CN101481680B - Production method of glucose oxidase - Google Patents
Production method of glucose oxidase Download PDFInfo
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- CN101481680B CN101481680B CN2009100051267A CN200910005126A CN101481680B CN 101481680 B CN101481680 B CN 101481680B CN 2009100051267 A CN2009100051267 A CN 2009100051267A CN 200910005126 A CN200910005126 A CN 200910005126A CN 101481680 B CN101481680 B CN 101481680B
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Abstract
The invention discloses a method for producing glucose oxidase, comprising the following steps: (1) obtaining crude enzyme liquid of glucose oxidase from 1) fermentation broth with high content of glucose oxidase by 2) centrifugating and concentrating the fermentation broth; (2) purifying and de-salting the glucose oxidase; and (3) forming freeze-drying finished products of glucose oxidase. The method for producing glucose oxidase has characteristics of simple technique, easy operation, high enzyme survival rate, high yield and low cost. The prepared glucose oxidase can be widely applied to food additive industry.
Description
Technical field
The present invention relates to a kind of production method of glucose oxidase.
Background technology
As important food fresh keeping foodstuff additive, do not see complete glucose oxidase production technique report at present, most of documents and materials reports only are a certain links of production technique, link property is poor each other, the characteristics that operability is not strong.This patent technology comparatively system has been reported the glucose oxidase preparation production method, has fermentative production cycle weak point, and production unit requires simple, workable characteristics, preparation-obtained glucose oxidase has stable performance, and catalysis speed is fast, acts on characteristics such as the substrate height is single-minded.
Summary of the invention
The purpose of this invention is to provide a kind of production method of glucose oxidase, technology is simple, easy to operate, enzyme is lived height, yield is high, production cost is low.
For achieving the above object, technical scheme provided by the present invention is: a kind of production method of glucose oxidase may further comprise the steps:
(1) acquisition of glucose oxidase crude enzyme liquid:
1. high-load glucose oxidase fermented liquid obtains:
But picking covers with the Penicillium notatum lawn that the malaga carbohydrate oxidase is selected, 30～32 ℃ of shaking culture 24h in the one-level substratum;
Be inoculated in the secondary medium 30～32 ℃ of shaking culture 24h then with 2～3% inoculum sizes;
Have in the fermentor tank of three-stage culture medium in being inoculated in 2～3% inoculum sizes at last, the control dissolved oxygen is more than 60%, and pH remains between 6.0～7.0, and fermentation 36～48h can obtain the glucose oxidase fermented liquid more than the enzyme 7.0U of being alive;
2. the centrifugal concentration of fermented liquid:
Fermented liquid is carried out centrifugal treating, 8000rpm, 20min collects supernatant liquor, and micro-filtration is removed residual molecule and thalline, and ultrafiltration and concentration reaches glucose oxidase fermented liquid more than the 20U until enzyme work;
(2) purification of glucose oxidase and desalination:
Under 4 ℃ of conditions, in ultrafiltration and concentration enzyme liquid, slowly add the ammonium sulfate solids particle, until forming 90% ammoniumsulphate soln, 4～6h saltouts, 12000r/min is centrifugal must to be precipitated, Tris-HCl buffered soln with pH 7.0 is collected protein precipitation, adds 8～10 times of deionized water dilutions, and desalination and concentration by ultrafiltration is handled;
(3) form glucose oxidase freeze-drying finished product:
Add protective material vacuum freezedrying 48 hours under-20 ℃ of conditions, promptly obtain the glucose oxidase dried frozen aquatic products.
The composition of one-level substratum, secondary medium and the three-stage culture medium of described step in 1. is: glucose 80g/L, peptone 5g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, calcium chloride 2g/L.
The advantage that the present invention has: the present invention produces glucose oxidase and has the advantages that technology is simple, easy to operate, enzyme is lived height, yield is high, production cost is low, obtains the glucose oxidase for preparing and can be widely used in the foodstuff additive industry.
1, the present invention comparatively system announcement the technological process of production of glucose oxidase dried frozen aquatic products, the oenoxydase for preparing has the yield height, the enzyme high characteristics of living.
2, this technical process is simple to operate, and strong operability is fit to scale operation, and preparation-obtained glucose oxidase is yellowish-white, and impurity is few, does not have any deleterious pharmaceutical chemicals, can be widely used in the industry of foodstuff additive amount.
Embodiment:
The invention will be further described below in conjunction with specific embodiment, to help understanding content of the present invention.
Embodiment
(1), the acquisition of glucose oxidase crude enzyme liquid:
1. high-load glucose oxidase fermented liquid obtains
But a, picking cover with malaga carbohydrate oxidase penicillium notatum lawn in the sterilising medium (glucose 80g/L, peptone 5g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, calcium chloride 2g/L) of 30ml (250ml specification triangular flask), 30～32 ℃ of shaking culture 24h;
B, be inoculated in 2～3% inoculum sizes and taken advantage of in 150ml (the 250ml specification triangular flask) sterilising medium (glucose 80g/L, peptone 5g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, calcium chloride 2g/L) 30～32 ℃ of shaking culture 24h then;
C, be inoculated in the substratum of having sterilized (glucose 80g/L, peptone 5g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, the calcium chloride 2g/L) fermentor tank with 2～3% inoculum sizes again, by revolution and air flow control dissolved oxygen is more than 60%, make by sodium hydroxide solution that pH remains between 6.0～7.0 in the fermentor tank, can obtain containing enzyme through 36～48h fermentation and live and be the above glucose oxidase fermented liquid of 7.0U (enzyme live definition: the enzyme amount of per minute catalyzed oxidation 1 μ mol glucose is defined as an enzyme activity unit).
2. the centrifugal concentration of fermented liquid
Centrifugally prepare fermented liquid (centrifugal condition 8000rpm 20min), removes mycelium and collects supernatant liquor, crosses microfiltration membrane and removes residual molecule and thalline, crosses ultra-filtration membrane and carries out concentration and reach more than the 20U until enzyme work.
(2), the progressive of glucose oxidase purified and desalination
Under 4 ℃ of conditions, slowly add ammonium sulfate in the ultrafiltration and concentration enzyme liquid until forming 90% ammoniumsulphate soln, 4～6h saltouts, 12000r/min is centrifugal must to be precipitated, collect protein precipitation with the dissolving of quantitative Tris-HCl (pH 7.0) buffering, add the deionized water dilution, handle via a certain amount of Hollow Fiber Ultrafiltration post film desalination and concentration.
(3), glucose oxidase freeze-drying finished product forms
Collection obtains desalination and concentration glucose oxidase enzyme liquid, adds protective material vacuum freezedrying 48 hours under-20 ℃ of conditions, promptly obtains the glucose oxidase dried frozen aquatic products.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, though the present invention discloses as above with preferred embodiment, yet be not in order to limit the present invention, any those skilled in the art, in not breaking away from the technical solution of the present invention scope, when the technology contents that can utilize above-mentioned announcement is made a little change or is modified to the equivalent embodiment of equivalent variations, in every case be the content that does not break away from technical solution of the present invention, according to technical spirit of the present invention to any simple modification that above embodiment did, equivalent variations and modification all still belong in the scope of technical solution of the present invention.
Claims (1)
1. production method of glucose oxidase may further comprise the steps:
(1) acquisition of glucose oxidase crude enzyme liquid:
1. high-load glucose oxidase fermented liquid obtains:
But picking covers with the Penicillium notatum lawn that the malaga carbohydrate oxidase is selected, 30～32 ℃ of shaking culture 24h in the one-level substratum;
Be inoculated in the secondary medium 30～32 ℃ of shaking culture 24h then with 2～3% inoculum sizes;
Have in the fermentor tank of three-stage culture medium in being inoculated in 2～3% inoculum sizes at last, the control dissolved oxygen is more than 60%, and pH remains between 6.0～7.0, and fermentation 36～48h can obtain the glucose oxidase fermented liquid more than the enzyme 7.0U of being alive;
2. the centrifugal concentration of fermented liquid:
Fermented liquid is carried out centrifugal treating, 8000rpm, 20min collects supernatant liquor, and micro-filtration is removed residual molecule and thalline, and ultrafiltration and concentration reaches glucose oxidase fermented liquid more than the 20U until enzyme work;
(2) purification of glucose oxidase and desalination:
Under 4 ℃ of conditions, in ultrafiltration and concentration enzyme liquid, slowly add the ammonium sulfate solids particle, until forming 90% ammoniumsulphate soln, 4～6h saltouts, 12000r/min is centrifugal must to be precipitated, Tris-HCl buffered soln with pH 7.0 is collected protein precipitation, adds 8～10 times of deionized water dilutions, and desalination and concentration by ultrafiltration is handled;
(3) form glucose oxidase freeze-drying finished product:
Add protective material vacuum freezedrying 48 hours under-20 ℃ of conditions, promptly obtain the glucose oxidase dried frozen aquatic products;
The composition of one-level substratum, secondary medium and the three-stage culture medium of described step in 1. is: glucose 80g/L, peptone 5g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, calcium chloride 2g/L.
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| CN2009100051267A CN101481680B (en) | 2009-01-19 | 2009-01-19 | Production method of glucose oxidase |
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| CN2009100051267A CN101481680B (en) | 2009-01-19 | 2009-01-19 | Production method of glucose oxidase |
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| CN101481680B true CN101481680B (en) | 2011-06-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502598A (en) * | 2017-10-19 | 2017-12-22 | 蒋文明 | A kind of preparation method of immobilized glucose oxidase |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101787360B (en) * | 2010-01-20 | 2012-01-04 | 云南万芳生物技术有限公司 | Tobacco processing active complex enzyme preparation for improving and enhancing tobacco quality and preparation method thereof |
| CN102154233A (en) * | 2010-12-31 | 2011-08-17 | 山东西王生化科技有限公司 | Method for preparing glucose oxidase from gluconate waste thallus |
| CN103397005B (en) * | 2013-08-07 | 2014-12-17 | 苏州昆蓝生物科技有限公司 | Production method of glucose oxidase |
| CN103966179B (en) * | 2014-05-27 | 2016-04-13 | 河北省微生物研究所 | The preparation method of glucose oxidase preparation and utilization thereof |
| CN106978404B (en) * | 2016-12-30 | 2020-06-26 | 徐州工程学院 | Method for preparing glucose oxidase enzyme preparation by submerged liquid fermentation and application |
| CN107164427A (en) * | 2017-06-15 | 2017-09-15 | 重庆大学 | A kind of method that enzyme process prepares carboxylated viscose rayon |
| CN107353049A (en) * | 2017-08-10 | 2017-11-17 | 黑龙江九穗谷农业科技发展有限公司 | A kind of preparation method of multiple-effect biological bacterium enzyme |
| CN110885801B (en) * | 2019-11-25 | 2021-04-30 | 中国海洋大学 | Glucose oxidase M5GOD and coding gene and application thereof |
| CN111011599A (en) * | 2019-12-20 | 2020-04-17 | 河南翔大生物科技有限公司 | Preparation method and application of additive for removing endotoxin in animal body |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1060026A (en) * | 1991-06-04 | 1992-04-08 | 湛江向阳制药厂 | Process for glucose oxidase cold-dry powder |
| WO2006123343A2 (en) * | 2005-05-18 | 2006-11-23 | Ramot At Tel Aviv University Ltd. | Biologically active metal-coated proteins |
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2009
- 2009-01-19 CN CN2009100051267A patent/CN101481680B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1060026A (en) * | 1991-06-04 | 1992-04-08 | 湛江向阳制药厂 | Process for glucose oxidase cold-dry powder |
| WO2006123343A2 (en) * | 2005-05-18 | 2006-11-23 | Ramot At Tel Aviv University Ltd. | Biologically active metal-coated proteins |
Non-Patent Citations (2)
| Title |
|---|
| 刘峰等.从土壤中快速筛选葡萄糖氧化酶产生菌及发酵工艺的优化.《生物技术》.2007,第17卷(第3期),第64-68页. * |
| 郭晓贤等.葡萄糖氧化酶产生菌的快速筛选及其产酶条件的研究.《饲料工业》.2007,第28卷(第8期),第17-20页. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502598A (en) * | 2017-10-19 | 2017-12-22 | 蒋文明 | A kind of preparation method of immobilized glucose oxidase |
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| CN101481680A (en) | 2009-07-15 |
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Application publication date: 20090715 Assignee: Shandong Sinder Animal Vaccine Co., Ltd. Assignor: Shandong Sinder Technology Co., Ltd. Contract record no.: 2014990000224 Denomination of invention: Production method of glucose oxidase Granted publication date: 20110622 License type: Exclusive License Record date: 20140421 |
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