CN108795892B - Method for preparing, separating and purifying glucose oxidase - Google Patents
Method for preparing, separating and purifying glucose oxidase Download PDFInfo
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Abstract
The invention belongs to the technical field of enzyme separation, and discloses a method for preparing, separating and purifying glucose oxidase, which comprises the following steps: step 1) culturing Aspergillus niger, step 2) preparing fermentation liquor, step 3) preparing crude enzyme liquid, and step 4) separating and purifying. The product obtained by the invention has high enzyme activity and the yield can reach more than 50 percent.
Description
Technical Field
The invention belongs to the technical field of enzyme separation, and particularly relates to a method for preparing, separating and purifying glucose oxidase.
Background
Glucose Oxidase (GOD) specifically catalyzes beta-D-glucose to be oxidized into 1, 5-gluconolactone and hydrogen peroxide in the presence of molecular oxygen, and then the gluconolactone is spontaneously combined with water to generate gluconic acid and hydrogen peroxide. Glucose oxidase is an important enzyme in the technical field of enzymes, and is widely applied to food processing, fermentation industry and medical detection.
The glucose oxidase is light yellow powder, is easily soluble in water, and is completely insoluble in diethyl ether, chloroform, butanol, pyridine, glycerol, ethylene glycol, etc. 50% acetone and 66% methanol can precipitate the catalase, and the general preparation contains catalase. The molecular weight is about 150000, and each gram of enzyme contains 2g of FAD. The maximum light absorption wavelengths of the enzymes were 377nm and 455 nm. No fluorescence under uv light but a distinctive green color after heat, acid or base treatment. The solid enzyme preparation is stable for at least two years at 0 ℃ and 8 years at-15 ℃. The stable pH value range of the glucose oxidase is 3-4, the optimal pH value is 5, and if no protective agent such as glucose exists, the pH value is more than 8 or less than 3, and the glucose oxidase can be quickly inactivated. The action temperature of the glucose oxidase is 30-60 ℃, the enzyme is not inhibited by EDTA, KCN and NaF, but HgCl2, AgCl, p-chloromercuribenzoic acid and phenylhydrazine are inhibited by the enzyme. In the presence of oxygen, glucose oxidase can remove glucose from a system, and in the presence of excess glucose, oxygen can be removed. Based on the principle, the glucose oxidase can be widely applied to the aspects of food manufacture, oxygen removal and rust prevention of instruments and the like. Such as: desugarizing is carried out in the egg processing process to prevent browning; deoxygenation of fruit juices, beer, food cans, and the like; used for preventing shrimp meat and dry and fresh food from oxidation; to produce gluconic acid and the like. In addition, glucose oxidase has also been used for the quantitative determination of glucose in several complex biochemical systems, in view of its high specificity of action. Such as: measuring the content of glucose in blood; the prepared diabetes test paper is used for clinical diagnosis.
Glucose oxidase is widely distributed in animals, plants and microorganisms, and because the extraction of the glucose oxidase from the animals and plants has certain limitation, the glucose oxidase is not abundant in enzyme amount, and the capability of producing the glucose oxidase of mould is strong under certain conditions, so that the main mould which is convenient for producing the glucose oxidase on a large scale is derived from the mould. The production methods of enzymes can be classified into extraction methods, fermentation methods, and chemical synthesis methods. Fermentation has been the main method for producing enzymes since the 50 th of the 20 th century, and it is intended to obtain enzymes required by people by utilizing the vital activities of cells, mainly microbial cells.
Glucose oxidase is generally produced by using a strain of the genus Mycoplasma as a producing bacterium. The enzyme production efficiency difference of the strains is large under different conditions, and the selection of the enzyme production strains to ferment under the condition suitable for the enzyme production of the strains is the key point of research. According to the literature, the mutation breeding of the penicillium notatum strain capable of producing glucose oxidase at high yield is reported in 2006, penicillium notatum scientific academy for Hebei province, a strain capable of producing glucose oxidase at high yield is obtained by taking penicillium notatum 8312 as an initial strain and carrying out ultraviolet mutation treatment, primary screening and secondary screening, and the strain is subjected to continuous 12 times of passage tests, so that the mutant strain is stable in hereditary character, the enzyme production level in a shake flask reaches 55.7U/mL, and is improved by 53.9% compared with the initial strain (36.2U/mL). The literature, "mutation breeding of glucose oxidase-producing aspergillus niger and research of calcium gluconate fermentation conditions, science and technology in the food industry in 2010" discloses that aspergillus niger strains producing glucose oxidase are subjected to mutagenesis by ultraviolet rays and cobalt 60 radioactive rays, a selective culture medium containing 2-deoxy-D-glucose is used for screening, and 7 tolerant strains are obtained in total, wherein the glucose oxidase activity of the U-69 strain producing the largest enzyme activity is 2.5 times that of the original strain. The fermentation conditions for synthesizing calcium gluconate by using the U-69 strain are studied, and the results show that the optimal fermentation conditions are 150g/L of glucose, 400g/L of calcium carbonate, 3-4 g/L of ammonium sulfate, pH5.5, 30mL of fermentation liquor in a 250mL triangular bottle, the inoculation amount is 10%, and the fermentation is carried out for 18 hours at the temperature of 30-34 ℃. In the fermentative production of glucose oxidase, glucose is generally used as a carbon source, which is used by the bacterial cells as an inducer of enzyme synthesis, while catabolites thereof repress biosynthesis, and therefore, controlling the glucose concentration in the fermentation medium has a great influence on the enzyme production by the strain. The applicant's previous research result ' a method for preparing glucose oxidase by microbial fermentation ', takes aspergillus niger as an original strain, lactobacillus casei is subjected to synergistic fermentation, and culture conditions are optimized, so that the enzyme activity is improved, and the fermentation time is shortened.
Disclosure of Invention
On the basis of the prior research, the invention continuously separates and purifies the fermentation liquor to obtain the enzyme preparation with higher purity.
The technical scheme of the invention is implemented by the following modes:
a method of preparing, isolating and purifying glucose oxidase comprising the steps of: step 1) culturing Aspergillus niger, step 2) preparing fermentation liquor, step 3) preparing crude enzyme liquid, and step 4) separating and purifying.
Further, the method comprises the following steps:
step 1) culturing Aspergillus niger: culturing Aspergillus niger seed liquid in a fermentation tank containing fermentation culture medium according to 6-8% of inoculum size, at 30-34 deg.C, under 0.02-0.03MPa, with air flow of 500-600L/h and culture time of 30-40 h;
step 2) preparing fermentation liquor: inoculating lactobacillus casei seed solution into a fermentation tank according to the inoculation amount of 8-10%, adding methanol, continuously culturing for 20-30h, and adding ammonia water to control pH to 7.2-7.8 to obtain fermentation liquor;
step 3) preparing a crude enzyme solution: centrifuging at 5000rpm for 5min, collecting thallus precipitate, grinding and pulverizing thallus precipitate, adding 0.1mol/L phosphoric acid buffer solution with weight of five times, leaching at 4 deg.C for 2 hr, centrifuging at 12000rpm for 20min at 4 deg.C, and collecting supernatant to obtain crude enzyme solution;
step 4), separation and purification: collecting the crude enzyme solution, passing through DEAE-Sepharose ion exchange chromatography column, collecting eluate, passing through Superdex-200 gel filtration chromatography column, eluting, collecting eluate, dialyzing, desalting, and freeze drying.
Preferably, the first and second electrodes are formed of a metal,
the addition amount of the methanol is 0.1-0.2 wt%.
Preferably, the first and second electrodes are formed of a metal,
the speed of the centrifugation is 2000rpm, and the time is 5 min.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the Aspergillus niger seed liquid comprises the following steps: inoculating Aspergillus niger on slant culture medium for culturing, inoculating into seed culture medium for culturing to obtain density of (1-2) × 108CFU/mL Aspergillus niger seed solution.
Preferably, the first and second electrodes are formed of a metal,
the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar.
Preferably, the first and second electrodes are formed of a metal,
the seed culture medium comprises the following components: 20g/L of sucrose, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and 0.01g/L of ferrous sulfate heptahydrate.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the lactobacillus casei seed liquid comprises the following steps: inoculating Lactobacillus casei into MRS agar culture medium, culturing, inoculating into MRS liquid culture medium, and seed culturing to obtain lactobacillus casei with density of (3-5) x 108CFU/mL of Lactobacillus casei seed solution.
The technical scheme of the invention brings a series of beneficial effects by improving the prior art, and mainly comprises the following aspects:
the glucose oxidase is prepared by mixing and fermenting two strains, the purity is high, and the yield can reach more than 50%;
the high-concentration carbon source is beneficial to thallus proliferation, but can block enzyme production, possibly influence the permeation of enzyme to the outside of cells, and also possibly influence the enzyme activity expression by the denaturation of protein due to acid concentration; according to the invention, the Aspergillus niger is subjected to propagation culture by adopting a high-concentration carbon source, when the Aspergillus niger is cultured for 30-40h, the biomass of the strain is obviously increased, the enzyme production capacity is improved at the moment, but the enzyme production effect is inhibited by high-concentration glucose, and the inoculated lactobacillus casei can utilize the glucose to compete with the Aspergillus niger, so that the enzyme production capacity is improved;
glucose oxidase generated by aspergillus niger can metabolize glucose into gluconic acid, and can generate a feedback inhibition effect on the synthesis of the glucose oxidase along with the increase of the concentration of a metabolite, and lactobacillus casei can utilize the gluconic acid as a carbon source, so that the inhibition effect is effectively removed;
according to the invention, the low-concentration methanol is added in the second stage culture, so that the permeability of cell membranes can be increased, and the secretion level can be improved; proper pH and calcium ions can influence enzyme synthesis and improve enzyme activity. The invention optimizes the factors through single factor test to determine the best enzyme production condition.
The invention comprises the following steps: the strains specifically used in the embodiments are aspergillus niger ATCC20611, lactobacillus casei ATCC 334; other strains of the same species that function similarly can also be used.
Drawings
FIG. 1: influence of methanol concentration on enzyme activity:
FIG. 2: influence of pH on enzyme activity;
FIG. 3: the influence of the second stage culture time on the enzyme activity.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
A method of preparing, isolating and purifying glucose oxidase comprising the steps of:
inoculating Aspergillus niger on slant culture medium for culturing, inoculating to seed culture medium, and culturing to obtain Aspergillus niger with density of 1 × 108CFU/mL Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the seed culture medium comprises the following components: 20g/L of sucrose, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and 0.01g/L of ferrous sulfate heptahydrate;
inoculating Lactobacillus casei into MRS agar culture medium for culturing, and inoculating into MRS liquid culture medium for culturingSeed culture to obtain density of 3 × 108CFU/mL of lactobacillus casei seed liquid;
culturing Aspergillus niger seed liquid in a fermentation tank containing 8% of fermentation medium at 32 deg.C under 0.02MPa for 35h with air volume of 500L/h;
inoculating lactobacillus casei seed solution into a fermentation tank according to the inoculation amount of 10%, adding 0.1wt% methanol, culturing for 25h, controlling pH to 7.5 by feeding ammonia water to obtain fermentation liquid,
centrifuging at 5000rpm for 5min, collecting thallus precipitate, grinding and pulverizing thallus precipitate, adding 0.1mol/L phosphoric acid buffer solution with weight of five times, leaching at 4 deg.C for 2 hr, centrifuging at 12000rpm for 20min at 4 deg.C, and collecting supernatant to obtain crude enzyme solution; using 0.05mol/L Tris-HCl buffer solution with pH7.1 to balance a DEAE-Sepharose ion exchange chromatographic column, taking a crude enzyme liquid to load, using 0.5mol/L NaCl (prepared by using 0.05mol/L Tris-HCl buffer solution with pH7.1) to carry out gradient elution, carrying out linear gradient elution, collecting and combining in parts, then using a Superdex-200 gel filtration chromatographic column overnight, using 0.05mol/L Tris-HCl buffer solution with pH7.1 to carry out elution with the flow rate of 0.3mL/min, collecting eluent, dialyzing and desalting, freezing and drying to obtain a pure product.
The fermentation medium comprises the following components: 50g/L of glucose, 30g/L of corn steep liquor, 20g/L of calcium carbonate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of urea, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate and the pH value of 7.0.
Example 2
A method of preparing, isolating and purifying glucose oxidase comprising the steps of:
inoculating Aspergillus niger on slant culture medium for culturing, inoculating to seed culture medium for culturing to obtain Aspergillus niger with density of 2 × 108CFU/mL Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the seed culture medium comprises the following components: 20g/L of cane sugar, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate and 0.01g/L of manganese sulfate monohydrateL, 0.01g/L ferrous sulfate heptahydrate;
inoculating Lactobacillus casei into MRS agar culture medium for culturing, inoculating into MRS liquid culture medium for seed culture to obtain lactobacillus casei with density of 5 × 108CFU/mL of lactobacillus casei seed liquid;
the first stage culture: culturing Aspergillus niger seed liquid in a fermentation tank containing fermentation medium at 6% inoculation amount at 30 deg.C under 0.03MPa for 40h with air volume of 600L/h;
and (3) second-stage culture: inoculating lactobacillus casei seed liquid into a fermentation tank according to the inoculation amount of 8%, adding 0.15wt% of methanol, continuing to culture for 30 hours, controlling the pH to be 7.8 by feeding ammonia water, centrifuging the obtained fermentation liquid at 5000rpm for 5 minutes, collecting thallus precipitates, grinding and crushing the thallus precipitates, adding five times of 0.1mol/L phosphoric acid buffer solution for leaching, leaching at 4 ℃ for 2 hours, centrifuging at 12000rpm for 20 minutes after completion, and taking supernatant, namely crude enzyme liquid; using 0.05mol/L Tris-HCl buffer solution with pH7.1 to balance a DEAE-Sepharose ion exchange chromatographic column, taking a crude enzyme liquid to load, using 0.5mol/L NaCl (prepared by using 0.05mol/L Tris-HCl buffer solution with pH7.1) to carry out gradient elution, carrying out linear gradient elution, collecting and combining the fractions, then using a Superdex-200 gel filtration chromatographic column for overnight, using 0.05mol/L Tris-HCl buffer solution with pH7.1 to carry out elution with the flow rate of 0.3mL/min, collecting eluent, dialyzing and desalting, freezing and drying to obtain the finished product.
The fermentation medium comprises the following components: 50g/L of glucose, 30g/L of corn steep liquor, 20g/L of calcium carbonate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of urea, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate and the pH value of 7.0.
Comparative example 1
A method of preparing, isolating and purifying glucose oxidase comprising the steps of:
inoculating Aspergillus niger on slant culture medium for culturing, inoculating to seed culture medium, and culturing to obtain Aspergillus niger with density of 1 × 108CFU/mL Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the above-mentionedThe seed culture medium comprises the following components: 20g/L of sucrose, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and 0.01g/L of ferrous sulfate heptahydrate;
culturing Aspergillus niger seed liquid in a fermentation tank containing 8% of inoculation amount of fermentation medium at 33 deg.C under 0.02MPa, air volume of 600L/h and culture time of 60h to obtain fermentation liquid, centrifuging at 5000rpm for 5min, collecting thallus precipitate, grinding and pulverizing the thallus precipitate, adding five times of 0.1mol/L phosphoric acid buffer solution, leaching at 4 deg.C for 2h, centrifuging at 4 deg.C and 12000rpm for 20min, and collecting supernatant to obtain crude enzyme liquid; using 0.05mol/L Tris-HCl buffer solution with pH7.1 to balance a DEAE-Sepharose ion exchange chromatographic column, taking a crude enzyme liquid to load, using 0.5mol/L NaCl (prepared by using 0.05mol/L Tris-HCl buffer solution with pH7.1) to carry out gradient elution, carrying out linear gradient elution, collecting and combining the fractions, then using a Superdex-200 gel filtration chromatographic column for overnight, using 0.05mol/L Tris-HCl buffer solution with pH7.1 to carry out elution with the flow rate of 0.3mL/min, collecting eluent, dialyzing and desalting, freezing and drying to obtain the finished product.
The fermentation medium comprises the following components: 50g/L of glucose, 30g/L of corn steep liquor, 20g/L of calcium carbonate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of urea, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate and the pH value of 7.0.
Comparative example 2
A method of preparing, isolating and purifying glucose oxidase comprising the steps of:
inoculating Aspergillus niger on slant culture medium for culturing, inoculating to seed culture medium, and culturing to obtain Aspergillus niger with density of 1 × 108CFU/mL Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the seed culture medium comprises the following components: 20g/L of sucrose, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and 0.01g/L of ferrous sulfate heptahydrate;
inoculating lactobacillus casei into MRS agar culture mediumCulturing, inoculating to MRS liquid culture medium, and culturing to obtain the final product with density of 3 × 108CFU/mL of lactobacillus casei seed liquid;
inoculating an Aspergillus niger seed liquid into a fermentation tank containing a fermentation culture medium according to an inoculation amount of 8% and an inoculation amount of 10% to culture, adding 0.1wt% of methanol, continuing to culture for 25h, controlling the pH to be 7.5 by feeding ammonia water, controlling the culture temperature to be 32 ℃, the tank pressure to be 0.02MPa, the air volume to be 500L/h and the culture time to be 60h to obtain a fermentation liquid, centrifuging for 5min at 5000rpm, collecting a thallus precipitate, grinding and crushing the thallus precipitate, adding 0.1mol/L phosphoric acid buffer solution with five-fold weight to leach for 2h at 4 ℃, centrifuging for 20min at 12000rpm after completion, and taking a supernatant to obtain a crude enzyme liquid; using 0.05mol/L Tris-HCl buffer solution with pH7.1 to balance a DEAE-Sepharose ion exchange chromatographic column, taking a crude enzyme liquid to load, using 0.5mol/L NaCl (prepared by using 0.05mol/L Tris-HCl buffer solution with pH7.1) to carry out gradient elution, carrying out linear gradient elution, collecting and combining the fractions, then using a Superdex-200 gel filtration chromatographic column for overnight, using 0.05mol/L Tris-HCl buffer solution with pH7.1 to carry out elution with the flow rate of 0.3mL/min, collecting eluent, dialyzing and desalting, freezing and drying to obtain the finished product.
The fermentation medium comprises the following components: 50g/L of glucose, 30g/L of corn steep liquor, 20g/L of calcium carbonate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.2g/L of urea, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate and the pH value of 7.0.
Example 3
Taking example 1 and comparative examples 1-2 as examples, the glucose concentration in the fermentation broth after 35h of fermentation and after the end of fermentation, the gluconic acid concentration after the end of fermentation and the biomass of the cells were determined, and the specific results are shown in table 1:
TABLE 1
| Group of | Glucose concentration g/L for 35h | Glucose concentration g/L after termination | The concentration of gluconic acid is g/L after the end | Bacterial biomass g/L |
| Example 1 | 17.8 | 0.6 | 0.1 | 87.1 |
| Comparative example 1 | 18.3 | 5.7 | 4.3 | 51.4 |
| Comparative example 2 | 11.2 | 1.3 | 0.7 | 69.3 |
As shown in Table 1, in example 1, the sequential inoculation mode is adopted, the biomass is highest and the concentration of gluconic acid is lowest after the fermentation is finished; glucose oxidase generated by aspergillus niger can metabolize glucose into gluconic acid, a feedback inhibition effect can be generated on the synthesis of the glucose oxidase along with the increase of the concentration of a metabolite, and lactobacillus casei can utilize the gluconic acid as a carbon source, so that the inhibition effect is effectively eliminated, and the enzyme production efficiency is improved.
Example 4
The enzyme activity test of the crude enzyme solution obtained by the invention comprises the following steps:
the absorbance was measured spectrophotometrically (absorbance at 460 nm). Specific enzyme activity data are shown in table 2:
TABLE 2
| Group of | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 |
| Enzyme activity U/ml | 231 | 249 | 92 | 138 |
Example 5
Effect of methanol concentration, pH and second stage incubation time on enzyme production efficiency:
the methanol concentrations wt% are respectively set to be 0, 0.05,0.1,0.15.0.2,0.25 and 0.3, taking example 1 as an example, the enzyme production activity is detected, as shown in figure 1, methanol can increase the permeability of cell membranes and improve the secretion level of enzymatic metabolites, so that the enzyme production activity is improved, the methanol is beneficial to rapidly increasing the enzyme production activity along with the increase of the methanol concentration, after 0.1wt%, the activity is slowly increased along with the increase of the concentration, and probably because the cell toxicity is easily generated due to the overhigh concentration.
Six gradients of pH5.5, 6,6.5,7,7.5,8 and 8.5 are respectively set, taking example 1 as an example, and enzyme production activity is detected, wherein as shown in figure 2, the pH is maintained between 7 and 8 to be most beneficial to the improvement of enzyme activity. As shown in fig. 3, the selection of the culture time in the second stage for 25-30 hours can achieve higher enzyme activity, and as the time is further increased, the enzyme activity is not improved but reduced, and it may be that the nutrient content in the culture solution is too low to support the continuous proliferation of the strain, which causes the reduction of activity and autolysis of part of the strain; shortens the culture time and saves the cost.
Example 6
Taking example 1 as an example, 1000ml of crude enzyme solution is selected to finally obtain 7.1mg of pure product, and the indexes of the crude enzyme solution and the pure product are detected as follows:
TABLE 3
| Index (I) | Total activity (Wan U) | The recovery rate of enzyme activity is% |
| Crude enzyme solution | 23.1 | 100％ |
| Pure product | 11.9 | 55.6 |
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (8)
1. A method of preparing, isolating and purifying glucose oxidase, comprising the steps of:
step 1) culturing Aspergillus niger: culturing Aspergillus niger seed liquid in a fermentation tank containing fermentation culture medium according to 6-8% of inoculum size, at 30-34 deg.C, under 0.02-0.03MPa, with air flow of 500-600L/h and culture time of 30-40 h;
step 2) preparing fermentation liquor: inoculating lactobacillus casei seed solution into a fermentation tank according to the inoculation amount of 8-10%, adding methanol, continuously culturing for 20-30h, and adding ammonia water to control pH to 7.2-7.8 to obtain fermentation liquor; the addition amount of the methanol is 0.1-0.2 wt%;
step 3) preparing a crude enzyme solution: centrifuging at 5000rpm for 5min, collecting thallus precipitate, grinding and pulverizing thallus precipitate, adding 0.1mol/L phosphoric acid buffer solution with weight of five times, leaching at 4 deg.C, centrifuging at 12000rpm for 20min at 4 deg.C, and collecting supernatant to obtain crude enzyme solution;
step 4), separation and purification: collecting the crude enzyme solution, passing through DEAE-Sepharose ion exchange chromatography column, collecting eluate, passing through Superdex-200 gel filtration chromatography column, eluting, collecting eluate, dialyzing, desalting, and freeze drying.
2. The method of claim 1, wherein the centrifugation is performed at 2000rpm for 5 min.
3. The process according to claim 1, characterized in that the leaching time is 2 h.
4. The method according to claim 1, wherein the Aspergillus niger seed solution is prepared by: inoculating Aspergillus niger on slant culture medium for culturing, inoculating into seed culture medium for culturing to obtain density of (1-2) × 108CFU/mL dark starterAnd (4) mildew seed liquid.
5. The method of claim 4, wherein the slant medium composition is: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar.
6. The method of claim 4, wherein the seed medium comprises: 20g/L of sucrose, 10g/L of yeast extract, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.02g/L of magnesium sulfate heptahydrate, 0.01g/L of manganese sulfate monohydrate and 0.01g/L of ferrous sulfate heptahydrate.
7. The method of claim 1, wherein the lactobacillus casei seed fluid is prepared by: inoculating Lactobacillus casei into MRS agar culture medium, culturing, inoculating into MRS liquid culture medium, and seed culturing to obtain lactobacillus casei with density of (3-5) x 108CFU/mL of Lactobacillus casei seed solution.
8. The method of claim 1, wherein the ion exchange chromatography column is a DEAE-Sepharose ion exchange chromatography column and the gel filtration chromatography column is a Superdex-200 gel filtration chromatography column.
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