CN1041327C - Fermentation method for prodn. of sodium fructose diphosphate - Google Patents

Abstract

The present invention relates to a microorganism fermentation production method for FDP, which can enhance the yield and the purity of FDP. The method comprises the following steps; dextrose and sodium dihydrogen phosphate which are used as matrixes are fermented by beer yeast, bromogeramine as an aldolase inhibitor is added to the dextrose and the sodium dihydrogen phosphate to promote the accumulation of the FDP, the fermentation liquor is directly extracted on an anion resin column after being diluted, impurities are eluted by dilute hydrochloric acid, and the FDP is crystallized and purified by ethanol to form FDP trisodium salt with eight crystal water molecules. In the present invention, the fermentation conversion rate reaches 88% when measured by the inorganic phosphorus consumption, the fermentation unit of the FDP reaches about 40000r, the product purity of the FDP reaches 99%, and the success ratio reaches 98.6% after tens of batches of continuous pilot scale productions; the present invention has stable technique and is suitable for industrial production.

Description

The Fructose Diphosphate fermentation method for producing

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.

Embodiment 1 is glucose 50kg, SODIUM PHOSPHATE, MONOBASIC 30kg, cereuisiae fermentum slurry 200kg, benzalkonium bromide 100g, add water to cumulative volume 900L, PH6.7,37 ℃ of ferment at constant temperature, stopped fermentation in 10 hours, 5 times of fermented liquid dilutions, last 717 resin anion(R.A) posts absorption is with 0.01M dilute hydrochloric acid wash-out impurity, 6% sodium-chlor is resolved FDP, desorbed solution filters through decolorizing with activated carbon, and filtrate is transferred PH5.5, add 2 times of amount volume of ethanol, the washing with alcohol FDP soup compound of 60% concentration, static crystallization 10 hours, crystallization is through centrifuge dehydration, oven dry below 37 ℃, its result: 1. Fa Jiao inorganic phosphorus transformation efficiency 85%; 2.FDP fermentation unit 36000 γ; 3. extract yield 85%; 4. crystal refining obtains FDP trisodium salt crystallization 20.5kg; 5. purity 99.5%.

Embodiment 2 is with glucose 60kg, SODIUM PHOSPHATE, MONOBASIC 35kg, cereuisiae fermentum slurry 250kg, benzalkonium bromide 150g, cumulative volume 900L, PH7.0,37 ℃ to 40 ℃ fermentations stopped fermenting in 12 hours, 7 times of fermented liquid dilutions, last 717 resin anion(R.A) posts absorption, with 0.008M dilute hydrochloric acid wash-out impurity, 6.5% sodium-chlor is resolved FDP, desorbed solution is through activated carbon decolorizing, filtrate is transferred PH5.6, adds 1.8 times of ethanol and separates out the FDP soup compound, with the washing with alcohol FDP soup compound of 60% concentration, leave standstill crystallization in 12 hours, crystallization is dried below 37 ℃ through the washing centrifuge dehydration, and its result is as follows:

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%.

Embodiment 3 is glucose 45kg, SODIUM PHOSPHATE, MONOBASIC 25kg, and cereuisiae fermentum slurry 180kg, benzalkonium bromide 200g, cumulative volume 1000L, PH7.0,37 ℃～40 ℃ fermentations stopped fermentation in 15 hours.6 times of last 717 resin anion(R.A) posts absorption of fermented liquid dilution, with 0.012M dilute hydrochloric acid wash-out impurity, resolve FDP with 5% sodium-chlor, desorbed solution is through activated carbon decolorizing and filter filtrate accent PH5.54, add 1.8 times of volume ethanol, the FDP soup compound of separating out left standstill crystallization 16 hours with the washing with alcohol of 60% concentration, and crystallization is through washing, centrifuge dehydration, oven dry below 37 ℃, its result is as follows:

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%.

Claims (1)

1,1, the fermentation method for producing of 6 hexose diphosphate trisodiums comprises that microbial fermentation is synthetic, ion exchange method is extracted, refining three steps of solvent crystallization, and it is characterized in that: (1) is in the microbial fermentation synthesis step, use beer yeast fermenting, use benzalkonium bromide

The control fermenting process wherein when the reaction beginning, is pressed benzalkonium bromide weight and fermentation

The long-pending ratio of liquid is that the ratio of 0.01～0.02% (w/v) adds benzalkonium bromide, in reaction

At last, add the benzalkonium bromide of 2～4 times of beginning dosages with termination reaction; (2) in the ion exchange method extraction step, with 717 resin anion(R.A)s absorption, 1,6 two phosphorus

Tart fruit sugar trisodium, the impurity that adsorbs with the dilute hydrochloric acid wash-out of 0.008～0.012M; (3) in the solvent crystallization purification step, desorbed solution is transferred PH5.5-5.6 with sodium hydroxide,

Aqueous ethanolic solution precipitation, washing Trisodium fructose 1,6-diphosphate pulpous state with 60～70% concentration

Thing and crystallization.

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