CN1690200A - Immobilization glucose amylase products and method for immobilization of glucose amylase - Google Patents
Immobilization glucose amylase products and method for immobilization of glucose amylase Download PDFInfo
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- CN1690200A CN1690200A CN 200410038809 CN200410038809A CN1690200A CN 1690200 A CN1690200 A CN 1690200A CN 200410038809 CN200410038809 CN 200410038809 CN 200410038809 A CN200410038809 A CN 200410038809A CN 1690200 A CN1690200 A CN 1690200A
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- glucoamylase
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Abstract
The invention discloses an immobilization glucoamylase goods and a method of fixation of glucoamylase with polyvinyl alcohol composite gel. The immobilization glucoamylase goods is obtained from cryo-forming the mixture of polyvinyl alcohol composite gel and glucoamylase in low temperature. And the content of polyvinyl alcohol in polyvinyl alcohol composite gel is 8 to 15 percent(mass percent ), the concentration of glucoamylase is 8 to 15 percent(mass percent), Every 100ml composite gel can bear the weight of 0.5 to 1.5 gram of glucoamylase enzyme powder. The polyvinyl alcohol immobilizing glucoamylase goods has perfect mechanical capability, operating stabilization and has a good industrial application foreground. It can apply in alcohol industry, food industry, wine brewing, the finely processed of starch and starch stuff, analyzing and measuring, and other related ermentation industry.
Description
Technical field:
The present invention relates to a kind of fixation glucose amylase goods and the diastatic method of fixing glucose.
Background technology:
Glucoamylase is widely used in starch syrup, glucose, wine brewing and the fermentation industry as a kind of important biological catalyst.At present, domestic industry all adopts the resolvase preparation in producing, and exists the product separation difficulty, causes the huge waste of raw material, has also increased production cost simultaneously.And the immobilized glucoamylase goods not only can separate catalyzer and product (or substrate) as solid-phase biocatalyst, but also can realize continuous operation and automatization control, have improved the quality and the yield of product.
The process for fixation of saccharifying enzyme mainly contains covalent bonds method, absorption method, entrapping method, co-immobilization and several process for fixation coupling.What wherein absorption method was used is less, mainly is because enzyme splits away off from carrier easily.It is more that the covalent bonds method is used, and immobilization efficiency is higher, but the cost of this kind method is higher, and difficulty is carried out large-scale industrial production.Entrapping method is a kind of enzyme immobilization method cheaply, simultaneously with method coupling such as other method one crosslinking, has not only improved the performance and the environment of carrier, and has improved the immobilization efficiency of enzyme.
The aperture of carrier can not be greater than the enzyme molecule when utilizing the using embedding immobilization saccharifying enzyme, in order to avoid saccharifying enzyme is revealed, on polyvinyl alcohol, enzyme loss alive is lower with the chemical crosslink technique immobilized glucoamylase for Thomas etc., but concerning polymer substrate starch, its apparent vigor is lower.What U.S. Pat 4794083 was introduced is to contain functional group NH
2-and the polymkeric substance of CO-after carbodiimide is handled, saccharifying enzyme is carried out immobilization.Polymkeric substance is synthetic by vinylformic acid, methacrylic acid, acrylamide, Methacrylamide etc.JP601805861 introduces be a kind of high-intensity be linking agent to guarantee to be applied to the enzyme immobilization material in the industrial production with saturated boric acid, mix with enzyme, freeze forming is mainly used in the immobilization of zymin and microbes producing cellulase cell.Remove this, the carrier that utilizes the using embedding immobilization glucoamylase commonly used also has Mierocrystalline cellulose, triacetate fiber, gelatin, carrageenin, sodium alginate, silk fibroin, polyacrylamide gel etc.In above-mentioned carrier, behind carrageenin, gelatin and the sodium alginate to embed saccharifying enzyme, in use support strength is relatively poor easily breaks, and causes the enzyme molecule to reveal.And behind the polyacrylamide immobilization saccharifying enzyme, the rate of recovery is higher though apparent enzyme is lived and enzyme is lived, and processing property is relatively poor, and the immobilization complicated operation, and raw material and preparation cost are also higher.
At present, the immobilization of relevant glucoamylase report is more, but is that the glucoamylase enzyme immobilization method of carrier is less with the polyvinyl alcohol.
Summary of the invention
The purpose of this invention is to provide that a kind of support strength is higher, excellent property, operational stability fixation glucose amylase goods preferably.
Another object of the present invention is to provide a kind of diastatic method of polyvinyl alcohol plural gel fixing glucose of utilizing, in producing with the deep processing, assay determination and other the relevant fermentation industry that are applied to alcohol industry, foodstuffs industry, wine brewing, starch and starchy material.
Fixation glucose amylase goods of the present invention are to be mixed by polyvinyl alcohol plural gel and glucoamylase enzyme liquid, through the cryogenic freezing moulding; Wherein the content of polyvinyl alcohol is 8～15% (quality percentage compositions) in the polyvinyl alcohol plural gel, and glucoamylase enzyme liquid concentration is 8～15% (quality percentage compositions).The glucoamylase enzyme powder of every 100mL plural gel carrying 0.5～1.5g, the enzyme amount of embedding just is 0.5～1.5% with respect to vehicle weight, promptly the polyvinyl alcohol plural gel carrier of 1g is fixed 100～1000 enzymes unit alive.The used glucoamylase of the present invention is the solid enzyme of 50000 units, removes by filter impurity.The selection of the concentration of glucoamylase enzyme liquid is a preferred higher concentration below the saturation solubility of this enzyme, and will consider the immobilization efficiency of this enzyme.
The diastatic method of fixing glucose of the present invention, may further comprise the steps, earlier 8～10g powdery glucoamylase is dissolved in the acetate-sodium acetate buffer of 100mLpH4.6～5.0, through 4～6 layers of gauze coarse filtration, at room temperature the glucoamylase enzyme liquid of 3～15mL10% is mixed with the 50mL polyvinyl alcohol plural gel then, (preventing to produce bubble) stirs, pour in the plate of 20cm, thickness is 3-4mm, this polyvinyl alcohol plural gel under-30 ℃～-10 ℃ freezing temp freezing crosslinked 24～48 hours, freezing crosslinked in preferred freezing temp be-24 ℃, preferably freezing time is 40～48 hours.Back to be formed is taken out nature and is thawed, and the fritter that is cut into (3～6) mm * (3～6) mm * (3～6) mm gets final product.
The diastatic method of fixing glucose of the present invention after also just glucoamylase enzyme liquid polyvinyl alcohol plural gel mixes, stirs, and pours in the plate of 20cm, and thickness is 1mm, and back to be formed is taken out nature and thawed, and is cut into little of 3mm * 6mm.
The present invention is in the building-up process of polyvinyl alcohol plural gel, apparent enzyme work has certain influence to fixation glucose amylase in the interpolation of dextran-40, when the addition of dextran-40 is 1%, the apparent enzyme of gained fixation glucose amylase is lived higher, and processing property is preferably arranged.
By the comparison of the embodiment of the invention and Comparative Examples, illustrate and utilize polyvinyl alcohol plural gel fixation glucose amylase goods that mechanical property, operational stability and prospects for commercial application are preferably arranged.The apparent enzyme work that also shows simultaneously the immobilized glucoamylase product is along with the embedding amount of saccharifying enzyme increases and increases, and the enzyme rate of recovery of living descends on the contrary.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1
Get concentration and be 8% polyvinyl alcohol plural gel (containing dextran-40) 50mL, be that (Wuxi is outstanding can biotechnology company limited of section for 10% glucoamylase at room temperature with 5mL concentration, 50,000 units) (glucoamylase dissolves with acetate-sodium acetate buffer of pH4.6 enzyme liquid, through the multilayer filtered through gauze) mix, stir, pour in the plate of 20cm, thickness is 3-4mm, under-24 ℃ condition freezing 48 hours, back to be formed is taken out nature and is thawed, and is cut into the fritter of 3mm * 3mm * 3mm.
Use Na
2S
2O
3Standard solution titration method (QB 1805.2-93 standard) is measured the enzyme of fixation glucose amylase and is lived.Carry out the batch hydrolysis experiment of starch simultaneously, measure the DE value, glucose content Tripotassium iron hexacyanide titration measuring.The result shows: the apparent enzyme of fixation glucose amylase is lived and is 1558u/gdrygel, and the enzyme rate of recovery 32% alive can be used 17～25 batches continuously, and the DE value is all greater than 95%.
Comparative example A: repeat embodiment 1, following difference is arranged: working concentration is 8% polyvinyl alcohol plural gel 50mL with 3mL, 10mL and 15mL concentration is that 10% glucoamylase enzyme liquid mixes.In embodiment 1, measure under the similarity condition, the result shows: the embedding amount be the apparent enzyme of the immobilized enzyme of 3mL live and the rate of recovery low than control experiment, the apparent enzyme of 10mL and the 15mL immobilized enzyme more above-mentioned height of living, but the rate of recovery is lower, and coacervation also may take place when big in the embedding amount.
Embodiment 2:
Repeat embodiment 1, following difference is arranged: using 50mL concentration is 10% polyvinyl alcohol plural gel, is that 10% glucoamylase enzyme liquid mixes with 5mL concentration at room temperature, and it is 1480u/gdrygel that the apparent enzyme of gained immobilized glucoamylase is lived.
Compare with the comparative example A, the continuous hydrolysis processing property is poor slightly because the voidage of carrier is lower, and the aperture is less, has also increased production cost in addition.
Embodiment 3:
Repeat embodiment 1, following difference is arranged: working concentration is 10% polyvinyl alcohol plural gel 50mL, be that 10% glucoamylase enzyme liquid mixes at room temperature with 5mL concentration, stir, pour plate into, thickness 1mm under-24 ℃ condition freezing 48 hours then, is cut into little of 3mm * 6mm after to be formed.The result shows: it is 2573.6u/gdrygel that the apparent enzyme of immobilized glucoamylase is lived higher, and the enzyme rate of recovery alive is 51.2%.
Compare with " embodiment 1 ", the more granular immobilized enzyme of its processing property is poor, easily causes coming off of enzyme molecule in operation process.
Comparative Examples B: repeat embodiment 3, following difference is arranged: working concentration is 10% polyvinyl alcohol plural gel 50mL, is that 10% glucoamylase enzyme liquid mixes with 3mL, 10mL and 15mL concentration.The result shows: along with the increase of enzyme embedding amount, the apparent enzyme of immobilized enzyme is lived and is increased thereupon.
Comparative Examples C: repeat embodiment 1, following difference is arranged: concentration is 6% polyvinyl alcohol plural gel 50mL, is that 10% glucoamylase enzyme liquid mixes with 5mL concentration at room temperature.The result shows: compare with " embodiment 3 ", the mechanical property of immobilized glucoamylase membrane carrier than concentration be 8～10% poor slightly, and be difficult for film forming.
Embodiment 4:
Repeat embodiment 1, following difference is arranged: concentration is 8% polyvinyl alcohol gel (no dextran-40) 50mL, is that 10% glucoamylase mixes with 5mL concentration at room temperature.The result shows: compare with " embodiment 1 ", repeat batch low slightly during hydrolyzed starch.If increase the addition of dextran, then can increase the viscosity of gel, and then have influence on the processing property of immobilized glucoamylase.It is better when its addition is the 1g/100g carrier.
Comparison by the embodiment of the invention and Comparative Examples, illustrate that the apparent enzyme work of immobilized glucoamylase product is along with the embedding amount of glucoamylase increases and increases, the enzyme rate of recovery alive descends on the contrary, and the adding of medium molecular dextran also has influence on the processing property of immobilized glucoamylase.
Claims (5)
1, a kind of fixation glucose amylase goods is characterized in that: it is mixed by polyvinyl alcohol plural gel and glucoamylase enzyme liquid, through the cryogenic freezing moulding; Wherein the content of polyvinyl alcohol is 8～15% (quality percentage compositions) in the polyvinyl alcohol plural gel, and glucoamylase enzyme liquid concentration is 8～15% (quality percentage compositions).The glucoamylase enzyme powder of every 100mL plural gel carrying 0.5～1.5g.
2, the diastatic method of a kind of fixing glucose, it is characterized in that: it may further comprise the steps, earlier 8～10g powdery glucoamylase is dissolved in the acetate-sodium acetate buffer of 100mLpH4.6～5.0, through 4～6 layers of gauze coarse filtration, at room temperature the glucoamylase enzyme liquid of 3～15mL10% is mixed with the 50mL polyvinyl alcohol plural gel then, (preventing to produce bubble) stirs, pour in the plate of 20cm, thickness is 3-4mm, this polyvinyl alcohol plural gel under-30 ℃～-10 ℃ freezing temp freezing crosslinked 24～48 hours, back to be formed is taken out nature and is thawed, and the fritter that is cut into (3～6) mm * (3～6) mm * (3～6) mm gets final product.
3, the diastatic method of fixing glucose according to claim 2 is characterized in that: freezing crosslinked middle freezing temp is-24 ℃, and freezing time is 40～48 hours.
4, the diastatic method of a kind of fixing glucose, it is characterized in that: it may further comprise the steps, earlier 8～10g powdery glucoamylase is dissolved in the acetate-sodium acetate buffer of 100mLpH4.6～5.0, through 4～6 layers of gauze coarse filtration, at room temperature the glucoamylase enzyme liquid of 3～15mL 10% is mixed with the 50mL polyvinyl alcohol plural gel then, (preventing to produce bubble) stirs, pour in the plate of 20cm, thickness is 1mm, this polyvinyl alcohol plural gel under-30 ℃～-10 ℃ condition freezing crosslinked 24～48 hours, back to be formed is taken out nature and is thawed, and little that is cut into 3mm * 6mm gets final product.
5, the diastatic method of fixing glucose according to claim 4 is characterized in that: freezing crosslinked middle freezing temp is-24 ℃, and freezing time is 40～48 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994489A (en) * | 2012-10-09 | 2013-03-27 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN108611339A (en) * | 2018-05-11 | 2018-10-02 | 中国农业科学院饲料研究所 | Glucoamylase TlGa15 and its gene and application |
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2004
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994489A (en) * | 2012-10-09 | 2013-03-27 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN102994489B (en) * | 2012-10-09 | 2014-01-08 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN108611339A (en) * | 2018-05-11 | 2018-10-02 | 中国农业科学院饲料研究所 | Glucoamylase TlGa15 and its gene and application |
| CN108611339B (en) * | 2018-05-11 | 2021-05-25 | 中国农业科学院北京畜牧兽医研究所 | Glucoamylase TlGa15 and gene and application thereof |
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