CN109234247A - A kind of glucose oxidase and preparation method thereof - Google Patents

A kind of glucose oxidase and preparation method thereof Download PDF

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CN109234247A
CN109234247A CN201811088698.1A CN201811088698A CN109234247A CN 109234247 A CN109234247 A CN 109234247A CN 201811088698 A CN201811088698 A CN 201811088698A CN 109234247 A CN109234247 A CN 109234247A
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glucose oxidase
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fermentation
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蔡海燕
郭杰
李觅
刘义会
孙中理
常少键
刘念
张磊
王超凯
张颖
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Sichuan Food Fermentation Industry Research and Design Institute
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

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Abstract

The present invention relates to a kind of glucose oxidases and preparation method thereof, belong to bio-fermentation engineering field；The following steps are included: (1) strain breeding thereof；(2) fermentation expands culture；(3) glucose oxidase is extracted；The present invention passes through the highest bacterial strain of enzyme activity after breeding mutagenic treatment, and the condition of culture by controlling each breeding phase, the calcium carbonate of suitable concentration is added in the concentration of glucose and culture medium of culture medium, in terms of strain excellent screening and suitable zymotechnique, the glucose oxidase of high enzyme activity of the invention (>=100U/mL) enzyme activity and high yield (yield >=20g/L) is prepared, preparation method of the invention has filled up the technological deficiency of the preparation low output of domestic glucose oxidase particularly suitable for industrialized production.

Description

A kind of glucose oxidase and preparation method thereof
Technical field
The invention belongs to bio-fermentation engineering fields, and in particular to a kind of glucose oxidase and preparation method thereof.
Background technique
Glucose oxidase is a kind of aerobic dehydrogenase.It can be catalyzed D-Glucose+O2D- gluconic acid (delta-lactone)+ H2O2Reaction, due to reaction generate H2O2Have sterilization functions, therefore there is excellent anti-microbial property.Glucose oxidase Purification contains the FAD of 2 molecules, as electron acceptor, removes O2Outside, also can be with 2,6, Dichlorophenol, indophenol reaction.The enzyme is to grape Sugar has specificity.Due to can quantitatively generate H2O2, therefore it is applied to biology extensively as the quantitative reagent of D-Glucose Chemical field and clinical examination.
Glucose oxidase market is very extensive, mainly in the following aspects application: 1, food additives；2, glucose Anti-corrosive fresh-keeping effect of the oxidizing ferment in animal product: fresh-keeping name your make, nontoxic residue-free increase delicious flavour；3, grape Effect of the carbohydrate oxidase in animal-breeding: removing in vivo, especially virulence factor in enteron aisle, and solid intestinal environment improves food It is intended to, increases feed intake, substitute antibiotics and probiotic, protect immune system, enhance liver function, it is residual to reduce drug in animal body It stays, improves livestock products quality；4, effect of the glucose oxidase in animal diseases control: can with Rapid reversal toxin poisoning, Especially mycotoxicosis；Curative effect can be increased with ancillary drug, accelerate recovery from illness.Such as, highly skilled doctor like find pleasure in, the products such as profit is strong Use in cultivation, remarkable benefit.
Country's fermentation method production glucose oxidase is difficult large-scale production at present, is primarily present following problems: zymogenic rate It is low, production technology is unstable etc.；Therefore carry out the researchs such as breeding high-yield bacterial strain, improvement zymotechnique, glucose oxidase is produced Industryization development has important meaning.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of glucose oxidase and preparation method thereof, passes through this method Glucose oxidase enzyme activity >=100U/mL of preparation, and the yield of glucose oxidase can be significantly improved.
Technical purpose of the invention is achieved through the following technical solutions:
One of the objects of the present invention is to provide a kind of preparation methods of glucose oxidase, comprising the following steps:
(1) strain breeding thereof
A, being put into slant medium to carry out constant temperature incubation to spore, cultivation temperature is generated by original strain is 26-28 DEG C, training Supporting the time is 120h, then washes down the spore from inclined-plane with sterile saline, is fitted into sterilizing bottle, mistake after shake well Spore suspension is made in filter, and the spore suspension concentration is 1 × 106-2 × 106 spore/mL；
B, spore suspension obtained by step A is subjected to induction mutation of bacterium processing, multiple plating mediums is then taken, by the spore Sub- suspension is spread evenly across in each plating medium with 0.2-0.3mL/ ware equivalent cultivates respectively, until growing bacterium colony；It is described Cultivation temperature is 25-27 DEG C, incubation time 72-120h；
C, it picks them separately each colony inoculation obtained by step B and carries out constant temperature incubation into different slant mediums, and carry out Label, until growing spore or mycelia, the cultivation temperature is 26~28 DEG C, incubation time 120h；Then by each spore or Mycelia carries out shake flask fermentation respectively, and fermentation temperature is 30 DEG C, fermentation time 48-60h, revolving speed 200r/min；Then by bottle Fermentation liquid is taken, the glucose oxidase enzyme activity in each fermentation liquid is measured, filters out the highest bacterial strain of enzyme activity；The fermentation medium PH value is 7-8；
(2) fermentation expands culture
The bacterial strain filtered out in step (1) is subjected to fermentation and expands culture, by weight, the component and proportion of culture medium Are as follows: 70~80 parts of glucose, 7~9 parts of sodium nitrate, 0.6~1 part of potassium chloride, 1~1.5 part of dipotassium hydrogen phosphate, magnesium sulfate 0.5~1 Part, 1-2 part of calcium carbonate, 900-1000 part of distilled water, condition of culture: liquid amount is 10~20ml/100ml, inoculum concentration for 3~ 7%, temperature is 30~35 DEG C, and revolving speed is 200~400r/min, and incubation time is 72~96h.
(3) glucose oxidase is extracted
Step (2) resulting culture is taken, under 0~5 DEG C, 4000~8000r/min speed conditions, centrifugation 10~ 15min, obtained supernatant are glucose oxidase liquid, and the glucose oxidase liquid is concentrated and dried, glucose is obtained Oxidizing ferment.
As a further optimization solution of the present invention, the slant medium in the step (1) and plating medium are Czapek's medium.
As a further optimization solution of the present invention, the mutagenic treatment in the step (1) is specially and hangs the spore Supernatant liquid is under 25-30W ultraviolet lamp, irradiation and stir process 15-20min at distance 15-20cm.
As a further optimization solution of the present invention, the component and proportion of the fermentation medium in the step (1) are as follows: Portugal Grape sugar 75 parts, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, distilled water 950 parts.
As a further optimization solution of the present invention, the bacterial strain filtered out in the step (1) is aspergillus niger or Penicillium notatum Strain.
As a further optimization solution of the present invention, the bacterial strain enzyme activity >=100U/mL filtered out in the step (1).
As a further optimization solution of the present invention, fermentation is adjusted using 10%HCl or 10%NaOH in the step (1) The pH of culture medium, it is preferred that the pH value of fermentation medium is 7.5.
As a further optimization solution of the present invention, the component and proportion of the culture medium in the step (2) are as follows: glucose 75 parts, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water, Condition of culture: liquid amount 15ml/100ml, inoculum concentration 5%, temperature are 30 DEG C, revolving speed 200r/min, and incubation time is 84h。
As a further optimization solution of the present invention, the drying temperature in the step (3) is 25-35 DEG C.
The second object of the present invention is to provide the glucose oxidase of more than one preparation method preparations.
Invention mechanism of the invention is:
The present invention passes through the highest bacterial strain of enzyme activity after breeding mutagenic treatment, and the culture item by controlling each breeding phase The calcium carbonate of suitable concentration is added in the concentration of glucose and culture medium of culture medium for part, screens from strain excellent and is suitable for fermentation Process aspect, has prepared the glucose oxidase of high enzyme activity and high yield of the invention, and preparation method of the invention is particularly suitable In industrialized production, the technological deficiency of the preparation low output of domestic glucose oxidase has been filled up.
The beneficial effects of the present invention are:
1, glucose oxidase prepared by the present invention is in enzyme activity >=100U/mL, yield >=20g/L；
2, preparation method of the present invention is simple to operation, particularly suitable for industrialized production.
Specific embodiment
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used It is further detailed in the present invention, should not be understood as limiting the scope of the invention, which is skilled in technique Some nonessential modifications and adaptations that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
A kind of preparation method of glucose oxidase, comprising the following steps:
(1) strain breeding thereof
A, being put into slant medium to carry out constant temperature incubation to spore, cultivation temperature is generated by original strain is 27 DEG C, culture Time is 120h, then washes down the spore from inclined-plane with sterile saline, is fitted into sterilizing bottle, filters after shake well Spore suspension is made, the spore suspension concentration is 1.5 × 106A spore/mL；
B, spore suspension obtained by step A is subjected to induction mutation of bacterium processing, multiple plating mediums is then taken, by the spore Sub- suspension is spread evenly across in each plating medium with 0.2mL/ ware equivalent cultivates respectively, until growing bacterium colony；The culture Temperature is 26 DEG C, incubation time 96h；
C, it picks them separately each colony inoculation obtained by step B and carries out constant temperature incubation into different slant mediums, and carry out Label, until growing spore or mycelia, the cultivation temperature is 27 DEG C, incubation time 120h；Then by each spore or mycelia Shake flask fermentation is carried out respectively, and fermentation temperature is 30 DEG C, fermentation time 54h, revolving speed 200r/min；Then fermentation is taken by bottle Liquid measures the glucose oxidase enzyme activity in each fermentation liquid, filters out the highest bacterial strain of enzyme activity；The fermentation medium pH value is 7.5；
(2) fermentation expands culture
The bacterial strain filtered out in step (1) is subjected to fermentation and expands culture, by weight, the component and proportion of culture medium Are as follows: 75 parts of glucose, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, steam by 2 parts of calcium carbonate 950 parts of distilled water, condition of culture: liquid amount 15ml/100ml, inoculum concentration 5%, temperature are 30 DEG C, revolving speed 200r/min, Incubation time is 84h.
(3) glucose oxidase is extracted
Step (2) resulting culture is taken, under 3 DEG C, 6000r/min speed conditions, is centrifuged 13min, obtained supernatant Liquid is glucose oxidase liquid, and the glucose oxidase liquid is concentrated and dried, glucose oxidase is obtained.
The above-mentioned slant medium stated in step (1) and plating medium are Czapek's medium.
Mutagenic treatment in above-mentioned steps (1) is specially by the spore suspension under 25W ultraviolet lamp, at distance 15cm Irradiate simultaneously stir process 15min.
The component and proportion of fermentation medium in above-mentioned steps (1) are as follows: 75 parts of glucose, 8 parts of sodium nitrate, potassium chloride 0.8 part, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water.
The bacterial strain filtered out in above-mentioned steps (1) is aspergillus niger or penicillium bacterial strain.
Bacterial strain enzyme activity >=the 100U/mL filtered out in above-mentioned steps (1).
The pH of fermentation medium is adjusted in above-mentioned steps (1) using 10%HCl or 10%NaOH, it is preferred that fermented and cultured The pH value of base is 7.5.
Drying temperature in above-mentioned steps (3) is 25-35 DEG C.
Embodiment 2
A kind of preparation method of glucose oxidase, comprising the following steps:
(1) strain breeding thereof
A, being put into slant medium to carry out constant temperature incubation to spore, cultivation temperature is generated by original strain is 26 DEG C, culture Time is 120h, then washes down the spore from inclined-plane with sterile saline, is fitted into sterilizing bottle, filters after shake well Spore suspension is made, the spore suspension concentration is 1 × 106A spore/mL；
B, spore suspension obtained by step A is subjected to induction mutation of bacterium processing, multiple plating mediums is then taken, by the spore Sub- suspension is spread evenly across in each plating medium with 0.2mL/ ware equivalent cultivates respectively, until growing bacterium colony；The culture Temperature is 25 DEG C, incubation time 72h；
C, it picks them separately each colony inoculation obtained by step B and carries out constant temperature incubation into different slant mediums, and carry out Label, until growing spore or mycelia, the cultivation temperature is 26 DEG C, incubation time 120h；Then by each spore or mycelia Shake flask fermentation is carried out respectively, and fermentation temperature is 30 DEG C, fermentation time 48h, revolving speed 200r/min；Then fermentation is taken by bottle Liquid measures the glucose oxidase enzyme activity in each fermentation liquid, filters out the highest bacterial strain of enzyme activity；The fermentation medium pH value is 7；
(2) fermentation expands culture
The bacterial strain filtered out in step (1) is subjected to fermentation and expands culture, by weight, the component and proportion of culture medium Are as follows: 70 parts of glucose, 7 parts of sodium nitrate, 0.6 part of potassium chloride, 1 part of dipotassium hydrogen phosphate, 0.5 part of magnesium sulfate, 1 part of calcium carbonate, distillation 900 parts of water, condition of culture: liquid amount 10ml/100ml, inoculum concentration 3%, temperature are 30 DEG C, revolving speed 200r/min, training Supporting the time is 72h.
(3) glucose oxidase is extracted
Step (2) resulting culture is taken, under 0 DEG C, 4000r/min speed conditions, is centrifuged 15min, obtained supernatant Liquid is glucose oxidase liquid, and the glucose oxidase liquid is concentrated and dried, glucose oxidase is obtained.
Slant medium and plating medium in above-mentioned steps (1) are Czapek's medium.
Mutagenic treatment in above-mentioned steps (1) is specially by the spore suspension under 25W ultraviolet lamp, at distance 15cm Irradiate simultaneously stir process 15min.
The component and proportion of fermentation medium in above-mentioned steps (1) are as follows: 75 parts of glucose, 8 parts of sodium nitrate, potassium chloride 0.8 part, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water.
The bacterial strain filtered out in above-mentioned steps (1) is aspergillus niger or penicillium bacterial strain.
Bacterial strain enzyme activity >=the 100U/mL filtered out in above-mentioned steps (1).
The pH of fermentation medium is adjusted in above-mentioned steps (1) using 10%HCl or 10%NaOH, it is preferred that fermented and cultured The pH value of base is 7.5.
Drying temperature in above-mentioned steps (3) is 25-35 DEG C.
Embodiment 3
A kind of preparation method of glucose oxidase, comprising the following steps:
(1) strain breeding thereof
A, being put into slant medium to carry out constant temperature incubation to spore, cultivation temperature is generated by original strain is 28 DEG C, culture Time is 120h, then washes down the spore from inclined-plane with sterile saline, is fitted into sterilizing bottle, filters after shake well Spore suspension is made, the spore suspension concentration is 2 × 106A spore/mL；
B, spore suspension obtained by step A is subjected to induction mutation of bacterium processing, multiple plating mediums is then taken, by the spore Sub- suspension is spread evenly across in each plating medium with 0.3mL/ ware equivalent cultivates respectively, until growing bacterium colony；The culture Temperature is 27 DEG C, incubation time 120h；
C, it picks them separately each colony inoculation obtained by step B and carries out constant temperature incubation into different slant mediums, and carry out Label, until growing spore or mycelia, the cultivation temperature is 28 DEG C, incubation time 120h；Then by each spore or mycelia Shake flask fermentation is carried out respectively, and fermentation temperature is 30 DEG C, fermentation time 60h, revolving speed 200r/min；Then fermentation is taken by bottle Liquid measures the glucose oxidase enzyme activity in each fermentation liquid, filters out the highest bacterial strain of enzyme activity；The fermentation medium pH value is 8；
(2) fermentation expands culture
The bacterial strain filtered out in step (1) is subjected to fermentation and expands culture, by weight, the component and proportion of culture medium Are as follows: 80 parts of glucose, 9 parts of sodium nitrate, 1 part of potassium chloride, 1.5 parts of dipotassium hydrogen phosphate, 1 part of magnesium sulfate, 2 parts of calcium carbonate, distilled water 1000 parts, condition of culture: liquid amount 20ml/100ml, inoculum concentration 7%, temperature are 35 DEG C, revolving speed 400r/min, culture Time is 96h.
(3) glucose oxidase is extracted
Step (2) resulting culture is taken, under 5 DEG C, 8000r/min speed conditions, is centrifuged 10min, obtained supernatant Liquid is glucose oxidase liquid, and the glucose oxidase liquid is concentrated and dried, glucose oxidase is obtained.
Slant medium and plating medium in above-mentioned steps (1) are Czapek's medium.
Mutagenic treatment in above-mentioned steps (1) is specially the distance 15- by the spore suspension under 25-30W ultraviolet lamp Irradiation and stir process 15-20min at 20cm.
The component and proportion of fermentation medium in above-mentioned steps (1) are as follows: 75 parts of glucose, 8 parts of sodium nitrate, potassium chloride 0.8 part, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water.
The bacterial strain filtered out in above-mentioned steps (1) is aspergillus niger or penicillium bacterial strain.
Bacterial strain enzyme activity >=the 100U/mL filtered out in above-mentioned steps (1).
The pH of fermentation medium is adjusted in above-mentioned steps (1) using 10%HCl or 10%NaOH, it is preferred that fermented and cultured The pH value of base is 7.5.
The component and proportion of culture medium in above-mentioned steps (2) are as follows: 75 parts of glucose, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water, condition of culture: liquid amount 15ml/ 100ml, inoculum concentration 5%, temperature are 30 DEG C, revolving speed 200r/min, incubation time 72h.
Drying temperature in above-mentioned steps (3) is 25-35 DEG C.
Comparative example 1
In addition in step (1) without induction mutation of bacterium processing, remaining is same as Example 1.
Comparative example 2
Except in step (1) each culture parameters it is smaller than minimum of the present invention in addition to, remaining is same as Example 1；I.e. inclined-plane is trained Feeding cultivation temperature is 24 DEG C (being less than 26 DEG C of minimum temperature), and incubation time is 115h (being less than 120h)；The culture of plate culture Temperature is 23 DEG C (being less than 25 DEG C of minimum temperature), and incubation time is 65h (being less than minimum time 72h)；The training of shake flask fermentation culture Supporting temperature is 28 DEG C (less than 30 DEG C), and incubation time is 40h (being less than minimum time 48h).
Comparative example 3
Except in step (1) each culture parameters it is higher than peak of the present invention in addition to, remaining is same as Example 1；I.e. inclined-plane is trained Feeding cultivation temperature is 30 DEG C (being greater than 28 DEG C of maximum temperature), and incubation time is 125h (being greater than 120h)；The culture of plate culture Temperature is 30 DEG C (being greater than 27 DEG C of maximum temperature), and incubation time is 125h (being greater than highest time 120h)；Shake flask fermentation culture Cultivation temperature is 32 DEG C (being greater than 30 DEG C), and incubation time is 65h (being greater than highest time 60h).
Comparative example 4
In addition to the component and proportion of the culture medium of fermentation expansion culture in step (2) are different, remaining is same as Example 1. Experimental example 1
The enzyme activity and yield for measuring the glucose oxidase of above-described embodiment 1-3 and comparative example 1-4 preparation, as a result such as table 1 It is shown.
The enzyme activity and yield of the glucose oxidase of each embodiment and comparative example of table 1 preparation
Enzyme activity (U/mL) Yield (g/L)
Embodiment 1 112 25
Embodiment 2 100 20
Embodiment 3 108 23
Comparative example 1 43 9
Comparative example 2 46 11
Comparative example 3 42 12
Comparative example 4 45 8
As shown in Table 1, the setting of each Parameter Conditions and nutrient media components and content of the invention, which has, reaches this experiment effect The necessity of fruit, if using other settings instead, glucose oxidase meeting fast deactivation, yield is rapidly reduced, prepared by the present invention Glucose oxidase has significant technical effect in enzyme activity and yield aspects.

Claims (10)

1. a kind of preparation method of glucose oxidase, it is characterised in that the following steps are included:
(1) strain breeding thereof
A, original strain is put into slant medium and carries out constant temperature incubation to spore is generated, cultivation temperature is 26-28 DEG C, when culture Between be 120h, then the spore is washed down with sterile saline from inclined-plane, is fitted into sterilizing bottle, after shake well filtering make At spore suspension, the spore suspension concentration is 1 × 106-2×106A spore/mL；
B, spore suspension obtained by step A is subjected to induction mutation of bacterium processing, then takes multiple plating mediums, the spore is hanged Supernatant liquid is spread evenly across in each plating medium with 0.2-0.3mL/ ware equivalent cultivates respectively, until growing bacterium colony；The culture Temperature is 25-27 DEG C, incubation time 72-120h；
C, it picks them separately each colony inoculation obtained by step B and carries out constant temperature incubation into different slant mediums, and mark, Until growing spore or mycelia, the cultivation temperature is 26~28 DEG C, incubation time 120h；Then by each spore or mycelia point Not carry out shake flask fermentation, fermentation temperature be 30 DEG C, fermentation time 48-60h, revolving speed 200r/min；Then fermentation is taken by bottle Liquid measures the glucose oxidase enzyme activity in each fermentation liquid, filters out the highest bacterial strain of enzyme activity；The fermentation medium pH value is 7-8；
(2) fermentation expands culture
The bacterial strain filtered out in step (1) is subjected to fermentation and expands culture, by weight, the component and proportion of culture medium are as follows: 70~80 parts of glucose, 7~9 parts of sodium nitrate, 0.6~1 part of potassium chloride, 1~1.5 part of dipotassium hydrogen phosphate, 0.5~1 part of magnesium sulfate, 1-2 parts of calcium carbonate, 900-1000 parts of distilled water, condition of culture: liquid amount is 10~20ml/100ml, and inoculum concentration is 3~7%, Temperature is 30~35 DEG C, and revolving speed is 200~400r/min, and incubation time is 72~96h；
(3) glucose oxidase is extracted
Step (2) resulting culture is taken, under 0~5 DEG C, 4000~8000r/min speed conditions, 10~15min is centrifuged, obtains To supernatant be glucose oxidase liquid, by the glucose oxidase liquid be concentrated and dried, obtain glucose oxidase.
2. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) Slant medium and plating medium be Czapek's medium.
3. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) Mutagenic treatment be specially by the spore suspension under 25-30W ultraviolet lamp, distance 15-20cm at irradiation simultaneously stir process 15-20min。
4. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) Fermentation medium component and proportion are as follows: 75 parts of glucose, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 2 parts of calcium carbonate, 950 parts of distilled water.
5. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) The bacterial strain filtered out is aspergillus niger or penicillium bacterial strain.
6. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) Bacterial strain enzyme activity >=the 100U/mL filtered out.
7. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (1) The pH of fermentation medium is adjusted using 10%HCl or 10%NaOH, it is preferred that the pH value of fermentation medium is 7.5.
8. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (2) Culture medium component and proportion are as follows: 75 parts of glucose, 8 parts of sodium nitrate, 0.8 part of potassium chloride, 1.3 parts of dipotassium hydrogen phosphate, sulfuric acid 0.8 part of magnesium, 2 parts of calcium carbonate, 950 parts of distilled water, condition of culture: liquid amount 15ml/100ml, inoculum concentration 5%, temperature are 30 DEG C, revolving speed 200r/min, incubation time 84h.
9. a kind of preparation method of glucose oxidase according to claim 1, which is characterized in that in the step (3) Drying temperature be 25-35 DEG C.
10. a kind of glucose oxidase prepared such as any one of claim 1-9 preparation method.
CN201811088698.1A 2018-09-18 2018-09-18 A kind of glucose oxidase and preparation method thereof Pending CN109234247A (en)

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