CN107384978A - A kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid - Google Patents
A kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid Download PDFInfo
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- CN107384978A CN107384978A CN201710577650.6A CN201710577650A CN107384978A CN 107384978 A CN107384978 A CN 107384978A CN 201710577650 A CN201710577650 A CN 201710577650A CN 107384978 A CN107384978 A CN 107384978A
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Abstract
The invention discloses a kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid, belong to technological field of biochemistry.The present invention includes preparing magnetic immobilized multi-enzyme system particle section and prepares the gluconic acid stage.The present invention, as raw material, adds magnetic immobilized multi-enzyme system particle, one kettle way enzymic catalytic reaction production gluconic acid directly using starch.The Starch Conversion rate of the present invention is 87.7%, and wherein 1g soluble starches can convert production 0.75g gluconic acid, improve the utilization rate of starch material；The present invention can conveniently realize the separation of catalyst enzyme and reaction solution by attraction effect, improve the recycling rate of waterused of enzyme, after reusing 6 times, the catalyzing hydrolysis vigor of enzyme remains in 70.8% or so, reduces production cost.Instant invention overcomes the shortcomings that the preparation of traditional enzyme process, have the advantages of preparation technology is simple, cost is low, with short production cycle, enzyme utilization rate is high and product purity is high.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of magnetic immobilized multi-enzyme system one kettle way prepares Portugal
The method of grape saccharic acid.
Background technology
Gluconic acid is also known as hexonic acid, be one kind in the wide variety of organic acid in the fields such as food, medicine and chemical industry.
Such as gluconic acid can be used as protein coagulation agent and food preservative；Calcium salt, ferrous salt, bismuth salt and other salts of gluconic acid
It is widely used in chemotherapy；The metal complex of gluconic acid is widely used as the screening agent of metal ion in alkaline system
Deng.The production method of gluconic acid mainly has microbe fermentation method, chemical catalytic oxidation method, electrolytic oxidation and enzymatic at present
4 kinds of methods such as method.
Microbe fermentation method is that the fermenting raw materials such as carbohydrate are converted into the one of gluconic acid by the metabolic pathway of microorganism
Kind method, but the yield of this method gluconic acid is limited by strain quality, concentration of substrate also has inhibitory action to strain, and
Due to there is the generation of the addition of mass propgation base and accessory substance, the product extraction to the later stage brings very burden, has a strong impact on production
Quality.
Chemical catalytic oxidation method includes two methods of homogeneous catalysis and heterogeneous catalysis, and homogeneous catalysis is one kind in alkalescence condition
Under, the oxidability of limiting catalyst, and then the method that glucose is oxidized to gluconic acid；And heterogeneous catalysis be with solid, liquid,
Gas three-phase catalytic glucose production gluconic acid, it is that metallic catalyst is fixed on carrier first, then is passed through into reaction solution
Oxygen, and then the method for producing gluconic acid.The technical process is easily controllable, but gluconic acid low yield, and this method is high
The production cost of volume and the complexity of effective catalyst preparation technology govern the method industrial applications always.
Electrolytic oxidation is by glucose solution by way of being passed through electric current in a cell and adding suitable electrolyte
Oxidation generation gluconic acid, although this method has accessory substance few, it is multiple to overcome advantage, highly energy-consuming, the techniques such as substrate suppression
It is miscellaneous, at present in the case of Chinese energy shortage, it is difficult to apply to industrialized production.
Enzyme catalysis method is mainly the method that catalytic material is reacted to production gluconic acid using enzymatic reagent, with other 3 kinds of sides
Method is compared, and prepared by enzyme catalysis method have the advantages that reaction condition is gentle, product purity is high, short preparation period.
Traditional enzyme catalysis method divides multistep to carry out, and is by starch materials first by alpha-amylase and saccharification enzyme effect
Glucose is hydrolyzed into, then gluconic acid, this method long preparation period, behaviour are converted glucose into by glucose oxidase effect
It is more to make complexity, process, seriously constrains its application in industrialized production.Recently have glucose oxidase and hydrogen peroxide
Enzyme co-immobilization produces gluconic acid, but this method can only use glucose as raw material, and cost is higher, is not suitable for the big rule of industry
Mould application.Studied in addition, having by carbohydrase and glucose oxidase co-immobilization, utilize Glucoamylase hydrolysis starch to recycle grape
The method that carbohydrate oxidase changes into the glucose of generation gluconic acid, but this method starch utilization ratio is low so that wastage of material
Seriously, the requirement of industrialized production can not be met.
The content of the invention
According to above the deficiencies in the prior art, the technical problems to be solved by the invention are to propose that a kind of magnetic immobilized is more
The method that enzyme system one kettle way prepares gluconic acid, by directly by the use of starchy material as raw material, carrying out starch to grape
The conversion process of saccharic acid, realize one kettle way catalysis and prepare gluconic acid.Instant invention overcomes the shortcomings that the preparation of traditional enzyme process, have
There is the advantages of preparation technology is simple, cost is low, with short production cycle, enzyme utilization rate is high and product purity is high.
In order to achieve the above object, the technical solution adopted by the present invention is：The present invention proposes a kind of magnetic immobilized multienzyme
Particle one kettle way prepares the method for gluconic acid, including prepares magnetic immobilized multi-enzyme system granule stage and prepare gluconic acid
Stage.
It is above-mentioned to prepare magnetic immobilized multi-enzyme system granule stage, comprise the following steps：
(1) magnetic nanoparticle, alpha-amylase, carbohydrase, catalase and glucose oxidase are added to pH
It is worth in the cushioning liquid for 4.0~8.0, and adds precipitating reagent and stir and evenly mix, 0.5~1h is placed under the conditions of 4~25 DEG C, is obtained
Mixture precipitation；
(2) crosslinking agent is added into step (1) mixture precipitation, 1.5~2.5h of oscillating reactions under the conditions of 4~25 DEG C,
Obtain magnetic immobilized multiple enzyme granulates mixture；
(3) solid particle in attraction magnetic immobilized multiple enzyme granulates mixture is used, abandoning supernatant, uses deionization
Water cleans solid particle, produces magnetic immobilized multi-enzyme system particle.
Alpha-amylase, carbohydrase, hydrogen peroxide in above-mentioned the step of preparing magnetic immobilized multi-enzyme system granule stage (1)
The ratio of the addition of enzyme and glucose oxidase is 10:10:10：1~3, the alpha-amylase, carbohydrase, hydrogen peroxide
The protein concentration of enzyme and glucose oxidase is 10g/L.
In above-mentioned the step of preparing magnetic immobilized multi-enzyme system granule stage (1) magnetic nanoparticle be particle diameter be 50~
600nm Fe3O4Particle, the addition of the magnetic nanoparticle is 2~10g/L.
Cushioning liquid is citric acid-citric acid in above-mentioned the step of preparing magnetic immobilized multi-enzyme system granule stage (1)
One kind in sodium buffer solution, dipotassium hydrogen phosphate-potassium phosphate buffer, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution.
Precipitating reagent is acetone, saturated ammonium sulfate in above-mentioned the step of preparing magnetic immobilized multi-enzyme system granule stage (1)
One kind in solution, n-butanol, acetonitrile and the tert-butyl alcohol, addition and alpha-amylase, carbohydrase, the hydrogen peroxide of the precipitating reagent
The volume ratio 1~2 of the addition total amount of enzyme and glucose oxidase:1.
Crosslinking agent is that glutaraldehyde or glucan gather in above-mentioned the step of preparing magnetic immobilized multi-enzyme system granule stage (2)
Aldehyde, the addition of the crosslinking agent is 0.5~3%.
Above-mentioned magnetic immobilized multi-enzyme system particle may be recovered recycling.
It is above-mentioned to prepare the gluconic acid stage, comprise the following steps：
Step 1: the starch slurry for being gelatinized and cooling down is added in reactor, regulation pH value is 4.0~6.0, adds system
The magnetic immobilized multi-enzyme system particle that standby magnetic immobilized multi-enzyme system granule stage is prepared, leads under the conditions of 30~60 DEG C
Gas agitating reacts 12~16h and obtains product, and mixing speed is 100~300r/min；
Step 2: with the magnetic immobilized multi-enzyme system particle in the product of attraction recovery above-mentioned steps one, will be anti-
Liquid filtering, concentration are answered, produces gluconic acid.
Starch is rice starch, farina, cornstarch and can in above-mentioned the step of preparing the gluconic acid stage one
One or more in soluble starch.
Preferably, starch is soluble starch in above-mentioned the step of preparing the gluconic acid stage one.
The addition of magnetic immobilized multiple enzyme granulates is 2~10% in above-mentioned the step of preparing the gluconic acid stage one.
In above-mentioned steps two in the preparation process of gluconic acid, can by the filtering of reaction solution, concentration, regulation pH value, point
It is gluconic acid or gluconate from, the product that is dried to obtain.
In the measure of magnetic immobilized multi-enzyme system hydrolysis vigor of the present invention, with what is handled without precipitating reagent and crosslinking agent
The hydrolysis vigor of enzyme liquid is 100%, calculates the enzyme activity rate of recovery of the magnetic immobilized multi-enzyme system of preparation.To prepare gluconic acid
The relative hydrolysis vigor of preceding magnetic immobilized multi-enzyme system is 100%, and the magnetic collected in reaction solution is calculated and fixes
Change the last phase of multi-enzyme system to hydrolysis activity.
The content of gluconic acid is measured using high performance liquid chromatography, and condition determination is methanol：Water=5:95, Portugal
Grape sugar acid solution is adjusted pH value to 2.7 with phosphoric acid, flow velocity 1mL/min, Detection wavelength 200nm, 30 DEG C of column temperature.
Present invention has the advantages that:The invention provides a kind of magnetic immobilized multi-enzyme system one kettle way to prepare gluconic acid
Method, the present invention is first by immobilization method by alpha-amylase, carbohydrase, glucose oxidase, catalase and magnetic
Property nano particle co-immobilization, is prepared for the magnetic immobilized multi-enzyme system particle with catalytic activity.First alpha-amylase and
Starch Hydrolysis is glucose by carbohydrase, then converts glucose into gluconic acid by glucose oxidase, and hydrogen peroxide
Enzyme can eliminate caused H in gluconic acid preparation process2O2To the damaging action of enzyme, above-mentioned catalytic process be all on one point on
It is carried out continuously, can is that glucose is then converted to gluconic acid by Starch Hydrolysis so by the common continuous action of multienzyme, realize
One kettle way prepares the process of gluconic acid.The present invention is using magnetic immobilized multi-enzyme system by one kettle way directly by starch
Catalytic material is prepared for gluconic acid, high conversion rate.The present invention by attraction effect can conveniently realize catalyst enzyme with
The separation of reaction solution, the recycling rate of waterused of enzyme is improved, reduce production cost.The present invention also has that preparation technology is simple, production
Cycle is short and the advantages of product purity is high so that the method that the present invention prepares gluconic acid has in commercial process
It is widely applied prospect.
Brief description of the drawings
The content expressed by this specification accompanying drawing and the mark in figure are briefly described below：
Fig. 1 is the configuration of surface figure of the magnetic immobilized multi-enzyme system particle of the present invention；
Fig. 2 is that magnetic immobilized multi-enzyme system particle reuses stability change figure in embodiments of the invention one；
Fig. 3 is the comparison diagram for being utilized respectively soluble starch and rice starch malaga saccharic acid amount of the present invention.
1, magnetic-particle in figure, 2, zymoprotein.
Embodiment
By the description to embodiment, to help those skilled in the art to have inventive concept of the invention, technical scheme
More complete, accurate and deep understanding.
Embodiment 1
The method that the magnetic immobilized multi-enzyme system one kettle way of the present embodiment prepares gluconic acid is more including magnetic immobilized
The preparatory phase of enzyme system particle and the preparatory phase of gluconic acid.
The preparatory phase of magnetic immobilized multi-enzyme system particle, specifically includes following steps：
(1) by 0.1g, particle diameter is 100nm Fe3O4The zymoprotein concentration that particle is added to 20mL is 10g/L and pH value is
In 6.0 mixing enzyme solutions, add the 40mL tert-butyl alcohols and place 0.5h under the conditions of 4 DEG C after mixing as precipitating reagent, vibration, its
The addition species and ratio of middle mixed enzyme are 10 parts of mesophilicα-diastases, 10 parts of carbohydrase, 1 part of glucose oxidase, 10 parts of mistakes
Hydrogen oxide enzyme；
(2) 2% crosslinking agent glutaraldehyde, the oscillating reactions 2h under the conditions of 4 DEG C are added into step (1)；
(3) attraction magnetic immobilized multi-enzyme system particle is used, abandoning supernatant, 3 particles are cleaned with deionized water,
Collect the magnetic immobilized multi-enzyme system particle prepared.
The preparatory phase of gluconic acid, specifically includes following steps：
Step 1: after 5g has been gelatinized into cooling, and the soluble starch slurry that pH value is 6.0 is added in reactor, then
The magnetic immobilized multi-enzyme system particle 0.2g prepared is added, under the conditions of 50 DEG C, 16h is reacted in air agitation, and mixing speed is
200r/min；
Step 2: reclaiming magnetic immobilized multi-enzyme system particle with attraction, by reacting liquid filtering, concentration, Portugal is obtained
Grape saccharic acid.
The enzyme activity rate of recovery for the magnetic immobilized multi-enzyme system particle that the present embodiment is prepared is 87.2%；The present embodiment Portugal
The last phase of magnetic immobilized multi-enzyme system is 95.4% to hydrolysis activity after the preparation reaction of grape saccharic acid；The present embodiment can
The conversion ratio of soluble starch is 87.7%%；3.75g gluconic acids are prepared in the present embodiment.
As shown in Fig. 2 the magnetic immobilized multi-enzyme system for reclaiming to obtain after reaction is continued to put into reaction by the present embodiment
Reacted in device, keep reaction condition constant, so repeat 6 secondary responses, magnetic immobilized is more after finally measuring reaction
The last phase of enzyme system is 70.8% to hydrolysis activity, and the conversion ratio of soluble starch is 85.1%, is finally prepared
3.56g gluconic acid.
Embodiment 2
The method that the magnetic immobilized multi-enzyme system one kettle way of the present embodiment prepares gluconic acid includes two magnetic immobilizeds
The preparatory phase of multi-enzyme system particle and the preparatory phase of gluconic acid.
The preparatory phase of magnetic immobilized multi-enzyme system particle, specifically includes following steps：
(1) by 0.1g, particle diameter is 100nm Fe3O4The zymoprotein concentration that particle is added to 20mL is 10g/L and pH value is
In 6.0 mixing enzyme solutions, add the 40mL tert-butyl alcohols and place 0.5h under the conditions of 4 DEG C after mixing as precipitating reagent, vibration, its
The addition species and ratio of middle mixed enzyme are 10 parts of mesophilicα-diastases, 10 parts of carbohydrase, 3 parts of glucose oxidases, 10 parts of mistakes
Hydrogen oxide enzyme；
(2) 2.5% crosslinking agent glutaraldehyde, the oscillating reactions 2h under the conditions of 4 DEG C are added into step (1)；
(3) attraction magnetic immobilized multi-enzyme system particle is used, abandoning supernatant, 3 particles are cleaned with deionized water,
Collect the magnetic immobilized multi-enzyme system particle prepared.
The preparatory phase of gluconic acid, specifically includes following steps：
Step 1: after 5g has been gelatinized into cooling, and the soluble starch slurry that pH value is 6.0 is added in reactor, then
The magnetic immobilized multi-enzyme system particle 0.2g prepared is added, under the conditions of 40 DEG C, 16h is reacted in air agitation, and mixing speed is
300r/min；
Step 2: reclaiming magnetic immobilized multi-enzyme system particle with attraction, by reacting liquid filtering, concentration, Portugal is obtained
Grape saccharic acid.
The enzyme activity rate of recovery for the magnetic immobilized multi-enzyme system particle that the present embodiment is prepared is 83.8%；The present embodiment Portugal
The last phase of magnetic immobilized multi-enzyme system is 93.4% to hydrolysis activity after the preparation reaction of grape saccharic acid；The present embodiment can
The conversion ratio of soluble starch is 85.0%；3.44g gluconic acids are prepared in the present embodiment.
Embodiment 3
The method that the magnetic immobilized multi-enzyme system one kettle way of the present embodiment prepares gluconic acid includes two magnetic immobilizeds
The preparatory phase of multi-enzyme system particle and the preparatory phase of gluconic acid.
The preparatory phase of magnetic immobilized multi-enzyme system particle, specifically includes following steps：
(1) by 0.24g, particle diameter is 100nm Fe3O4The zymoprotein concentration that particle is added to 30mL is 10g/L and pH value is
In 6.0 mixing enzyme solutions, add the 60mL tert-butyl alcohols and place 0.5h under the conditions of 4 DEG C after mixing as precipitating reagent, vibration, its
The addition species and ratio of middle mixed enzyme are 10 parts of mesophilicα-diastases, 10 parts of carbohydrase, 1 part of glucose oxidase, 10 parts of mistakes
Hydrogen oxide enzyme；
(2) 2.5% crosslinking agent glutaraldehyde, the oscillating reactions 2h under the conditions of 4 DEG C are added into step (1)；
(3) attraction magnetic immobilized multi-enzyme system particle is used, abandoning supernatant, 3 particles are cleaned with deionized water,
Collect the magnetic immobilized multi-enzyme system particle prepared.
The preparatory phase of gluconic acid, specifically includes following steps：
Step 1: after 10g has been gelatinized into cooling, and the rice starch slurry that pH value is 5.0 is added in reactor, then add
Enter the magnetic immobilized multi-enzyme system particle 0.5g of preparation, under the conditions of 50 DEG C, 16h is reacted in air agitation, and mixing speed is
200r/min；
Step 2: reclaiming magnetic immobilized multi-enzyme system particle with attraction, by reacting liquid filtering, concentration, Portugal is obtained
Grape saccharic acid.
The enzyme activity rate of recovery for the magnetic immobilized multi-enzyme system particle that the present embodiment is prepared is 86.4%；The present embodiment Portugal
The last phase of magnetic immobilized multi-enzyme system is 90.9% to hydrolysis activity after the preparation reaction of grape saccharic acid；The present embodiment can
The conversion ratio of soluble starch is 69.5%；5.31g gluconic acids are prepared in the present embodiment.
Embodiment 4
The present embodiment keeps the preparatory phase condition of the magnetic immobilized multi-enzyme system in embodiment 1 constant, by glucose
Soluble starch used in the preparatory phase of acid is changed to rice starch, and other conditions are same as Example 1, and magnetic is consolidated after measuring reaction
Surely the last phase for changing multi-enzyme system is 92.8% to hydrolysis activity, and the conversion ratio of rice starch is 70.1%, is finally prepared into
To 2.69g gluconic acids.
Compared with Example 1, the last phase of magnetic immobilized multi-enzyme system is to hydrolysis activity, shallow lake after reaction for the present embodiment
The conversion ratio of powder and the gluconic acid yield being finally prepared are all relatively low.
It is illustrated in figure 3 and is utilized respectively Starch Conversion rate during soluble starch and rice starch malaga saccharic acid, from
It can be seen from the figure that rice starch conversion ratio is low compared with soluble starch conversion ratio, and reason is that rice starch belongs to raw starch, its
In the proteinaceous impurities that contain it is more, and when being gelatinized slurry viscosity it is also larger, these reasons cause in end reaction liquid
Magnetic immobilized multi-enzyme system last phase to hydrolysis activity, the conversion ratio of starch and the gluconic acid yield that is prepared
It is all relatively low.
Embodiment 5
Magnetic immobilized multi-enzyme system particle in embodiment 1 is changed to identical enzyme concentration and enzyme activity ratio by the present embodiment
Resolvase enzyme liquid, it is added in the reactor that the soluble starch that the pH value that 5g has been gelatinized and cooled down is 6.0 is starched, in 50 DEG C of bars
16h, air agitation reaction, mixing speed 200r/min are reacted under part；Magnetic immobilized multi-enzyme system is reclaimed with attraction
Particle, by reacting liquid filtering, concentration, obtain gluconic acid.The conversion ratio for measuring soluble starch after reacting is 73.6%, finally
2.17g gluconic acids are prepared.
Compared with the present embodiment, the conversion ratio of starch and the gluconic acid amount being finally prepared all obtain very embodiment 1
It is big to improve, and the enzyme in embodiment 1 can recycle and reuse, and reduce the influence that enzyme liquid purifies to final products.This reality
It is resolvase malaga saccharic acid to apply example, because the stability of enzyme is poor, needs to continuously adjust the pH value of reaction in course of reaction, so that
Reaction persistently goes on, but the conversion ratio of final starch and gluconic acid yield are all relatively low；High starch is obtained in embodiment 1
Conversion ratio and high glucose acid yield, reason is to have used magnetic immobilized multi-enzyme system as catalyst, after immobilization
Enzyme specific ionization enzyme in gluconic acid production process is catalyzed stability it is more preferable, and reduce in preparation process continuously adjust it is anti-
The operating procedure of liquid pH value is answered, enormously simplify technological process；The efficient effect of magnetic immobilized multi-enzyme system is also resided in four
Kind of enzyme flocks together, and realizes one kettle way and prepares gluconic acid, and four kinds of enzymes can be with coordinative role, on one point in catalytic process
On realize conversion of the starch to gluconic acid, these all substantially increase the catalytic action efficiency of enzyme.
The present invention is exemplarily described above, it is clear that present invention specific implementation is not subject to the restrictions described above,
As long as employing the improvement of the various unsubstantialities of inventive concept and technical scheme of the present invention progress, or not improved this is sent out
Bright design and technical scheme directly applies to other occasions, within protection scope of the present invention.The protection of the present invention
Scope should be determined by the scope of protection defined in the claims.
Claims (9)
1. a kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid, it is characterised in that including preparing magnetic
Immobilized multienzyme system particle section and prepare the gluconic acid stage.
2. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 1 prepares gluconic acid, its feature exist
In：
It is described to prepare magnetic immobilized multi-enzyme system granule stage, comprise the following steps：
(1) magnetic nanoparticle, alpha-amylase, carbohydrase, catalase and glucose oxidase are added into pH value is
In 4.0~8.0 cushioning liquid, and add precipitating reagent and stir and evenly mix, 0.5~1h is placed under the conditions of 4~25 DEG C, is mixed
Thing precipitates；
(2) crosslinking agent is added into step (1) mixture precipitation, 1.5~2.5h of oscillating reactions, is obtained under the conditions of 4~25 DEG C
Magnetic immobilized multiple enzyme granulates mixture；
(3) it is clear with deionized water with the solid particle in attraction magnetic immobilized multiple enzyme granulates mixture, abandoning supernatant
Solid particle is washed, produces magnetic immobilized multi-enzyme system particle；
It is described to prepare the gluconic acid stage, comprise the following steps：
Step 1: the starch for being gelatinized and cooling down is added in reactor, regulation pH value is 4.0~6.0, adds preparation magnetic
The magnetic immobilized multi-enzyme system particle that immobilized multienzyme system granule stage is prepared, the air agitation under the conditions of 30~60 DEG C
12~16h of reaction obtains product, and mixing speed is 100~300r/min；
Step 2: with attraction reclaim above-mentioned steps one product in magnetic immobilized multi-enzyme system particle, by reaction solution
Filtering, concentration, produce gluconic acid.
3. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：Alpha-amylase in the step of of preparing magnetic immobilized multi-enzyme system granule stage (1), carbohydrase, catalase with
And the ratio of the addition of glucose oxidase is 10:10:10：1~3, the alpha-amylase, carbohydrase, catalase with
And the protein concentration of glucose oxidase is 10g/L.
4. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：Magnetic nanoparticle is that particle diameter is 50~600nm in the step of of preparing magnetic immobilized multi-enzyme system granule stage (1)
Fe3O4Particle, the addition of the magnetic nanoparticle is 2~10g/L.
5. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：Cushioning liquid buffers for citric acid-sodium citrate in the step of of preparing magnetic immobilized multi-enzyme system granule stage (1)
One kind in liquid, dipotassium hydrogen phosphate-potassium phosphate buffer, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution.
6. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：In the step of of preparing the magnetic immobilized multiple enzyme granulates system stage (1) precipitating reagent be acetone, saturated ammonium sulfate solution,
One kind in n-butanol, acetonitrile and the tert-butyl alcohol, the addition of the precipitating reagent and alpha-amylase, carbohydrase, catalase with
And the volume ratio 1~2 of the addition total amount of glucose oxidase:1.
7. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：Crosslinking agent is glutaraldehyde or glucan polyacetals in the step of of preparing the magnetic immobilized multiple enzyme granulates system stage (2), institute
The addition for stating crosslinking agent is 0.5~3%.
8. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：Starch is rice starch, farina, cornstarch and soluble shallow lake in described the step of preparing the gluconic acid stage one
One or more in powder.
9. the method that magnetic immobilized multi-enzyme system one kettle way according to claim 2 prepares gluconic acid, its feature exist
In：The addition of magnetic immobilized multiple enzyme granulates is 2~10% in described the step of preparing the gluconic acid stage one.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410853A (en) * | 2018-03-23 | 2018-08-17 | 吉林农业大学 | A method of preparing gluconic acid using co-immobilization enzyme system |
| CN110904164A (en) * | 2019-12-02 | 2020-03-24 | 武汉新华扬生物股份有限公司 | Biocatalysis method for preparing gluconate |
| CN111621482A (en) * | 2020-06-30 | 2020-09-04 | 浙江工业大学 | Glufosinate-ammonium dehydrogenase mutant, gene engineering bacteria and one-pot multi-enzyme synchronous directed evolution method |
| CN113881661A (en) * | 2021-09-29 | 2022-01-04 | 淮阴工学院 | Method for immobilizing enzyme by magnetic nanoparticles based on carboxymethyl starch modification |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602710A (en) * | 2013-09-30 | 2014-02-26 | 山东西王糖业有限公司 | Method of preparing calcium gluconate by composite immobilized enzyme |
-
2017
- 2017-07-15 CN CN201710577650.6A patent/CN107384978A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602710A (en) * | 2013-09-30 | 2014-02-26 | 山东西王糖业有限公司 | Method of preparing calcium gluconate by composite immobilized enzyme |
Non-Patent Citations (5)
| Title |
|---|
| FUHUA ZHAO等: "Co-immobilization of multi-enzyme on control-reduced graphene oxide by non-covalent bonds: an artificial biocatalytic system for the one-pot production of gluconic acid from starch", 《GREEN CHEMISTRY》 * |
| JIANDONG CUI等: "Hybrid Cross-Linked Lipase Aggregates with Magnetic Nanoparticles: A Robust and Recyclable Biocatalysis for the Epoxidation of Oleic Acid", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| SACHIN TALEKAR等: "A tri-enzyme magnetic nanobiocatalyst with one pot starch hydrolytic activity", 《CHEMICAL ENGINEERING JOURNAL》 * |
| 贺玉兰等: "葡萄糖氧化酶与过氧化氢酶的共固定化研究", 《食品科技》 * |
| 顾旭炯等: "硅藻土共固定化淀粉酶和糖化酶的研究", 《饲料工业》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410853A (en) * | 2018-03-23 | 2018-08-17 | 吉林农业大学 | A method of preparing gluconic acid using co-immobilization enzyme system |
| CN110904164A (en) * | 2019-12-02 | 2020-03-24 | 武汉新华扬生物股份有限公司 | Biocatalysis method for preparing gluconate |
| CN111621482A (en) * | 2020-06-30 | 2020-09-04 | 浙江工业大学 | Glufosinate-ammonium dehydrogenase mutant, gene engineering bacteria and one-pot multi-enzyme synchronous directed evolution method |
| CN111621482B (en) * | 2020-06-30 | 2022-04-29 | 浙江工业大学 | Glufosinate-ammonium dehydrogenase mutant, gene engineering bacteria and one-pot multi-enzyme synchronous directed evolution method |
| CN113881661A (en) * | 2021-09-29 | 2022-01-04 | 淮阴工学院 | Method for immobilizing enzyme by magnetic nanoparticles based on carboxymethyl starch modification |
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