CN1041327C - Fermentation method for prodn. of sodium fructose diphosphate - Google Patents
Fermentation method for prodn. of sodium fructose diphosphate Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 37
- 230000004151 fermentation Effects 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229930091371 Fructose Natural products 0.000 title claims description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 title claims description 5
- 239000005715 Fructose Substances 0.000 title claims description 5
- 239000001177 diphosphate Substances 0.000 title claims description 5
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 title claims description 3
- 235000011180 diphosphates Nutrition 0.000 title claims description 3
- 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 title 1
- 229910052708 sodium Inorganic materials 0.000 title 1
- 239000011734 sodium Substances 0.000 title 1
- 229960002233 benzalkonium bromide Drugs 0.000 claims abstract description 27
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000002425 crystallisation Methods 0.000 claims abstract description 19
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
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- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical compound [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 claims 2
- FOBPTJZYDGNHLR-UHFFFAOYSA-N diphosphorus Chemical compound P#P FOBPTJZYDGNHLR-UHFFFAOYSA-N 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
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- 235000019819 trisodium diphosphate Nutrition 0.000 claims 1
- MLIKYFGFHUYZAL-UHFFFAOYSA-K trisodium;hydron;phosphonato phosphate Chemical compound [Na+].[Na+].[Na+].OP([O-])(=O)OP([O-])([O-])=O MLIKYFGFHUYZAL-UHFFFAOYSA-K 0.000 claims 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 13
- 239000011574 phosphorus Substances 0.000 abstract description 13
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 13
- 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 abstract description 12
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- 238000009776 industrial production Methods 0.000 abstract description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 abstract description 2
- 239000003112 inhibitor Substances 0.000 abstract 1
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- CEHGLSKJDFICTB-PWVOXRODSA-K D-Fructose 1,6-bisphosphate trisodium salt Chemical compound [Na+].[Na+].[Na+].[O-]P(=O)([O-])OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)COP(O)([O-])=O CEHGLSKJDFICTB-PWVOXRODSA-K 0.000 description 14
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 6
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- 238000010521 absorption reaction Methods 0.000 description 4
- 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 description 4
- 229950006790 adenosine phosphate Drugs 0.000 description 4
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- 238000004879 turbidimetry Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 159000000009 barium salts Chemical class 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
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- 238000010586 diagram Methods 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
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- 238000005516 engineering process Methods 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
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
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- -1 vitriol Chemical class 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 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 description 1
- 206010009866 Cold sweat Diseases 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
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- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- Saccharide Compounds (AREA)
Abstract
本发明是一种能够提高FDP产率和纯度的FDP的微生物发酵生产方法。以葡萄糖，磷酸二氢钠为基质用啤酒酵母发酵，加入溴苄烷铵作为醛缩酶抑制剂，促进FDP的积累，发酵液稀释后直接上阴离子树脂柱提取，用稀盐酸洗脱杂质，用乙醇结晶纯化FDP，成为含八个结晶水FDP三钠盐。本发明发酵转化率以无机磷消耗计达到88%，FDP发酵单位达到40000r左右，FDP产品纯度达99%经数拾批次连续中试生产成功率98.6%，工艺稳定，适合于工业生产。The invention is a microbial fermentation production method of FDP capable of improving FDP yield and purity. Use glucose and sodium dihydrogen phosphate as the substrate to ferment with brewer’s yeast, add benzalkonium bromide as an aldolase inhibitor, and promote the accumulation of FDP. FDP was purified by ethanol crystallization and became the trisodium salt of FDP containing eight crystal waters. The fermentation conversion rate of the present invention reaches 88% based on inorganic phosphorus consumption, the FDP fermentation unit reaches about 40000r, the purity of FDP product reaches 99%, and the success rate of continuous pilot production after dozens of batches is 98.6%. The process is stable and suitable for industrial production.
Description
The invention belongs to the microbial fermentation technology field, is a kind of Trisodium fructose 1,6-diphosphate, i.e. the fermentation method for producing of 1.6 hexose diphosphate trisodium salts.
1.6 hexose diphosphate salt Fructose-1.6-Diphosphate is called for short FDP, wherein the FDP trisodium salt is a kind of sick first aid good medicine such as Acute Myocardial Infarction, cardiac insufficiency, coronary heart disease, myocardial ischemia outbreak, shock that is applied to.The fermentation method for producing of FDP mainly contains the method that method that Italian FOX card agate (FOCAMA) biochemical pharmacy company proposes and Japanese TOCHIKURA etc. propose.The method of FOSCAMA is to ferment together with matrix such as fresh yeast, glucose, Sodium phosphate dibasic, phosphoric acid, toluene, protein is removed with the salt Acid precipitation in the fermentation back, filtrate transfers PH to neutral, add calcium chloride and become the FDP calcium precipitation, water repetitive scrubbing throw out is removed soluble impurity, is transformed into sodium salt then, ultrafiltration is lyophilized into dry powder.The method of TOCHIKURA is with (AMP) 1g, dry yeast 15g such as phosphate buffered saline buffer 0.33M, glucose 27g, adenylic acids, cultivates 4 hours for 37 ℃, and based on the consumption of sugar, transformation efficiency is 85%.Handle with ion exchange method, obtain 23g purity and be 75% FDP barium salt, add adenylic acid (AMP), Triphosaden (ATP), guanylic acid or other purine derivatives in the fermentation and cultivate, can improve output with the phosphoric acid salt tile.The shortcoming of FOSCAMA method is, uses the O for toluene yeast, and the toluene volatility is big, and is toxic.When being processed into the FDP calcium precipitation with calcium chloride, other phosphate impurities have coprecipitation, are difficult for Ex-all, cause the FDP product purity not high.In addition, the very easily moisture absorption of the dried frozen aquatic products of FDP is difficult for packing and preserves, and freeze-drier is also complicated.The method shortcoming of TOCHIKURA is, materials such as the AMP that adds in the fermentation, ATP, guanylic acid are more, and cost is higher, ferments 4 hours, and the time is shorter, and it is big that FDP transforms fluctuation, and industrial production is difficult to grasp.Extract with ion exchange method, purity has only 75% as a result, and not only purity is low, and to obtain product be the FDP barium salt.
The object of the present invention is to provide a kind of fermentation method for producing that can improve FDP trisodium salt productive rate and product purity.
FDP trisodium fermentation method for producing provided by the invention comprises steps such as microbial fermentation is synthetic, ion exchange method is extracted, solution crystal process is refining.Wherein, as matrix, synthetic with beer yeast fermenting with phosphoric acid salt, glucose, as zymohexase vigor control agent, the control fermenting process is to improve the productive rate of FDP trisodium salt with benzalkonium bromide; Fermented liquid is directly gone up the resin anion(R.A) post and is extracted after the deionized water dilution, with impurity such as dilute hydrochloric acid wash-out phosphoric acid salt; Then,, use the alcohol solvent crystallization, promptly obtain to contain the FDP trisodium salt of eight molecular crystal water according to the PH scope of FDP concentration and sodium chloride concentration adjustment FDP trisodium salt.
In the fermentation step of aforesaid method, the component concentration of fermentation suspension is (g)/volume (ml) meter by weight, and its ratio is as follows: glucose 5 ± 1%, SODIUM PHOSPHATE, MONOBASIC 3 ± 1%, (or with other phosphoric acid salt such as phosphoric acid, Sodium phosphate dibasic etc.).Cereuisiae fermentum slurry 20 ± 5%, benzalkonium bromide 0.01～0.02% adds water to cumulative volume 100ml, and wherein fermented substrate liquid is transferred PH to 6.5～7.0 with sodium hydroxide, and batching finishes back adjustment temperature in 37 ℃ ± 3 ℃, carries out static fermentation.Measure the inorganic phosphorus drop-out value during the fermentation with the control course of fermentation, when treating that inorganic phosphorus drops to about 80% (being generally 80 ± 3%) of former phosphorus, add excessive benzalkonium bromide (for 2～4 times of initial consumption) again and stop fermentation, whole fermentation time generally is no less than 8 hours.
In the aforesaid method refining step, can be with fermented liquid with the dilution of 5～10 times deionized water, clarification naturally is adsorbed FDP with 717 resin anion(R.A)s on the method for surperficial siphon (Chinese Chemicals catalogue, nineteen ninety version) post.With the dilute hydrochloric acid wash-out impurity of 0.008～0.012M, wash the reaction of phosphate radical in the effluent liquid near (comparing with the dilute hydrochloric acid liquid that does not strip) till the feminine gender behind the upper prop, be 5～7% sodium chloride aqueous solution parsing FDP with concentration always then.With mensuration FDP content, and measure impurity such as phosphoric acid salt, vitriol, calcium salt, carry out intermediate controlled.
In the purification step of aforesaid method, can add about 0.5% decolorizing with activated carbon in the desorbed solution of FDP, desuperheating is former, filters.Filtrate is transferred PH to 5.5～5.6 with sodium hydroxide, under agitation add 1.6～2 times of volume of ethanol, making FDP be pulpous state separates out, separate soup compound, and be 60～70% ethanol with concentration, the repetitive scrubbing soup compound is till wherein muriate is qualified (being that chlorine ion concentration is no more than 1000ppm), then the FDP soup compound is moved to static or dynamic crystallization in the crystallizing tank, be generally more than 10 hours.To FDP crystallization concentration is that 60 ± 2% aqueous ethanolic solution grinds washing, is 95% alcohol immersion at last with concentration, centrifuge dripping, and crystallization is in oven dry below 37 ℃, i.e. and acquisition contains the FDP trisodium salt of 8 molecular crystal water, and total recovery reaches more than 80%,
The measuring method of controlling index is as follows among the present invention:
(1) determination of inorganic phosphorus: molybdenum blue colorimetric method.
(2) FDP assay enzyme process: Italian method provides.
(3) FDP assay neutralisation (PH titration): calculate FDP content according to the alkali that consumes between PH4.0～9.5.
(4) vitriol inspection: barium sulfate turbidimetry.
(5) muriate inspection: silver chloride turbidimetry.
(6) calcium salt inspection: caoxalate turbidimetry.
The present invention adds benzalkonium bromide in fermented liquid mechanism of action is to suppress the zymohexase position in the glycolysis-passage, and may be little to the active influence of hexokinase and phosphofructokinase and other enzyme.But the amount that adds benzalkonium bromide is wanted suitably, too many activity of zymohexase is suppressed fully, so that fermentation stops, and FDP no longer transforms.The add-on of benzalkonium bromide will suit, and stay a metabolism passage, a part of carbohydrate metabolism is continued transform the required energy ATP of FDP to provide.
From known carbohydrate metabolism approach as can be seen, the generation of FDP is not simple enzymatic reaction, but be accompanied by energy consumption, a glucose molecule phosphorylation generates FDP and need consume two molecule ATP, and the metabolism of a FDP molecule can be produced four molecule ATP to pyruvic acid, thereby have only a part of consumption FDP could satisfy generation two molecule FDP institute energy requirements, be essential so allow a part of FDP to decompose.
About glucose and phosphoric acid salt ingredient proportion, their weight ratio is 0.72: 1 from chemical reaction, is 1.5: 1 from biosynthesizing.But in real attenuation, consider energy expenditure in the factor aspect synthetic such as protein, cell, therefore, the consumption of glucose is often got about two times of phosphatic weight.
Benzalkonium bromide is to represent by the quantitative variation of zymohexase decomposition FDP to the effect of vigor of zymohexase among the present invention.The quantitative variation system of FDP causes that optical density(OD) that DPNH is varied to oxidized form of nicotinamide-adenine dinucleotide changes that △ E determines when decomposing FDP quantitatively.
6.22 * 10
3Be the molar absorptivity of DPNH at 430 μ m.
2 molecule number for a part FDP consumption nadide.
In the vitality test system of zymohexase, add a certain amount of benzalkonium bromide, the vigor of zymohexase is suppressed, and is shown in Figure 1, and the 0-0 line represents to add the situation of benzalkonium bromide, and △-△ line represents not add the situation of benzalkonium bromide.
Inorganic phosphorus variation per-cent serves as that the basis relatively gets with former phosphorus among the present invention, and Fig. 2 illustrates in glucose, phosphoric acid salt, yeast fermentation system, adds the variation that the different benzalkonium bromides of measuring cause inorganic phosphorus.Among the figure, *-* line represents the appropriate amount benzalkonium bromide.The 0-0 line is represented excessive benzalkonium bromide, and △-△ line represents not add benzalkonium bromide.
The PH scope of FDP trisodium salt is along with the change in concentration of FDP itself changes, and PH is about 5.6～6.0 during common 10% concentration, and in addition, FDP has a large amount of sodium-chlor to exist from the solution that resin is resolved, and the concentration of sodium-chlor also affects the PH of FDP.Therefore will be according to the concentration of FDP and the PH of sodium chloride concentration adjusting desorbed solution.PH according to the FDP as a result that records is appropriate to transfer 5.5～5.6, and such PH is with the ethanol sedimentation crystallization time, and FDP just in time becomes trisodium salt and crystallizes out.PH is too high, and FDP becomes tetra-na salt to separate out instability, the easy flavescence of colourity.PH is low excessively, and FDP becomes disodium salt to separate out, and is difficult for into solid crystal, and product is clamminess, and crystal water is unfixing, and stability is not good.
The present invention compared with prior art has following advantage:
1. transformation efficiency height.Add benzalkonium bromide in the fermentation FDP is accumulated in a large number, the unit concentration of FDP can reach more than 30000 γ.If do not add benzalkonium bromide, the accumulation of FDP only remains on general substrate level, is about 100～500 γ, does not generally have industry to extract and is worth.
2. purity height.Since extract with ion exchange method, dilute hydrochloric acid wash-out separating impurity, and refining through solvent crystallization, the purity of FDP reaches more than 99%.
3. the process stabilizing security is good.Because shifting to new management mechanisms of FDP is clear and definite, can in time add benzalkonium bromide during the fermentation, make FDP transform hit the target, guarantee high and stable yields.In addition, benzalkonium bromide can also suppress microbial contamination, brings very favourable condition for fermentation and refinement.
Fig. 1 is the influence synoptic diagram of benzalkonium bromide to aldolase activity.
Fig. 2 is that benzalkonium bromide influences synoptic diagram to inorganic phosphorus conversion in the fermenting process.
1. Fa Jiao inorganic phosphorus transformation efficiency 80%.2.FDP fermentation unit 35000 γ; 3. extract yield 86%; 4. crystal refining obtains FDP trisodium salt crystallization 20.8kg; 5. purity 99.4%.
1. Fa Jiao inorganic phosphorus transformation efficiency 83%; 2. fermentation unit (FDP) 30000 γ; 3. extract yield 85.5%, 4. obtains crystallization FDP trisodium salt 18.9kg, and 5. purity 99.7%.
Above technology is through repeating 70 batch quantity statistics: inorganic phosphorus transforms 83 ± 5.1%, and fermentation unit is in FDP (μ g/ml) 35000 ± 5000 γ, extract yield 84.96 ± 14%, product weight 19.1 ± 4.0kg, product purity 99 ± 1.0%.
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| CN1322000C (en) * | 2003-01-20 | 2007-06-20 | 赵建安 | Diabetin-1, 6-diphosphonie acid as well as preparation method and application |
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| CN1042953C (en) * | 1995-07-10 | 1999-04-14 | 暨南大学 | Preparation method of magnesium fructose diphosphate |
| CN1057299C (en) * | 1998-08-28 | 2000-10-11 | 重庆医科大学 | Sodium magnesium D-fructose-1,6-diphosphate and its preparation method and application |
| CN102154399A (en) * | 2010-12-30 | 2011-08-17 | 张家港市华天药业有限公司 | Production process flow of fructose diphosphate sodium |
| CN109223709B (en) * | 2018-10-25 | 2020-12-08 | 北京华靳制药有限公司 | Preparation method of fructose diphosphate sodium injection |
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| US4920049A (en) * | 1986-08-06 | 1990-04-24 | Bisso Guglielmo M | Process for the continuous production of fructose-1,6-di-phosphate by employ of immobilized yeast |
| EP0385486A1 (en) * | 1989-03-03 | 1990-09-05 | Unitika Ltd. | Process for producing fructose-1,6-diphosphate |
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| US4920049A (en) * | 1986-08-06 | 1990-04-24 | Bisso Guglielmo M | Process for the continuous production of fructose-1,6-di-phosphate by employ of immobilized yeast |
| EP0385486A1 (en) * | 1989-03-03 | 1990-09-05 | Unitika Ltd. | Process for producing fructose-1,6-diphosphate |
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| CN1322000C (en) * | 2003-01-20 | 2007-06-20 | 赵建安 | Diabetin-1, 6-diphosphonie acid as well as preparation method and application |
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