CN103255181B - Method for catalyzing inulin fructose and cassava glucose to produce high-concentration sorbitol and gluconic acid by using immobilized movement fermented monas - Google Patents
Method for catalyzing inulin fructose and cassava glucose to produce high-concentration sorbitol and gluconic acid by using immobilized movement fermented monas Download PDFInfo
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- CN103255181B CN103255181B CN201210039707.4A CN201210039707A CN103255181B CN 103255181 B CN103255181 B CN 103255181B CN 201210039707 A CN201210039707 A CN 201210039707A CN 103255181 B CN103255181 B CN 103255181B
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Abstract
The invention relates to a method for catalyzing inulin fructose and cassava glucose to produce high-concentration sorbitol and gluconic acid by using immobilized movement fermented monas. The method comprises specific steps that: (1) a high-concentration equimolar inulin fructose and cassava glucose mixed hydrolysate is prepared with an enzymatic method; (2) cell immobilization of recombination movement fermented monas is carried out; and (3) the immobilized recombination movement fermented monas is used for simultaneously catalyzing fructose and glucose, such that a mixed product comprising high-concentration sorbitol and gluconic acid is obtained. The method provided by the invention has the advantages that: one glucoamylase is used for synchronously hydrolyzing inulin and cassava starch, such that equimolar fructose and glucose are produced; raw material cost is low, enzyme application amount is reduced, and production process is simplified; immobilized cells are circulated in catalyzing fructose and glucose to produce sorbitol and gluconic acid, such that catalyst dose is reduced, and continuous production of cell catalyst is avoided; the high-concentration sorbitol and gluconic acid mixed liquid is obtained, such that subsequent product separation cost is reduced.
Description
[technical field]
The present invention relates to biology manufacture and the biorefinery field of bio-based chemical, specifically, be that to take inulin and tapioca (flour) be raw material, utilize the method for the volumetric molar concentration inulin fructoses such as restructuring zymomonas mobilis catalysis and tapioca (flour) glucose production high density sorbyl alcohol and gluconic acid.
[background technology]
Sorbyl alcohol is a kind of broad-spectrum industrial chemicals, is mainly used in the production and preparation of sweeting agent, wetting agent, adjusting material, tenderizer etc.The industrial production technology of sorbyl alcohol is to adopt 50% D-Glucose slurry catalytic hydrogenation process at present, and it is a highly energy-consuming that still this chemical method is produced sorbyl alcohol, cost recovery is high and produce the technique of environmental pollution.And biological process production sorbyl alcohol is mainly to utilize dextrose fructose redox enzymatic conversion fructose and glucose in zymomonas mobilis periplasmic space to generate, reaction process is simple, and reaction conditions is gentle, without high-tension apparatus, and save energy, and environmentally friendly.But these two kinds of matrix of fructose and glucose are on the high side with respect to product, therefore use some cheap alternative materials can greatly reduce the cost that biological process is produced sorbyl alcohol.
Inulin is the mixture by a class natural fruit glycan, is to pass through β-2 by fructose units, and the carbohydrate that 1-glycosidic link is formed by connecting and stops with glucose unit, is mainly present in the stem tuber of the plants such as jerusalem artichoke, witloof.At present, the hydrolysis of inulin is mainly to utilize inulinase, cuts off β-2 of inulin, and 1-glycosidic link, is hydrolyzed to fructose and glucose.Inulin can be used for producing fructose, ethanol, single cell protein, Unicell Oils and Fats, citric acid, levan and other biological chemical products.And tapioca (flour) is also widely used in producing the products such as dry lactic acid, citric acid at present.Utilize these reproducible, cheap, abundant biomass materials of inulin, jerusalem artichoke or tapioca (flour) to produce biochemicals and there is good advantage.
In recent years, the research that utilizes jerusalem artichoke or inulin to produce sorbyl alcohol has some reports.After the people such as Dong-man fix the zymomonas mobilis of inulinase and osmotic treated altogether, catalysis jerusalem artichoke and glucose are produced sorbyl alcohol.The people such as Bao Jie points out to utilize the acid hydrolysis liquid of jerusalem artichoke to add after appropriate glucose in addition, with zymomonas mobilis catalysis jerusalem artichoke hydrolyzed solution, can obtain sorbyl alcohol and gluconic acid (patent No. 200910199317).But, in these researchs, first cell is carried out after toluene, cetyl trimethylammonium bromide or metal ion treatment to catalytic test, think the fresh cell without processing, at direct catalytic production sorbyl alcohol and gluconic acid, be to have a large amount of by product ethanol to produce, and cannot carry out production efficiency and the production cost that iterative cycles utilization can affect whole process.Meanwhile, the concentration that these researchs obtain sorbyl alcohol and gluconic acid is lower, has increased the cost of subsequent products separation.We attempt new fresh cell being come to gluconic acid and the sorbyl alcohol of catalysis high density through immobilization, and raw materials cost is low, has simplified production technique; Reduced the cost of subsequent product separation.
[summary of the invention]
The object of the invention is to utilize business saccharifying enzyme to be hydrolyzed tapioca (flour) and inulin simultaneously, under high inulin and tapioca (flour) substrate content, obtain and the fructose of volumetric molar concentration and the mixed hydrolysis liquid of glucose such as contain, then adopt the gluconic acid of immobilized fresh zymomonas mobilis catalytic production high density and the method for sorbyl alcohol.
Design of the present invention: from the method for inulin and tapioca (flour) production high concentration glucose acid and sorbyl alcohol, specifically, first by tapioca (flour) liquefaction, then lower the temperature and adjust pH.Utilize after business Glucoamylase hydrolysis inulin certain hour, add hydrolysis of inulin liquid to carry out common saccharification the starch after liquefaction, to obtain the fructose of volumetric molar concentrations such as containing and the mixed hydrolysis liquid of glucose.Have the zymomonas mobilis of dextrose fructose oxydo-reductase to carry out cell fixation expression, then the fructose of volumetric molar concentration such as the immobilized zymomonas mobilis catalysis of recycle and the mixed solution of glucose are produced sorbyl alcohol and gluconic acid.
The object of the invention is to be achieved through the following technical solutions:
Utilize a method for immobilization zymomonas mobilis catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid, its concrete steps are,
(1) enzyme process of the equimolar inulin fructose of high density and cassava glucose mixed hydrolysis liquid preparation;
First, the inulin that is 50% by mass concentration enzymolysis 48 hours under 60 ℃, the condition of pH 4.0, glucoamylase enzyme amount 1U/g inulin, then add at 90 ℃ of liquefaction tapioca (flour) (massfraction is 50%) of 1 hour, under 60 ℃, the condition of pH 4.0, continue hydrolysis 12 hours, the fructose of the volumetric molar concentrations such as acquisition and the mixed hydrolysis liquid of glucose.Wherein the mass ratio of inulin and tapioca (flour) is determined according to the content of starch in the content of Polylevulosan in inulin and tapioca (flour), and principle is fructose and the glucose that the volumetric molar concentration such as obtains after abundant hydrolysis;
In the enzyme process preparation of the equimolar inulin fructose of described high density and cassava glucose mixed hydrolysis liquid, saccharifying enzyme for enzymolysis inulin is mixed type business saccharifying enzyme, in this enzyme liquid, contain the plurality of enzymes albumen such as saccharifying enzyme and inulinase, but the enzyme liquid in the present invention is not limited to business saccharifying enzyme, also comprise saccharifying enzyme and inulinase mixed solution etc.;
(2) cell fixation of restructuring zymomonas mobilis;
The zymomonas mobilis that expression is had to a dextrose fructose oxydo-reductase is cultivated after 18 hours under stuffiness, 30 ℃, pH6.0,150rpm, inoculum size 10% (v/v) condition, and centrifugal acquisition wet cell is also suspended into 150g/L with ultrapure water.Then by the sodium alginate soln heating for dissolving of the PVA solution and 0.125% (w/v) of 12.5% (w/v), after being cooled to room temperature, mix (PVA solution and cell solution volume ratio 4: 1) with cell suspending liquid, mixing solutions is fully mixed to 1 hour under magnetic agitation effect.Then, mixing solutions is added drop-wise in saturated boric acid solution and (is contained 0.3mol/L calcium chloride) by peristaltic pump, and stir 3 hours.Making immobilized cell granulations mean diameter is 4 millimeters, and by a large amount of washed with de-ionized water to remove the boric acid solution of particle surface, i.e. the zymomonas mobilis cell of being fixed;
Described restructuring zymomonas mobilis is for expressing the zymomonas mobilis that has dextrose fructose oxydo-reductase, and its dextrose fructose oxydo-reductase enzyme work is than exceed 2 times in wild mushroom;
(3) utilize immobilization zymomonas mobilis while catalysis fructose and glucose to obtain the mixed solution that contains high density sorbyl alcohol and gluconic acid;
In reactor, add the mixed hydrolysis liquid and the immobilized zymomonas mobilis cell that prepare, under the condition of pH 6.4,39 ℃ of temperature, 150rpm, carry out catalysis, obtain the mixed solution that contains high density sorbyl alcohol and gluconic acid.After each catalysis finishes, carry out solid-liquid separation, liquid carries out next step sorbyl alcohol and the separated and purifying of gluconic acid, and solid cell particle is repeatedly cycled to used in catalyzed reaction.
Described is the mixing solutions of sodium alginate of the PVA and 0.125% (w/v) of 12.5% (w/v) for the fixing solution of zymomonas mobilis.Utilize separately sodium alginate to fix fresh zymomonas mobilis, particle easily expands to dissolve and causes catalytic process to carry out; And PVA and acid reaction speed are slow, add the moulding that a small amount of sodium alginate contributes to PVA particle, the mass ratio that wherein PVA and sodium alginate are comparatively suitable is 10: 1.
Described initial fructose and the total sugar concentration 500g/L of glucose, fructose and glucose concn are respectively 250g/L, can suppress the generation of ethanol under this high glucose concentration; Cell concn is selected 10g/L, and under this concentration, catalysis can be carried out smoothly, thereby has reduced the usage quantity of cell catalyst.
It is described after conversion finishes each time, reaction system is carried out to solid-liquid separation, liquid carries out the product separation of sorbyl alcohol and gluconic acid, immobilized cell granulations is stayed in reactor, rejoin fresh hydrolyzed solution, carry out catalytic process next time, whole process immobilized cell can carry out circulation catalyzed reaction 1-4 time.
Compared with prior art, positively effect of the present invention is:
(1) to take inulin and tapioca (flour) be raw material production sorbyl alcohol and gluconic acid in the present invention, and matrix is with low cost, and wide material sources are abundant, take full advantage of renewable resources, can meet the market requirement that gluconic acid and sorbyl alcohol constantly expand;
(2) the present invention, for the saccharifying enzyme of hydrolytic inulin and tapioca (flour), is a business-like cheap saccharifying enzyme, can meet the needs of large-scale production;
(3) the present invention, by the hydrolysis combination of inulin and tapioca (flour), realizes synchronous glycosylation process, reduces reactor and uses, and saves production cost;
(4) the present invention carrys out new fresh cell sorbyl alcohol and the gluconic acid of catalysis high density through immobilization, not only reduces cell handling procedure, has reduced the consumption of catalyzer, and has avoided the continuous production of cell catalyst;
(5) sorbyl alcohol that the present invention obtains containing high density and the mixed solution of gluconic acid, reduced the cost of subsequent product separation.
[accompanying drawing explanation]
Fig. 1 embodiment 1 immobilized cell catalytic process graphic representation.
[embodiment]
Below provide the present invention to utilize the embodiment of the method for immobilization zymomonas mobilis catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid.
Embodiment 1
Refer to accompanying drawing 1, the zymomonas mobilis that expression is had to a dextrose fructose oxydo-reductase is cultivated after 18 hours under stuffiness, 30 ℃, pH 6.0,150rpm, inoculum size 10% (v/v) condition, and centrifugal acquisition wet cell is also suspended into 150g/L with ultrapure water.Then by the sodium alginate soln heating for dissolving of the PVA solution and 0.125% (w/v) of 12.5% (w/v), after being cooled to room temperature, mix (PVA solution and cell solution volume ratio 4: 1) with cell suspending liquid, mixing solutions is fully mixed to 1 hour under magnetic agitation effect.Then, mixing solutions is added drop-wise in saturated boric acid solution and (is contained 0.3mol/L calcium chloride) by peristaltic pump, and stir 3 hours.Making immobilized cell granulations mean diameter is 4 millimeters, and by a large amount of washed with de-ionized water to remove the boric acid solution of particle surface, i.e. the zymomonas mobilis cell of being fixed.In the reactor of 500 ml volumes, add 300 milliliters of hydrolyzed solutions and immobilized zymomonas mobilis particle, at pH 6.4 (14mol/L sodium hydroxide control pH), 39 ℃, 150rpm, carry out catalyzed reaction.In whole process, the concentration of sorbyl alcohol is 180g/L, and the transformation efficiency of sorbyl alcohol is 97.33%, and the productive rate of sorbyl alcohol is every gram of cell 0.492g per hour.
Raw material involved in the present invention is not limited to inulin and tapioca (flour), can also be the raw material of cassava, W-Gum class, the levan raw materials such as jerusalem artichoke, witloof.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (4)
1. a method of utilizing immobilization zymomonas mobilis while catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid, is characterized in that, concrete steps are
(1) enzyme process of the equimolar inulin fructose of high density and cassava glucose mixed hydrolysis liquid preparation;
First, the inulin that is 50% by mass concentration enzymolysis 48 hours under 60 ℃, the condition of pH4.0, glucoamylase enzyme amount 1U/g inulin, then add at 90 ℃ of liquefaction tapioca (flour) of 1 hour, under 60 ℃, the condition of pH4.0, continue hydrolysis 12 hours, the fructose of the volumetric molar concentrations such as acquisition and the mixed hydrolysis liquid of glucose;
(2) cell fixation of restructuring zymomonas mobilis;
The zymomonas mobilis that expression is had to a dextrose fructose oxydo-reductase is cultivated after 18 hours under stuffiness, 30 ℃, the condition of pH6.0,150rpm, inoculum size 10% (v/v), centrifugal acquisition wet cell is also suspended into 150g/L with ultrapure water, then by the sodium alginate soln heating for dissolving of the polyvinyl alcohol solution of 12.5% (w/v) and 0.125% (w/v), after being cooled to room temperature, mix with cell suspending liquid, wherein PVA solution and cell solution volume ratio 4:1 fully mix 1 hour by mixing solutions under magnetic agitation effect; Then, mixing solutions is added drop-wise in saturated boric acid solution by peristaltic pump, and stirs 3 hours; Making immobilized cell granulations mean diameter is 4 millimeters, and by washed with de-ionized water to remove the boric acid solution of particle surface, i.e. the zymomonas mobilis cell of being fixed;
(3) utilize immobilization zymomonas mobilis while catalysis fructose and glucose to obtain the mixed solution that contains high density sorbyl alcohol and gluconic acid;
In reactor, add the mixed hydrolysis liquid and the immobilized zymomonas mobilis cell that prepare, under the condition of pH6.4,39 ℃ of temperature, 150rpm, carry out catalysis, obtain the mixed solution that contains high density sorbyl alcohol and gluconic acid; After each catalysis finishes, carry out solid-liquid separation, liquid carries out next step sorbyl alcohol and the separated and purifying of gluconic acid, and solid cell particle is repeatedly cycled to used in catalyzed reaction;
After conversion finishes each time, reaction system is carried out to solid-liquid separation, liquid carries out the product separation of sorbyl alcohol and gluconic acid, immobilized cell granulations is stayed in reactor, rejoin fresh hydrolyzed solution, carry out catalytic process next time, whole process immobilized cell can carry out circulation catalyzed reaction 1-4 time.
2. a kind of simultaneously method of catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid of immobilization zymomonas mobilis of utilizing as claimed in claim 1, it is characterized in that, in described step (2), described restructuring zymomonas mobilis is for expressing the zymomonas mobilis that has dextrose fructose oxydo-reductase, and its dextrose fructose oxydo-reductase enzyme work is than exceed 2 times in wild mushroom.
3. a kind of simultaneously method of catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid of immobilization zymomonas mobilis of utilizing as claimed in claim 1, it is characterized in that, in described step (2), it for the fixing solution of zymomonas mobilis, is the mixing solutions of sodium alginate of the PVA and 0.125% (w/v) of 12.5% (w/v); The mass ratio that PVA and sodium alginate are comparatively suitable is 10:1.
4. a kind of simultaneously method of catalysis inulin fructose and cassava glucose production high density sorbyl alcohol and gluconic acid of immobilization zymomonas mobilis of utilizing as claimed in claim 1, it is characterized in that, in described step (3), the total sugar concentration 500g/L of initial fructose and glucose, be that fructose and glucose concn are respectively 250g/L, under this high glucose concentration, can suppress the generation of ethanol, cell concn is selected 10g/L.
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