CN103524569A - 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 used for removing the processing method of oligomeric isomaltose product (IMO-500) glucose.
Background technology
Oligomeric isomaltose is called as " bifidus factor ".Through research for many years, show, bifidus bacillus has many nourishing functions, and as the oligomeric isomaltose of bifidus factor, has received people's concern.The few oligomeric isomaltose of occurring in nature exists with unbound state, therefore can not 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 oligomeric isomaltose product that China produces is that functional component is the primary products of 50% (IMO-500) mostly, has had a strong impact on its function and has tired and commercial value.And primary products are made to more than 90% oligomeric isomaltose of (IMO-900) of functional component, its economic benefit increases substantially.Therefore, improve the purity of oligomeric isomaltose, become the key issue that technique need to solve.
Bibliographical information about oligomeric isomaltose purifying products comprises: document 1:Science and Technology of Food industry, 2002,23 (5): 27-30 is 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 yeast fermentation method to carry out purifying to the oligomeric isomaltose product of low-purity.By yeast is carried out to screening experiment, select a strain and only utilize glucose and maltose, and do not utilize the yeast of oligomeric isomaltose.Utilize this bacterium to carry out purifying to oligomeric isomaltose product, final purity to 99%.Above method is higher to the efficiency of oligomeric isomaltose purifying products, but strain improvement overlong time, reacted bacterial classification is difficult for reusing again.Document 3:Journal of South China Agricultural University, 2005,26 (4): 102-105 utilizes 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 been used for reference the enzyme catalysis process in viable cell, and intracellular Substance Transformation is usually directed to the concerted catalysis process of plurality of enzymes, but this method used, is resolvase, is difficult for reclaiming, if the fixing Research Significance that will be in production application of enzyme is larger.Inspired by this, the structure that in simulation is biological, multienzyme system is carried out multienzyme system will efficiently be opened up new way for what realize multienzyme catalytic process.
Immobilized glucose oxidase and catalatic bibliographical information comprise: document 1: biotechnology journal, 1987,3 (1): 46-52 utilizes that diazonium salt covalent linkage is legal jointly to be received GOD and CAT and on Sepharose, prepare the two enzyme systems of immobilization.Document 2:Macromolecular Bioscience, 2004,4 (10): 950-956 is fixed on GOD and CAT on the polymeric film that is close in anion-exchange membrane jointly, in order to the production of gluconic acid.Document 3:Journal of Microbiology and Biotechnology, 2007,17 (6): 960-967 is fixed on florisil by GOD and CAT, and studied the fixing difference of fixing and order simultaneously.Result shows, no matter enzyme live aspect or cost consumption aspect, the fixing method of order is all better than fixing simultaneously.Compare with fixing separately GOD, optimum condition does not almost have change.And reuse with catalytic efficiency aspect, fixed system super independent fixing enzyme far away altogether.Document 4:Journal of Applied Polymer Science, 2004,91 (6), 4057-4063 is fixed on GOD and CAT on the fine multipolymer cytolemma of propylene of modification.
Above pertinent literature has been introduced immobilized glucose oxidase and catalatic different methods and application.Although being devoted to realize two enzymes, fixes current above strategy, but still the carrier of the immobilized multienzyme that urgently exploitation is gentle, efficient, easy, suitability is strong.So far, in document, yet there are no adopt layer-by-layer in conjunction with bionical silication, bionical bonding thought cellular-type immobilized glucose oxidase and catalase for removing the relevant report of the glucose of oligomeric isomaltose product.
Summary of the invention
The independent control that the object of the invention is (1) various enzymes of existing for current co-immobilization multi-enzyme system is poor; (2) the immobilization position of various enzymes is random, the more difficult object that forms substrate product chain that reaches; (3) being in contact with one another of multienzyme may cause the affected shortcoming of catalytic performance separately, and a kind of method of cellular-type immobilized multienzyme simple to operate, with low cost is provided, and this immobilized multienzyme is used for removing the glucose of oligomeric isomaltose product.It is to 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:
A processing method of removing the glucose in oligomeric isomaltose product, comprises the following steps:
1) preparation of immobilized multienzyme: the 1. preparation of the calcium carbonate microparticle containing glucose oxidase (GOD) of sodium polystyrene sulfonate (PSS) doping: the CaCl that 1. gets 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under agitation adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 15-30min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. the PSS solution 15ml that adds 2mg/ml in the whole calcium carbonate microparticles that obtain upward, vibration 15min, centrifugal, remove supernatant, washing, obtains the calcium carbonate microparticle that PSS wraps up; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 4-8h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after, to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6-12h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, being fixed multienzyme.
2) immobilized multienzyme is removed the glucose in oligomeric isomaltose product: the immobilized multienzyme of above-mentioned preparation is all put in the oligomeric isomaltose product solution of 200mg/ml of 3ml, pH=2-7, keep 30-50 ℃ of reaction 12-24h, add 80-110mg calcium hydroxide, generate calglucon, centrifuging purifying; Finally obtain removing the oligomeric isomaltose solution of glucose.
The related amount of substance of processing method that above-mentioned immobilized multienzyme is removed the glucose in oligomeric isomaltose product all can expand or dwindle by corresponding proportion is whole.
The Tris-HCl damping fluid compound method of described pH=8.5 is as follows: take Tris solid 1.2g, obtain 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%), with the volumetric flask adding distil water constant volume of 1000ml, obtain the HCl solution of 0.37g/L, afterwards mother liquor 1 by volume: mother liquor 2=20:3 mixes the two, and obtain after proofreading and correct 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 adds pH=7 phosphate buffered saline buffer again after dissolving completely, clarifying, mix; Its material proportion is volume ratio: TMOS:HCL solution: phosphoric acid buffer=1:19:200.
The preparation method of the norepinephrine hydrochloride solution of described 2mg/mL is as follows: take norepinephrine solid 200mg, obtain the norepinephrine hydrochloride 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 of 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, Xu FX, Du Y, Zhang ZL.J Mater Chem B, 2013,10:29. mentions its enzyme activity maximum can only reach 238.93U/g.And the immobilized enzyme of preparation can be used for removing the glucose in oligomeric isomaltose, and clearance reaches 80.73%, repeats 6 times, and 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 purchased from Hubei Ju Shunhong biochemical industry company limited.
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 liquid making method of testing pH=7 used is as follows: mother liquor 1: take NaH
2pO
42H
2o solid 0.1mol, 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, 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: mother liquor 2=2:3 mixes the two, and obtain after proofreading and correct with pH meter.
The Tris-HCl damping fluid compound method of testing pH=8.5 used is as follows: mother liquor 1: take Tris solid 1.2g, obtain 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%), with the volumetric flask adding distil water constant volume of 1000ml, obtain the HCl solution of 0.37g/L (about 10mmol/L), mother liquor 1 by volume afterwards: mother liquor 2=20:3 mixes the two, and obtains after proofreading and correct with pH meter.
The preparation method of norepinephrine hydrochloride solution who tests 2mg/mL used is as follows: take norepinephrine solid 200mg, obtain the norepinephrine hydrochloride 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, 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, 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, with the volumetric flask adding distil water constant volume of 1000ml, obtain 0.5mol/LNaCl solution; Take PSS solid 1000mg, with the volumetric flask of 500ml, add the PSS solution that mother liquor 1 constant volume obtains 2mg/mL; Take PDADMAC liquid 1000mg, with the volumetric flask of 500ml, add the PDADMAC solution that 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 adds pH=7 phosphate buffered saline buffer again after dissolving completely, clarifying, mix; Its material proportion is volume ratio: TMOS:HCL solution: phosphoric acid buffer=1:19:200.
The oligomeric isomaltose product solution compound method of 200mg/ml of testing pH=2-7 used is as follows: take oligomeric isomaltose product (IMO-500) 20g, obtain the oligomeric isomaltose product solution of 200mg/ml with the volumetric flask adding distil water constant volume of 100ml; With the HCI solution of 1mol/L and the NaOH solution of 1mol/L, regulate afterwards the pH value of the oligomeric isomaltose product solution of 200mg/ml, with pH meter, proofread and correct.
Example 1:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 59.9%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 41.8%.
Example 2:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 69.8%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 50.4%.
Example 3:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 80.7%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 70.2%.
Example 4:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 77.7%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 66.3%.
Example 5:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 63.4%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 52.7%.
Example 6:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 65.22%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 53.9%.
Example 7:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 76.3%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 65.7%.
Example 8:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 78.5%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 67.2%.
Example 9:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose reaches 62.7%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 51.5%.
Example 10:
1. get the CaCl of 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under magnetic stirring apparatus (600r/min), adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 20min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. to the PSS solution 15ml that adds 2mg/ml in above-mentioned whole calcium carbonate microparticles, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains the calcium carbonate microparticle of PSS parcel; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 6h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, 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 ℃) reaction 12h, add 100mg calcium hydroxide, generate calglucon, centrifuging purifying.By the component of liquid glucose after HPLC method detection separation and purification, use afterwards the clearance of glucose clearance (Degradation Rate)=1-glucose at residual night residual volume/raw glucose cubage glucose, the clearance of glucose is 70.2%.
More than operation repeats 6 times, and the clearance of glucose is still higher, is 61.1%.
Embodiments of the invention only, for to illustrating of inventing, are not considered as the restriction to invention protection domain.
Unaccomplished matter of the present invention is known technology.
Claims (4)
1. immobilized multienzyme is removed a processing method for the glucose in oligomeric isomaltose product, it is characterized by and comprises the following steps:
1) preparation of immobilized multienzyme: the 1. preparation of the calcium carbonate microparticle containing glucose oxidase (GOD) of sodium polystyrene sulfonate (PSS) doping: the CaCl that 1. gets 0.33mol/L
22H
2o solution 10ml, adds 300mgPSS and 800UGOD therein, then under agitation adds the Na of the 0.33mol/L of 10ml
2cO
3solution, makes it continue to stir 20s, by its standing 15-30min, shakes up, centrifugal, removes supernatant liquor, and washing, obtains calcium carbonate microparticle; 2. the PSS solution 15ml that adds 2mg/ml in the whole calcium carbonate microparticles that obtain upward, vibration 15min, centrifugal, remove supernatant, washing, obtains the calcium carbonate microparticle that PSS wraps up; Poly Dimethyl Diallyl Ammonium Chloride (PDADMAC) the solution 15ml that adds 2mg/ml in the calcium carbonate microparticle wrapping up to PSS afterwards, vibration 15min is centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of an organic bilayer of PSS and PDADMAC, is called for short (PSS/PDADMAC)
1-CaCO
3; Afterwards to (PSS/PDADMAC)
1-CaCO
3in add the PSS solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains (PSS/PDADMAC) of PSS parcel
1-CaCO
3; (PSS/PDADMAC) wrapping up to PSS afterwards
1-CaCO
3in add the PDADMAC solution 15ml of 2mg/ml, vibration 15min, centrifugal, remove supernatant liquor, washing, obtains being enclosed with the calcium carbonate microparticle of two organic bilayers of 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. again to the norepinephrine hydrochloride solution 15ml that adds 2mg/ml in above-mentioned whole hybrid microspheres, continue to stir 4-8h, centrifugal, remove supernatant liquor, then with the Tris-HCl damping fluid of pH=8.5, be washed till supernatant liquor colourless, centrifugal, remove supernatant liquor, obtain complex microsphere; 5. after, to the Tris-HCl damping fluid 20ml and the 4000U catalase (CAT) that add pH=8.5 in above-mentioned whole complex microspheres, under magnetic stirring apparatus, continue to stir 6-12h, centrifugal, remove supernatant liquor, with the dilute hydrochloric acid of 2mmol/L, remove template, washing afterwards, being fixed multienzyme;
2) immobilized multienzyme is removed the glucose in oligomeric isomaltose product: the immobilized multienzyme of above-mentioned preparation is all put in the oligomeric isomaltose product solution of 200mg/ml of 3ml, pH=2-7, keep 30-50 ℃ of reaction 12-24h, add 80-110mg calcium hydroxide, generate calglucon, centrifuging purifying; Finally obtain removing the oligomeric isomaltose solution of glucose.
2. immobilized multienzyme as claimed in claim 1 is removed the processing method of the glucose in oligomeric isomaltose product, the Tris-HCl damping fluid compound method that it is characterized by described pH=8.5 is as follows: take Tris solid 1.2g, obtain 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%), with the volumetric flask adding distil water constant volume of 1000ml, obtain the HCl solution of 0.37g/L, afterwards mother liquor 1 by volume: mother liquor 2=20:3 mixes the two, and obtain after proofreading and correct with pH meter.
3. immobilized multienzyme as claimed in claim 1 is removed the processing method of the glucose in oligomeric isomaltose product, it is characterized by 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 adds pH=7 phosphate buffered saline buffer again after dissolving completely, clarifying, mix; Its material proportion is volume ratio: TMOS:HCL solution: phosphoric acid buffer=1:19:200.
4. immobilized multienzyme as claimed in claim 1 is removed the processing method of the glucose in oligomeric isomaltose product, the preparation method of norepinephrine hydrochloride solution who it is characterized by described 2mg/mL is as follows: take norepinephrine solid 200mg, obtain the norepinephrine hydrochloride 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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| CN112760317A (en) * | 2021-04-07 | 2021-05-07 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by biomimetic silicified microcapsule immobilized multienzyme |
| CN112795598A (en) * | 2019-10-25 | 2021-05-14 | 中国科学院天津工业生物技术研究所 | Method for producing inositol based on silicon mineralized microcapsule immobilized multienzyme |
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