CN103524569B - Technological method for removing glucose in isomaltooligosaccharide product by using multiple immobilized enzymes - Google Patents
Technological method for removing glucose in isomaltooligosaccharide product by using multiple immobilized enzymes Download PDFInfo
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Abstract
The invention discloses a technological method for removing glucose in an isomaltooligosaccharide product by using multiple immobilized enzymes. The technological method is used for realizing multiple enzyme separated immobilization by taking calcium carbonate as a template and combining bionic silicification and bionic bonding thoughts on the basis of a layer-by-layer self-assembly technology so as to remove the glucose in the isomaltooligosaccharide product. The technological method is simple in preparation process, mild in condition and little in enzyme activity loss; the prepared immobilized enzymes are used for removing the glucose in the isomaltooligosaccharide product at a high removal rate up to over 80%, and are relatively high in repeated utilization ratio; a carrier is cheap and easy to obtain.
Description
Technical field
The invention belongs to immobilized enzyme Application Areas, particularly a kind of immobilized multienzyme is for removing the processing method of glucose in oligomeric isomaltose product (IMO-500).
Background technology
Oligomeric isomaltose is called as " bifidus factor ".Show through research for many years, bifidus bacillus has many nourishing functions, and receives the concern of people as the oligomeric isomaltose of bifidus factor.The few oligomeric isomaltose of occurring in nature exists with unbound state, does not therefore directly obtain by extraction separation and purification process.Due to the many integral parts as amylopectin, dextrose and polysaccharide etc. of oligomeric isomaltose, so are direct, the most most economical approach by these raw material production oligomeric isomaltoses.The primary products of the oligomeric isomaltose product that China produces to be functional component be mostly 50% (IMO-500), have had a strong impact on its function and have tired and commercial value.And primary products are made the oligomeric isomaltose of functional component more than 90% (IMO-900), its economic benefit increases substantially.Therefore, improve the purity of oligomeric isomaltose, namely become the key issue that technique needs to solve.
Bibliographical information about oligomeric isomaltose purifying products comprises: document 1:Science and Technology of Foodindustry, 2002,23 (5): 27-30 by after yeast saccharomyces cerevisiae As2.109 domestication, for fermentation method separation and purification oligomeric isomaltose.Document 2: Food science, 2004,25 (supplementary issue): 82-85 utilize the oligomeric isomaltose product of yeast fermentation method to low-purity to carry out purifying.By carrying out screening experiment to yeast, selecting a strain and only utilizing glucose and maltose, and not utilizing the yeast of oligomeric isomaltose.Utilize this bacterium to carry out purifying to oligomeric isomaltose product, final purity can reach 99%.The efficiency of above method to oligomeric isomaltose purifying products is higher, but strain improvement overlong time, reacted bacterial classification is not easily reused again.Document 3:Journal of South China Agricultural University, 2005,26 (4): 102-105 utilize glucose oxidase and catalatic synergy purification oligomeric isomaltose product, and isomaltose purity brings up to 85.28% by 62.78%.This method is simple, but can not recycle after resolvase reaction, causes larger waste.This method has used for reference the enzyme catalysis process in viable cell, and intracellular Substance Transformation generally involves the concerted catalysis process of multiple enzyme, but this method uses resolvase, not easily reclaims, if enzyme fix will Research Significance in production application larger.Inspire by this, the structure that in simulation is biological, multienzyme system carries out multienzyme system by for realize multienzyme catalytic process efficiently carry out open up new way.
Immobilized glucose oxidase and catalatic bibliographical information comprise: document 1: biotechnology journal, 1987,3 (1): 46-52 utilize diazonium salt covalent bonding method jointly to be received by GOD and CAT on Sepharose prepares immobilized bi-enzyme system.Document 2:Macromolecular Bioscience, GOD and CAT to be fixed on and to be close on the polymeric film of anion-exchange membrane, in order to the production of gluconic acid by 2004,4 (10): 950-956 jointly.Document 3:Journal of Microbiology andBiotechnology, GOD and CAT is fixed on florisil by 2007,17 (6): 960-967, and have studied the fixing difference fixing with order simultaneously.Result shows, and no matter in enzyme is lived or in cost consumption, the fixing method of order is all better than fixing simultaneously.Compared with GOD fixing separately, optimum condition does not almost have variation.And reuse with catalytic efficiency in, the altogether far super independent enzyme fixed of fixed system.Document 4:Journal of Applied PolymerScience, GOD and CAT is fixed on the fine multipolymer cytolemma of propylene of modification by 2004,91 (6), 4057-4063.
Above pertinent literature describes immobilized glucose oxidase and catalatic different methods and application.Although at present above strategy is devoted to realize pair enzyme and is fixed, but still urgently develop the carrier of gentleness, the immobilized multienzyme that efficient, easy, suitability is strong.So far, yet there are no employing layer-by-layer in document in conjunction with bionical silication, bionical bonding thought cellular-type immobilized glucose oxidase and catalase for removing the relevant report of the glucose in oligomeric isomaltose product.
Summary of the invention
The independent control that the object of the invention is (1) the various enzyme existed for current co-immobilization multi-enzyme system is poor; (2) various enzyme immobilizatio position is random, the more difficult object reaching formation substrate product chain; (3) contacting with each other of multienzyme may cause the affected shortcoming of respective catalytic performance, provides a kind of method of cellular-type immobilized multienzyme simple to operate, with low cost, and is used for removing the glucose in oligomeric isomaltose product by this immobilized multienzyme.It take calcium carbonate as template, on the basis of layer-by-layer, realizes multienzyme cellular-type be fixed for removing the glucose in oligomeric isomaltose product in conjunction with bionical silication and bionical bonding thought.The method used carrier is cheap and easy to get, cost is lower, and cellular-type immobilized multienzyme to remove the efficiency of glucose higher and can reuse.
Technical scheme of the present invention is:
Immobilized multienzyme removes a processing method for the glucose in oligomeric isomaltose product, comprises the following steps:
1) preparation of immobilized multienzyme: 1. sodium polystyrene sulfonate (PSS) preparation of calcium carbonate microparticle containing glucose oxidase (GOD) of adulterating: the CaCl 1. getting 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, then under agitation adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 15-30min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. the PSS solution 15ml of 2mg/ml is added in the whole calcium carbonate microparticles obtained upward, vibration 15min, centrifugal, remove supernatant, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 4-8h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, under magnetic stirring apparatus, Keep agitation 6-12h, centrifugal, remove supernatant liquor, remove template with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme.
2) immobilized multienzyme removes the glucose in oligomeric isomaltose product: be all put in by the immobilized multienzyme of above-mentioned preparation in the oligomeric isomaltose product solution of the 200mg/ml of 3ml, pH=2-7,30-50 DEG C is kept to react 12-24h, add 80-110mg calcium hydroxide, generate calglucon, centrifuging purifying; Finally obtain the oligomeric isomaltose solution removing glucose.
Involved by the processing method of the glucose in above-mentioned immobilized multienzyme removal oligomeric isomaltose product, amount of substance all can expand by corresponding proportion entirety or reduce.
The Tris-HCl buffer method of described pH=8.5 is as follows: take Tris solid 1.2g, namely obtains the Tris solution of 1.2g/L with the volumetric flask adding distil water constant volume of 1000ml; Mother liquor 2: taking concentrated hydrochloric acid 1g(massfraction is 37%), namely obtain the HCl solution of 0.37g/L with the volumetric flask adding distil water constant volume of 1000ml, afterwards mother liquor 1 by volume: the two mixes by mother liquor 2=20:3, and obtain after correcting with pH meter.
Being prepared as follows of described silicon precursor solution: at room temperature by TMOS(methyl silicate) join in the HCL solution of 0.37g/L, TMOS dissolves completely, clarification after add pH=7 phosphate buffered saline buffer again, mixing; Its material proportion is volume ratio: TMOS:HCL solution: phosphoric acid buffer=1:19:200.
The preparation method of the norepinephrine HCI solution of described 2mg/mL is as follows: take norepinephrine solid 200mg, namely obtains the norepinephrine HCI solution of 2mg/mL with the Tris-HCl damping fluid constant volume that the volumetric flask of 100ml adds above-mentioned pH=8.5.
The invention has the beneficial effects as follows:
1. the present invention is on layer-by-layer basis, and in conjunction with bionical silication and bionical bonding thought cellular-type immobilized glucose oxidase and catalase, preparation technology is simple, and mild condition is little to loss of enzyme activity.
2. the immobilized enzyme of preparation is for removing the glucose in oligomeric isomaltose product, and glucose clearance can reach more than 80%, and repeat usage is higher; (the enzyme rate of recovery alive is 72.85%.Present method immobilized enzyme is also higher, reaches 2668U/g.And document: Min DD, Zhang XD, He W, Zhang Y, Li PW, Zhang MM, Liu JN, Liu SJ, XuFX, Du Y, Zhang ZL.J Mater Chem B, 2013,10:29. mention that its enzyme activity is maximum can only reach 238.93U/g.And the immobilized enzyme of preparation can be used for removing the glucose in oligomeric isomaltose, clearance reaches 80.73%, and repeat 6 times, clearance still can reach 70.22%.）
3. carrier is cheap and easy to get.
Embodiment
The present invention TMOS used is purchased from Tianjin City Chemical Agent Research Institute.
The present invention's norepinephrine used is huge along grand biochemical industry company limited purchased from Hubei.
The glucose oxidase that the present invention is used and catalase are all purchased from sigma company.
Phosphate buffer soln of the present invention is Na
2hPO
4-NaH
2pO
4buffered soln.
The phosphate buffered saline method of testing pH=7 used is as follows: mother liquor 1: take NaH
2pO
42H
2o solid 0.1mol, namely obtains the NaH of 0.1mol/L with the volumetric flask adding distil water constant volume of 1000ml
2pO
4solution; Mother liquor 2: take Na
2hPO
412H
2o solid 0.1mol, namely obtains the Na of 0.1mol/L with the volumetric flask adding distil water constant volume of 1000ml
2hPO
4solution, afterwards mother liquor 1 by volume: the two mixes by mother liquor 2=2:3, and obtain after correcting with pH meter.
The Tris-HCl buffer method of testing pH=8.5 used is as follows: mother liquor 1: take Tris solid 1.2g, namely obtains the Tris solution of 1.2g/L (about 10mmol/L) with the volumetric flask adding distil water constant volume of 1000ml; Mother liquor 2: taking concentrated hydrochloric acid 1g(massfraction is 37%), namely the HCl solution of 0.37g/L (about 10mmol/L) is obtained with the volumetric flask adding distil water constant volume of 1000ml, mother liquor 1 by volume afterwards: the two mixes by mother liquor 2=20:3, and obtain after correcting with pH meter.
The preparation method testing the norepinephrine HCI solution of 2mg/mL used is as follows: take norepinephrine solid 200mg, namely obtains the norepinephrine HCI solution of 2mg/mL with the Tris-HCl damping fluid constant volume that the volumetric flask of 100ml adds above-mentioned pH=8.5.
Test the CaCl of 0.33mol/L used
22H
2o solution and Na
2cO
3solution preparation method is as follows: take CaCl
22H
2o solid 0.33mol, namely obtains the CaCl of 0.33mol/L with the volumetric flask adding distil water constant volume of 1000ml
22H
2o solution; Take Na
2cO
3solid 0.33mol, namely obtains the Na of 0.33mol/L with the volumetric flask adding distil water constant volume of 1000ml
2cO
3solution.
PDADMAC solution and the PSS solution preparation method of testing 2mg/mL used are as follows: mother liquor 1: take NaCl solid 0.5mol, namely obtain 0.5mol/LNaCl solution with the volumetric flask adding distil water constant volume of 1000ml; Take PSS solid 1000mg, add with the volumetric flask of 500ml the PSS solution that namely mother liquor 1 constant volume obtains 2mg/mL; Take PDADMAC liquid 1000mg, add with the volumetric flask of 500ml the PDADMAC solution that namely mother liquor 1 constant volume obtains 2mg/mL.
Test silicon precursor solution preparation method used as follows: at room temperature by TMOS(methyl silicate, analytical pure) join in the HCL solution of 1.2g/L, TMOS dissolves completely, clarification after add pH=7 phosphate buffered saline buffer again, mixing; Its material proportion is volume ratio: TMOS:HCL solution: phosphoric acid buffer=1:19:200.
The oligomeric isomaltose product solution compound method of testing the 200mg/ml of pH=2-7 used is as follows: take oligomeric isomaltose product (IMO-500) 20g, namely obtains the oligomeric isomaltose product solution of 200mg/ml with the volumetric flask adding distil water constant volume of 100ml; Regulate the pH value of the oligomeric isomaltose product solution of 200mg/ml afterwards by the HCI solution of 1mol/L and the NaOH solution of 1mol/L, correct with pH meter.
Example 1:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=2) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 59.9%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 41.8%.
Example 2:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=3) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 69.8%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 50.4%.
Example 3:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 80.7%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 70.2%.
Example 4:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4.5) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 77.7%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 66.3%.
Example 5:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=6) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 63.4%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 52.7%.
Example 6:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (30 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 65.22%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 53.9%.
Example 7:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (35 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 76.3%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 65.7%.
Example 8:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (45 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 78.5%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 67.2%.
Example 9:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (50 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose reaches 62.7%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 51.5%.
Example 10:
1. the CaCl of 0.33mol/L is got
22H
2o solution 10ml, adds 300mgPSS and 800UGOD wherein, under magnetic stirring apparatus (600r/min), then add the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. in above-mentioned whole calcium carbonate microparticle, the PSS solution 15ml of 2mg/ml is added, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, particle is disperseed again, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 6h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme, freeze-drying is weighed as 103.3mg.
The immobilized multienzyme of above-mentioned preparation is put in the oligomeric isomaltose product solution (3ml, pH=4) of 200mg/ml, keeps temperature (40 DEG C) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose afterwards, the clearance of glucose is 70.2%.
More than operate repetition 6 times, the clearance of glucose is still higher, is 61.1%.
Embodiments of the invention are only illustrating invention, are not considered as the restriction to invention protection domain.
Unaccomplished matter of the present invention is known technology.
Claims (3)
1. immobilized multienzyme removes a processing method for the glucose in oligomeric isomaltose product, it is characterized by and comprises the following steps:
1) preparation of immobilized multienzyme: 1. sodium polystyrene sulfonate (PSS) preparation of calcium carbonate microparticle containing glucose oxidase (GOD) of adulterating: the CaCl getting 0.33mol/L
22H
2o solution 10ml, adds 300mg PSS and 800U GOD wherein, then under agitation adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes its Keep agitation 20s, by its standing 15-30min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. the PSS solution 15ml of 2mg/ml is added in the whole calcium carbonate microparticles obtained upward, vibration 15min, centrifugal, remove supernatant, washing, obtain the calcium carbonate microparticle of PSS parcel; Backward PSS parcel calcium carbonate microparticle in add Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain the calcium carbonate microparticle of the organic bilayer being enclosed with PSS and PDADMAC, be called for short (PSS/PDADMAC)
1-CaCO
3; Backward (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtain (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) of backward PSS parcel
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of two organic bilayers being enclosed with PSS and PDADMAC, is called for short (PSS/PDADMAC)
2-CaCO
3; 3. by whole (PSS/PDADMAC) of above-mentioned preparation
2-CaCO
3be scattered in silicon precursor solution 15ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains hybrid microspheres; 4. in above-mentioned whole hybrid microspheres, the norepinephrine HCI solution 15ml of 2mg/ml is added again, Keep agitation 4-8h, centrifugal, remove supernatant liquor, be then washed till supernatant liquor with the Tris-HCl damping fluid of pH=8.5 colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. in above-mentioned whole complex microsphere, add Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) of pH=8.5 after, Keep agitation 6-12h under magnetic stirring apparatus, centrifugal, remove supernatant liquor, template is removed with the dilute hydrochloric acid of 2mmol/L, after washing, being fixed multienzyme;
2) immobilized multienzyme removes the glucose in oligomeric isomaltose product: be all put in by the immobilized multienzyme of above-mentioned preparation in the oligomeric isomaltose product solution of the 200mg/ml of 3ml, pH=2-7,30-50 DEG C is kept to react 12-24h, add 80-110mg calcium hydroxide, generate calglucon, centrifuging purifying; Finally obtain the oligomeric isomaltose solution removing glucose;
Being prepared as follows of described silicon precursor solution: at room temperature TMOS (methyl silicate) is joined in the HCL solution of 0.37g/L, TMOS dissolves completely, clarification after add pH=7 phosphate buffered saline buffer again, mixing; Its material proportion is volume ratio: TMOS:HCl solution: phosphoric acid buffer=1:19:200.
2. immobilized multienzyme as claimed in claim 1 removes the processing method of the glucose in oligomeric isomaltose product, the Tris-HCl buffer method that it is characterized by described pH=8.5 is as follows: mother liquor 1: take Tris solid 1.2g, namely obtains the Tris solution of 1.2g/L with the volumetric flask adding distil water constant volume of 1000ml; Mother liquor 2: take the concentrated hydrochloric acid 1g that massfraction is 37%, namely obtains the HCl solution of 0.37g/L with the volumetric flask adding distil water constant volume of 1000ml, afterwards mother liquor 1 by volume: the two mixes by mother liquor 2=20:3, and obtains after correcting with pH meter.
3. immobilized multienzyme as claimed in claim 1 removes the processing method of the glucose in oligomeric isomaltose product, the preparation method that it is characterized by the norepinephrine HCI solution of described 2mg/mL is as follows: take norepinephrine solid 200mg, namely obtains the norepinephrine HCI solution of 2mg/mL with the Tris-HCl damping fluid constant volume that the volumetric flask of 100ml adds above-mentioned pH=8.5.
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| CN102851273A (en) * | 2012-10-16 | 2013-01-02 | 河北工业大学 | Biomimetic immobilization method of multienzyme system |
| CN102943069A (en) * | 2012-11-27 | 2013-02-27 | 北京化工大学 | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt |
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| CN102943069A (en) * | 2012-11-27 | 2013-02-27 | 北京化工大学 | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt |
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