (AMP) to inosinic acid, with chemical synthesis process, compare, reduce the requirement to equipment, reduced the input of fixed capital;
Two, the present invention adopt in Nucleotide industry to cross production thing adenylic acid (AMP) be substrate, substrate conveniently is easy to get (account for Nucleotide total amount 25%), in liberal supply, can realize the catalyzed conversion under high density, and can make substrate reach the transformation efficiency more than 99.99%, be convenient to follow-up separating-purifying; The inosinic acid demand is in short supply, therefore take adenylic acid (AMP) as substrate, generates inosinic acid through adenylic deaminase catalysis significant;
Three, the present invention is fixed to resin surface by adsorption by adenylic deaminase, process for fixation is simple and easy to do, improved the catalytic stability of adenylic deaminase, and the immobilization cost is low, with resolvase catalysis, compare, this enzyme immobilization technology makes the optimal pH of adenylic deaminase broaden, and is more applicable for the catalytic process of adenylic acid (AMP) deamination;
Four, adenylic deaminase of the present invention can repeatedly recycle by the process for fixation in the present invention, be conducive to reduce costs, and do not use cross-linking reagent in immobilization process, in production process, impurity is few, make production more green, and be conducive to reduce later stage separation and Extraction cost.
The accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 has shown the change curve of the catalytic reaction activity of the adenylic deaminase that employing Amberlite IRA-900 resin is fixing with reaction batch.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
Reagent materials in following embodiment etc., if no special instructions, be commercially available purchase product.
embodiment 1
The pre-treatment of resin: Amberlite IRA-900 resin is carried out to pre-treatment according to following method steps:
Step 1a, take 200g Amberlite IRA-900 resin in the 1L Erlenmeyer flask, adds the 600ml dehydrated alcohol, soaked overnight, adopt deionized water wash in elutriant without alcohol residue, suction filtration then;
Step 1b, spend the night the resin obtained in step 1a by 600ml 1M NaOH solution soaking, adopt deionized water wash to elutriant to be pH neutrality, then suction filtration;
Step 1c, spend the night the resin obtained in step 1b by 600ml 1M HCl solution soaking, adopt deionized water wash to elutriant to be pH neutrality, then suction filtration;
Step 1d, spend the night the resin obtained in step 1c by 600ml 1M NaOH solution soaking, adopt deionized water wash to elutriant to be pH neutrality, then suction filtration;
Step 1e, spend the night the resin obtained in step 1d by 600ml 0.5M NaCl solution soaking, adopt deionized water wash to elutriant to be pH neutrality, and then suction filtration, obtain pretreated AmberliteIRA-900 resin.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-900 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add acetate buffer solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (21.98U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 50rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 1.25h, OD265nm no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch, so reuses the immobilization desaminase, and after using for the 30th time, batch reaction times extends to 1.40h.
Fig. 1 has shown the change curve of the catalytic reaction activity of the adenylic deaminase that employing Amberlite IRA-900 resin is fixing with reaction batch, as can be seen from Figure 1, increase along with reaction batch, the catalytic reaction activity of adenylic deaminase reduces gradually, after 30 secondary responses, the catalytic reaction activity of adenylic deaminase is reduced to approximately 89.29%.
embodiment 2
The pre-treatment of resin: Amberlite IRA-93 resin is carried out to pre-treatment according to the method identical with embodiment 1.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-93 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add acetate buffer solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (13.17U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 50rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 2.5h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 3.21h.
embodiment 3
The pre-treatment of resin: Amberlite IRA-400 resin is carried out to pre-treatment according to the method identical with embodiment 1.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-400 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add acetate buffer solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (14.37U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 60rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 3.25h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 4.51h.
embodiment 4
The pre-treatment of resin: according to the method similar to embodiment 1, Amberlite IRA-900 resin is carried out to pre-treatment, the NaOH that different is in step 1b-1d and the concentration of HCl solution are 2M, and the concentration of the NaCl solution in step 1e is 1M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-900 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add buffered with bicarbonate solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (20.98U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), make adenylic acid (AMP) at 40 ℃, under 80rpm mechanical stirring condition, carry out deamination reaction, by dripping the 3M hydrochloric acid soln, to control pH be approximately 5.6.After about 1.28h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 1.45h.
embodiment 5
The pre-treatment of resin: Amberlite IRA-93 resin is carried out to pre-treatment according to the method similar to embodiment 1.The NaOH that different is in step 1b-1d and the concentration of HCl solution are 2.5M, and the concentration of the NaCl solution in step 1e is 1.5M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-93 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add buffered with bicarbonate solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (13.27U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 50rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 2.5h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 3.21h.
embodiment 6
The pre-treatment of resin: according to the method similar to embodiment 1, Amberlite IRA-400 resin is carried out to pre-treatment, the NaOH that different is in step 1b-1d and the concentration of HCl solution are 1.5M, and the concentration of the NaCl solution in step 1e is 1M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-400 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add borate buffer solution, regulating pH is 5～6), triangular flask is placed in to 20 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (14.37U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 60rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 3.25h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 4.52h.
embodiment 7
The pre-treatment of resin: according to the method similar to embodiment 1, Amberlite IRA-900 resin is carried out to pre-treatment, the NaOH that different is in step 1b-1d and the concentration of HCl solution are 3M, and the concentration of the NaCl solution in step 1e is 2M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-900 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add Tris-HCl buffered soln, regulating pH is 5～6), triangular flask is placed in to 25 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (22.98U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 100rpm, carry out deamination reaction, is 5～6 by dripping 5M acetic acid solution control pH.After about 1.15h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 1.29h.
embodiment 8
The pre-treatment of resin: Amberlite IRA-93 resin is carried out to pre-treatment according to the method similar to embodiment 1.The NaOH that different is in step 1b-1d and the concentration of HCl solution are 3M, and the concentration of the NaCl solution in step 1e is 3M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-93 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add Tris-HCl buffered soln, regulating pH is 5～6), triangular flask is placed in to 30 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (13.56U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 50rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 2.48h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 3.26h.
embodiment 9
The pre-treatment of resin: according to the method similar to embodiment 1, Amberlite IRA-400 resin is carried out to pre-treatment, the NaOH that different is in step 1b-1d and the concentration of HCl solution are 2M, and the concentration of the NaCl solution in step 1e is 3M.
Catalyzed reaction: take above-mentioned pretreated Amberlite IRA-400 resin 10g in the triangular flask of 100ml, then add 50ml adenylic deaminase solution (10U/ml, wherein add Tris-HCl buffered soln, regulating pH is 5～6), triangular flask is placed in to 35 ℃, reclaim immobilized enzyme after immobilization 2h on the shaking table of 150rpm, adopt deionized water to be washed immobilized enzyme, until live residual without albumen and enzyme in elutriant.The immobilized enzyme (15.37U/g) obtained is joined to 100ml (100g/L in the adenylic acid (AMP) solution of 40 ℃ of preheatings, pH5～6), making adenylic acid (AMP) at 40 ℃, under the mechanical stirring condition of 60rpm, carry out deamination reaction, is 5～6 by dripping 1M hydrochloric acid soln control pH.After about 3.10h, OD265 no longer descends, (liquid-phase condition: moving phase is the 0.02M ammonium dihydrogen phosphate to Liquid Detection, contain 3.5% methyl alcohol (v/v), flow velocity is 1ml/min, chromatographic column is the hydrophilic post of C18, and the detector wavelength is 254nm) less than the inosinic acid signal, the transformation efficiency of adenylic acid (AMP) is 99.99 above %.Immobilized enzyme leaches rear employing deionized water wash three times, puts into next batch.So reuse the immobilization desaminase, after using for the 30th time, batch reaction times extends to 4.48h.
Similar to embodiment 4, difference be in catalytic reaction process during immobilization adenylic deaminase solution used be 30ml, immobilized enzyme unit's enzyme is lived as 17.86U/g.The 1st batch of reaction times is about 1.51h, and the 30th batch of reaction times is about 1.71h.
embodiment 11
Similar to embodiment 5, difference is that to regulate pH with the 3M acetic acid solution in catalytic reaction process be 5～6.
embodiment 12
Similar to embodiment 6, difference is in catalytic reaction process, to make adenylic acid (AMP) carry out deamination reaction under 55 ℃.The 1st batch of reaction times is about 2.51h, and the 30th batch of reaction times is about 3.49h.
embodiment 13
Similar to embodiment 1, difference is to reclaim immobilized enzyme in catalytic reaction process under 30 ℃, and immobilized enzyme unit's enzyme is lived as 23.56U/g.The 1st batch of reaction times is about 1.17h, and the 30th batch of reaction times is about 1.31h.
embodiment 14
Similar to embodiment 8, difference is that to regulate pH with the 6M acetic acid solution in catalytic process be 5～6.
Similar to embodiment 1, difference is, in step 1b-1d, resin is carried out to the H that pretreated soda acid is respectively 2M
2sO
4the NaOH solution of solution and 2M, the KCl solution that in step 1e, ion exchange resin conversion hydrochloride solution used is 1M.
embodiment 16
Similar to embodiment 2, difference is, in step 1b-1d, resin is carried out to the H that pretreated soda acid is respectively 3M
2sO
4the NaOH solution of solution and 3M, the KCl solution that in step 1e, ion exchange resin conversion hydrochloride solution used is 3M.
embodiment 17
Similar to embodiment 1, when difference is catalyzed reaction, the concentration of adenylic acid (AMP) solution used is 150g/L.
embodiment 18
Similar to embodiment 8, when difference is catalyzed reaction, the concentration of adenylic acid (AMP) solution used is 250g/L.
embodiment 19
Similar to embodiment 11, when difference is catalyzed reaction, the concentration of adenylic acid (AMP) solution used is 300g/L.
Above specific description of embodiments of the present invention does not limit the present invention, and those skilled in the art can make according to the present invention various changes or distortion, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (8)
1. the production method of an inosinic acid, described method comprises and adopts the adenylic deaminase catalysis adenylic acid (AMP) that is fixed in resin anion(R.A) to generate inosinic acid.
2. production method according to claim 1, is characterized in that, described resin anion(R.A) is skeleton for take styrene polymer or acrylate copolymer, take the resin that primary amine groups, tertiary amine groups or quaternary amine base be functional group;
Preferably, described resin anion(R.A) is for take styrene polymer as skeleton, take the season amino resin that is functional group; Further preferably, described resin anion(R.A) is for take vinylbenzene-divinylbenzene as skeleton, the resin that the tertiary amino of take is functional group; Perhaps take vinylbenzene-divinylbenzene as skeleton, the resin that the quaternary ammonium group of take is functional group;
Further preferably, described resin anion(R.A) is Amberlite IRA-900, Amberlite IRA-93 or Amberlite IRA-400 resin.
3. production method according to claim 1 and 2, is characterized in that, described production method specifically comprises:
Step 1, carry out pre-treatment to resin anion(R.A), and adenylic deaminase is fixed in to resin anion(R.A);
Step 2, be dissolved to the substrate adenylic acid (AMP) in deionized water, then add the immobilization adenylic deaminase obtained in step 1, makes the adenylic acid (AMP) reaction generate inosinic acid.
4. production method according to claim 3, is characterized in that, described step 1 specifically comprises:
Step 1a, adopt alcoholic solution to soak, wash resin anion(R.A), suction filtration, and then through strong acid solution immersion, washing, suction filtration, then through strong base solution immersion, washing, suction filtration; And then adopt the immersion of neutral salt acid salt solution, washing, suction filtration, obtain chlorion type resin anion(R.A);
Preferably, described alcoholic solution is anhydrous methanol or dehydrated alcohol, preferably dehydrated alcohol;
Preferably, described strong acid solution is HCl or H
2sO
4solution; The concentration of described strong acid solution is 1～3M preferably, more preferably 1M; The HCl solution of the preferred 1M of described strong acid solution;
Preferably, described strong base solution is NaOH or KOH solution; The concentration of described strong base solution is 1～3M preferably, more preferably 1M; The NaOH solution of the preferred 1M of described strong base solution;
Preferably, described neutral salt acid salt solution is NaCl or KCl solution; The concentration of described neutral salt acid salt solution is 0.5～3M, preferably 1M; The NaCl solution of the preferred 1M of described neutral salt acid salt solution;
Step 1b, be placed in by the chlorion type resin anion(R.A) and the adenylic deaminase solution that obtain the immobilization of carrying out adenylic deaminase on shaking table, 20～35 ℃ of set temperatures, and washing after taking out, be fixed on resin adenylic deaminase;
Preferably, before step 1b, adenylic deaminase solution is placed in to buffered soln, regulating pH is 5～8, and preferably pH is 5～6; Preferably, described buffered soln is acetate buffer solution, buffered with bicarbonate solution, borate buffer solution or Tris-HCl buffered soln.
5. according to the described production method of claim 3 or 4, it is characterized in that, described step 2 specifically comprises:
The substrate adenylic acid (AMP) is dissolved in deionized water, regulating pH is 5～8, preferably pH is 5～6,40～55 ℃ of preheating 10～30min, preferred 10min, then add the immobilization adenylic deaminase obtained in step 1, then 40～55 ℃ of insulations, 50～120 rev/mins of stirrings, make the adenylic acid (AMP) reaction generate inosinic acid;
Preferably, the substrate adenylic acid (AMP) is dissolved in deionized water, adopts the HCl solution of 1～3M or the acetic acid solution of 3～6M, preferably the HCl solution of 3M is regulated pH.
6. according to the described production method of any one in claim 1-5, it is characterized in that, the concentration of described substrate adenylic acid (AMP) is 100～300g/L.
7. according to the described production method of any one in claim 1-6, it is characterized in that, described production method also comprises the regeneration step of resin anion(R.A).
8. production method according to claim 7, it is characterized in that, described regeneration comprises with alkali cleaning exhaustion of yin ion exchange resin, and then adopts NaCl solution, preferably the NaCl solution of 1M soaked, washing, suction filtration, change the oh type resin anion(R.A) into chlorion type resin anion(R.A).
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