CN111500473A - Method for producing low-temperature glucose oxidase by microbial fermentation - Google Patents

Method for producing low-temperature glucose oxidase by microbial fermentation Download PDF

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CN111500473A
CN111500473A CN202010504825.2A CN202010504825A CN111500473A CN 111500473 A CN111500473 A CN 111500473A CN 202010504825 A CN202010504825 A CN 202010504825A CN 111500473 A CN111500473 A CN 111500473A
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王保全
谢燕霞
李常云
崔学儒
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Hongkui Biological China Co ltd
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    • 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 invention discloses a method for producing low-temperature glucose oxidase by microbial fermentation, which comprises the following steps: s1, strain pretreatment, S2, strain mutagenesis, S3, seeding tank cultivation, S4, fermentation tank cultivation, S5, tank emptying, S6 and enzyme extraction; the method has the advantages that the strain with good low-temperature activity is cultured by using the glucose oxidase prepared by the method and matching the mode of adding the mutagen with the mode of gradually reducing the temperature before the culture in the seeding tank, so that the obtained enzyme activity is more suitable for active movement in a low-temperature environment, the process is accelerated by the mutagenesis mode, the production efficiency is greatly improved, the strain with good low-temperature enzyme activity is obtained, the time for converting the strain into the low-temperature activity is shortened, and the production efficiency is greatly improved.

Description

Method for producing low-temperature glucose oxidase by microbial fermentation
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing low-temperature glucose oxidase by microbial fermentation.
Background
Glucose oxidase, GOD for short, is an important industrial enzyme in the food industry, is widely applied to the aspects of deoxidation of foods such as wine, beer, fruit juice, milk powder and the like, flour improvement, food browning prevention and the like, and also has wide application in rapid detection of foods and biosensors. Glucose oxidase widely exists in the nature, but the extraction difficulty is higher;
the existing processing technology is mostly produced by a microbial fermentation mode, and the patent with the application number of CN201510084670.0 indicates the advantages of low-temperature glucose oxidase, and because the low-temperature enzyme has high enzyme activity and high catalytic efficiency at low temperature, the time of the treatment process can be greatly shortened, and expensive heating or cooling cost can be saved; the energy-saving method has considerable advantages in energy saving; the activity of the low-temperature enzyme can be lost through mild heat treatment, the low-temperature or proper-temperature treatment does not influence the quality of the product, and the low-temperature glucose oxidase has greater advantages compared with the existing normal-temperature glucose oxidase based on the advantages, so that the cultivation of the low-temperature glucose oxidase is an economical development direction;
the oxidase culture method disclosed in CN201510084670.0 is capable of culturing low-temperature glucose oxidase suitable for low temperature, but during the acclimatization process, no corresponding inducer is added, so that the strain generation rate is slow, the production rate is affected, the acclimatization rate of the strain is low, the yield of the product is low, and the method is not particularly suitable for mass production.
Disclosure of Invention
The invention aims to solve the problems, designs a method for producing low-temperature glucose oxidase by microbial fermentation, and solves the problems that in the existing oxidase culture method, in the domestication process, a corresponding inducer is not added firstly, so that the strain generation rate is slow, the production rate is influenced, secondly, the domestication rate of the strain is low, the yield of products is low, and the method is not particularly suitable for batch production and preparation.
The technical scheme of the invention for realizing the aim is as follows: a method for producing low-temperature glucose oxidase by microbial fermentation comprises the following steps: s1, strain pretreatment, S2, strain mutagenesis, S3, seeding tank cultivation, S4, fermentation tank cultivation, S5, tank emptying, S6 and enzyme extraction;
s1: eluting spores on the inclined plane of a penicillium chrysogenum strain by using a proper amount of sterile normal saline, placing the spores in a sterilized triangular flask with glass beads in advance, oscillating the spores on a shaking table for 30-40 min, filtering hyphae to disperse single spore suspension by using sterilized absorbent cotton, counting the hyphae by using a blood counting chamber, and diluting the hyphae to 100/ml spore suspension;
s2: first, dibutyl phosphate mutagenesis was performed: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, adding 0.2ml of dibutyl sulfate (the volume concentration is 50%), oscillating for different times, adding 0.5ml of sodium thiosulfate (85%) to terminate the reaction, diluting the gradient to 10 < -1 > -10 < -6 >, taking 0.2ml of spore suspension with 3 gradients (10 < -4 > -10 < -6 >) in each dose, coating the spore suspension on a plate culture medium, inverting the spore suspension to culture in an incubator at 30 ℃ for 1-2 days, calculating the number of bacterial colonies, drawing a lethality curve, selecting a single bacterial colony with a large blue circle on a screening plate, inoculating the single bacterial colony to an inclined plane, and culturing until the spores are generated;
secondly, low-temperature mutagenesis is carried out: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, continuously adding ice blocks into the flask, performing low-temperature acclimation step by step at the acclimation temperature from high to low, wherein the acclimation temperature is from 30 ℃ to 25 ℃ to 20 ℃ to 15 ℃ to 10 ℃, so that the strain grows well in a low-temperature environment, determining the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium after acclimation is finished, and selecting the strain with high enzyme activity for storage; after the culture is finished, screening out strains with high activity and low temperature activity;
s3: the pressure of the tank is 0.1-0.12MPa, the culture temperature is 25-20 ℃, and the ventilation rate is 32-35m3H, stirring speed is 60-120r/min, and pH is controlled to be 6.0-6.5; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing;
s4: the pressure of the tank is 0.1-0.12Mpa, the culture temperature is 20-15 ℃, the stirring speed is 240-; ventilation quantity: 0-20h is 32-35m3H, 20-22h is 38-40m3The/h, 22 h-tank is 25-30m3H; when the pH value rises to 6.0, feeding is started, and the pH value is controlled to be 5.0-6.0；
S5: culturing in a fermentation tank for 48-78h, slowly increasing enzyme activity, and placing the thallus into the tank when the thallus begins to autolyze partially;
s6: centrifuging the fermentation liquor at 4000-8000 rpm to collect liquid, collecting supernate, namely crude enzyme liquid, further concentrating, separating and purifying the crude enzyme liquid, and preparing low-temperature glucose oxidase preparations with different activities, purities and dosage forms.
The plate medium (12% glucose, NaNO) used for the dibutyl phosphate mutagenesis in step S231％、K2HPO41.5％、KCl0.1％、MgSO40.1 percent of agar and 3 to 4 percent of agar; ).
The liquid medium (12% glucose, CaCO) subjected to the low-temperature mutagenesis in the step S231-5%, KI 0.5%, dibutyl phosphate 0.5 mol/L, agar 3-4%, pH6.0).
The seed tank medium (glucose 25%, peptone 20%, CaCO 35%, KI 1%, MgSO40.8%, pH6.5) in said step S3.
The culture temperature in the step S3 is preferably 20 ℃, and the ventilation rate is 35m3The stirring speed is 90 r/min.
The step S4 fermenter Medium (sucrose 24%, peptone 1%, CaCO)31％、K2HPO40.5％、KCl0.5％、MgSO40.2％、NaNO32％，pH6.5)。
The culture temperature in the step S4 is preferably 15 ℃, and the stirring speed is 240 r/min.
And in the step S5, the culture is put into a tank and cultured for 72 hours.
The feed medium (sucrose 25%, corn steep liquor 20%, KCl 1%, pH5.5) in step S4.
The centrifugal rate of the centrifuge in the step S6 is preferably 5000-6000 rpm.
The name manufactured by the technical scheme of the invention has the beneficial effect.
Detailed Description
The embodiment is characterized by comprising the following steps: step S1, strain pretreatment, step S2, strain mutagenesis, step S3, seedsCulturing in a tank, step S4, culturing in a fermentation tank, step S5, placing in a tank, step S6, and extracting enzyme; s1: eluting spores on the inclined plane of a penicillium chrysogenum strain by using a proper amount of sterile normal saline, placing the spores in a sterilized triangular flask with glass beads in advance, oscillating the spores on a shaking table for 30-40 min, filtering hyphae to disperse single spore suspension by using sterilized absorbent cotton, counting the hyphae by using a blood counting chamber, and diluting the hyphae to 100/ml spore suspension; s2: first, dibutyl phosphate mutagenesis was performed: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, adding 0.2ml of dibutyl sulfate (the volume concentration is 50%), oscillating for different times, adding 0.5ml of sodium thiosulfate (85%) to terminate the reaction, diluting the gradient to 10 < -1 > -10 < -6 >, taking 0.2ml of spore suspension with 3 gradients (10 < -4 > -10 < -6 >) in each dose, coating the spore suspension on a plate culture medium, inverting the spore suspension to culture in an incubator at 30 ℃ for 1-2 days, calculating the number of bacterial colonies, drawing a lethality curve, selecting a single bacterial colony with a large blue circle on a screening plate, inoculating the single bacterial colony to an inclined plane, and culturing until the spores are generated; secondly, low-temperature mutagenesis is carried out: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, continuously adding ice blocks into the flask, performing low-temperature acclimation step by step at the acclimation temperature from high to low, wherein the acclimation temperature is from 30 ℃ to 25 ℃ to 20 ℃ to 15 ℃ to 10 ℃, so that the strain grows well in a low-temperature environment, determining the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium after acclimation is finished, and selecting the strain with high enzyme activity for storage; after the culture is finished, screening out strains with high activity and low temperature activity; s3: the pressure of the tank is 0.1-0.12MPa, the culture temperature is 25-20 ℃, and the ventilation rate is 32-35m3H, stirring speed is 60-120r/min, and pH is controlled to be 6.0-6.5; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing; s4: the pressure of the tank is 0.1-0.12Mpa, the culture temperature is 20-15 ℃, the stirring speed is 240-; ventilation quantity: 0-20h is 32-35m3H, 20-22h is 38-40m3The/h, 22 h-tank is 25-30m3H; feeding when the pH value rises to 6.0, and controlling the pH value to be 5.0-6.0; s5: culturing in a fermentation tank for 48-78h, slowly increasing enzyme activity, and placing the thallus into the tank when the thallus begins to autolyze partially; s6: centrifuging the fermentation liquor at 4000-8000 rpm to collect liquid, wherein the collected supernatant is the crude enzymeFurther concentrating, separating and purifying the crude enzyme liquid to prepare low-temperature glucose oxidase preparations with different activities, purities and dosage forms; the glucose oxidase prepared by the method is used for culturing the strain with good low-temperature activity by using a mode of adding a mutagen and a mode of gradually reducing the temperature before the seeding tank is cultured, so that the obtained enzyme activity is more suitable for active movement in a low-temperature environment, the process is accelerated by using the mutagenesis mode, the production efficiency is greatly improved, the strain with good low-temperature enzyme activity is obtained, the time for converting the strain into the low-temperature activity is shortened, and the production efficiency is greatly improved.
Example (b): firstly, preparing glucose oxidase according to the following method, comprising the following steps:
firstly, strain pretreatment, secondly, strain mutagenesis, thirdly, seeding tank cultivation, fourthly, fermentation tank cultivation, fifthly, tank placement, sixthly, enzyme extraction;
firstly, the method comprises the following steps: and (3) eluting spores on the inclined plane of the penicillium chrysogenum strain by using a proper amount of sterile normal saline, placing the spores in a sterilized triangular flask with glass beads in advance, oscillating the spores on a shaking table for 30-40 min, filtering hyphae to disperse single spore suspension by using sterilized absorbent cotton, and counting the mycelia by using a blood counting chamber. Diluting to 100 spores/ml;
II, secondly: first, dibutyl phosphate mutagenesis was performed: 5ml of spore suspension was added to a 25ml triangular flask, and 0.2ml of dibutyl sulfate (50% by volume) was added thereto, followed by shaking and addition of 0.5ml of sodium thiosulfate (85%) to terminate the reaction. The dilution gradient is 10 < -1 > to 10 < -6 >. Taking 0.2ml of spore suspension with gradient of (10-4-10-6) 3 of each dose, and coating on plate culture medium (glucose 12%, NaNO)31％、K2HPO41.5％、KCl0.1％、MgSO40.1 percent of agar and 3 to 4 percent of agar; ) And (5) inversely culturing in an incubator for 1-2 days at 30 ℃. The number of colonies was counted and a lethality curve was plotted. Selecting a single colony with a large blue circle on a screening plate, inoculating the single colony on an inclined plane, and culturing until spores are generated;
secondly, low-temperature mutagenesis is carried out: taking 5ml of spore suspension, adding into a triangular flask with the volume of 25ml, and addingContinuously adding ice blocks into the bottle, performing low-temperature acclimation step by step at an acclimation temperature from high to low, from 30 ℃ to 25 ℃ to 20 ℃ to 15 ℃ to 10 ℃ to ensure that the strain grows well in a low-temperature environment, determining the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium when the strain can stably grow at a low temperature, selecting the strain with high enzyme activity for storage, and performing liquid culture medium (12% of glucose and CaCO)31-5 percent of KI, 0.5 percent of dibutyl phosphate, 0.5 mol/L percent of agar and 3-4 percent of pH6.0 percent;
thirdly, the method comprises the following steps: seeding tank culture medium (glucose 25%, peptone 20%, CaCO 35%, KI 1%, MgSO40.8%, pH6.5), tank pressure 0.1-0.12MPa, culture temperature 20 deg.C, and ventilation 35m3H, stirring speed is 90r/min, and pH is controlled to be 6.0-6.5; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing;
fourthly, the method comprises the following steps: fermenter Medium (sucrose 24%, peptone 1%, CaCO)31％、K2HPO40.5％、KCl0.5％、MgSO40.2％、NaNO32 percent, pH6.5), the pressure of the tank is 0.1-0.12Mpa, the culture temperature is 15 ℃, the stirring speed is 240r/min, and the pH is controlled to be 5.0-6.0; ventilation quantity: 0-20h is 32-35m3H, 20-22h is 38-40m3The/h, 22 h-tank is 25-30m3H; when the pH value rises to 6.0, feeding is started, and the culture medium (25% of sucrose, 20% of corn steep liquor, KCl 1%, pH value is 5.5) is fed and controlled to be 5.0-6.0;
fifthly: culturing in a fermentation tank for 72h, slowly increasing enzyme activity, and putting the thallus into the tank after partial autolysis;
sixthly, the method comprises the following steps: centrifuging the fermentation liquor at 5000-6000 rpm to collect liquid, collecting supernatant as crude enzyme liquid, further concentrating, separating and purifying the crude enzyme liquid, and preparing low-temperature glucose oxidase preparations with different activities, purities and dosage forms;
in summary, it can be seen that the glucose oxidase prepared by the above method is used for culturing a strain with good low-temperature activity by adding a mutagen in a manner of gradually reducing the temperature before the seeding tank is cultured, so that the obtained enzyme activity is more suitable for active movement in a low-temperature environment, and the process is accelerated by the mutagenesis, so that the production efficiency is greatly improved, the strain with good low-temperature enzyme activity is obtained, the time for converting the strain into low-temperature activity is shortened, and the production efficiency is greatly improved.
The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.

Claims (10)

1. The method for producing the low-temperature glucose oxidase by microbial fermentation is characterized by comprising the following steps of: s1, strain pretreatment, S2, strain mutagenesis, S3, seeding tank cultivation, S4, fermentation tank cultivation, S5, tank emptying, S6 and enzyme extraction;
s1: eluting spores on the inclined plane of a penicillium chrysogenum strain by using a proper amount of sterile normal saline, placing the spores in a sterilized triangular flask with glass beads in advance, oscillating the spores on a shaking table for 30-40 min, filtering hyphae to disperse single spore suspension by using sterilized absorbent cotton, counting the hyphae by using a blood counting chamber, and diluting the hyphae to 100/ml spore suspension;
s2: first, dibutyl phosphate mutagenesis was performed: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, adding 0.2ml of dibutyl sulfate (the volume concentration is 50%), oscillating for different times, adding 0.5ml of sodium thiosulfate (85%) to terminate the reaction, diluting the gradient to 10 < -1 > -10 < -6 >, taking 0.2ml of spore suspension with 3 gradients (10 < -4 > -10 < -6 >) in each dose, coating the spore suspension on a plate culture medium, inverting the spore suspension to culture in an incubator at 30 ℃ for 1-2 days, calculating the number of bacterial colonies, drawing a lethality curve, selecting a single bacterial colony with a large blue circle on a screening plate, inoculating the single bacterial colony to an inclined plane, and culturing until the spores are generated;
secondly, low-temperature mutagenesis is carried out: adding 5ml of spore suspension into a triangular flask with the volume of 25ml, continuously adding ice blocks into the flask, performing low-temperature acclimation step by step at the acclimation temperature from high to low, wherein the acclimation temperature is from 30 ℃ to 25 ℃ to 20 ℃ to 15 ℃ to 10 ℃, so that the strain grows well in a low-temperature environment, determining the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium after acclimation is finished, and selecting the strain with high enzyme activity for storage; after the culture is finished, screening out strains with high activity and low temperature activity;
s3: the pressure of the tank is 0.1-0.12MPa, the culture temperature is 25-20 ℃, and the ventilation rate is 32-35m3H, stirring speed is 60-120r/min, and pH is controlled to be 6.0-6.5; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing;
s4: the pressure of the tank is 0.1-0.12Mpa, the culture temperature is 20-15 ℃, the stirring speed is 240-; ventilation quantity: 0-20h is 32-35m3H, 20-22h is 38-40m3The/h, 22 h-tank is 25-30m3H; feeding when the pH value rises to 6.0, and controlling the pH value to be 5.0-6.0;
s5: culturing in a fermentation tank for 48-78h, slowly increasing enzyme activity, and placing the thallus into the tank when the thallus begins to autolyze partially;
s6: centrifuging the fermentation liquor at 4000-8000 rpm to collect liquid, collecting supernate, namely crude enzyme liquid, further concentrating, separating and purifying the crude enzyme liquid, and preparing low-temperature glucose oxidase preparations with different activities, purities and dosage forms.
2. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the plate medium (12% glucose, NaNO) used in dibutyl phosphate mutagenesis in step S231％、K2HPO41.5％、KCl0.1％、MgSO40.1 percent of agar and 3 to 4 percent of agar; ).
3. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the liquid medium (glucose 12%, CaCO) subjected to low-temperature mutagenesis in step S231-5%, KI 0.5%, dibutyl phosphate 0.5 mol/L, agar 3-4%, pH6.0).
4. The method for producing low-temperature glucose oxidase by microbial fermentation of claim 1, wherein the seeding tank culture medium (glucose 25%, peptone 20%, CaCO 35%, KI 1%, MgSO40.8%, pH6.5) in step S3.
5. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the culture temperature in step S3 is preferably 20 ℃ and the ventilation rate is 35m3The stirring speed is 90 r/min.
6. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the step S4 fermentation medium (sucrose 24%, peptone 1%, CaCO)31％、K2HPO40.5％、KCl0.5％、MgSO40.2％、NaNO32％，pH6.5)。
7. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the culture temperature in step S4 is preferably 15 ℃, and the stirring speed is 240 r/min.
8. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein step S5 is performed for 72 hours in a tank-open manner.
9. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the feeding medium (sucrose 25%, corn steep liquor 20%, KCl 1%, pH5.5) in step S4.
10. The method for producing low-temperature glucose oxidase by microbial fermentation according to claim 1, wherein the centrifuge centrifugation rate in step S6 is preferably 5000-6000 rpm.
CN202010504825.2A 2020-06-05 2020-06-05 Method for producing low-temperature glucose oxidase by microbial fermentation Pending CN111500473A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630166A (en) * 2015-02-16 2015-05-20 大连大学 Method for producing low-temperature glucose oxidase by virtue of microbial fermentation
CN107488640A (en) * 2017-09-18 2017-12-19 山东隆科特酶制剂有限公司 A kind of resistance to oxidation low temperature glucose oxidase and its production method and application
WO2019233083A1 (en) * 2018-06-04 2019-12-12 中国农业科学院饲料研究所 Glucose oxidase god mutant and gene and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630166A (en) * 2015-02-16 2015-05-20 大连大学 Method for producing low-temperature glucose oxidase by virtue of microbial fermentation
CN107488640A (en) * 2017-09-18 2017-12-19 山东隆科特酶制剂有限公司 A kind of resistance to oxidation low temperature glucose oxidase and its production method and application
WO2019233083A1 (en) * 2018-06-04 2019-12-12 中国农业科学院饲料研究所 Glucose oxidase god mutant and gene and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
刘春莹 等: "一株海洋低温葡萄糖氧化酶担子菌的诱变育种及其酶学性质研究", 《中国酿造》, vol. 38, no. 5, 31 December 2019 (2019-12-31), pages 5 *

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