CN107488640B - Oxidation-resistant low-temperature glucose oxidase and production method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to low-temperature glucose oxidase as well as a production method and application thereof. The glucose oxidase is produced from Aspergillus niger CH870, and the preservation number is CGMCC No. 14138. The enzyme activity of fermentation liquor of the glucose oxidase produced by the strain fermentation reaches above 2300U/ml, the produced glucose oxidase is stable in the range of pH2.0-8.0, the optimum reaction temperature is 20 ℃, the stability is good, the glucose oxidase has oxidation resistance, and the residual enzyme activity is 100% under the condition of 30mmol of hydrogen peroxide; under the condition of 200mmol of hydrogen peroxide, the residual enzyme activity is 90.8 percent, and the application is wide.

Description

Oxidation-resistant low-temperature glucose oxidase and production method and application thereof

The technical field is as follows:

the invention belongs to the technical field of bioengineering, and particularly relates to low-temperature glucose oxidase with improved oxidation resistance, and a production method and application thereof.

Background art:

glucose Oxidase (GOD) can specifically oxidize beta-D-glucose into gluconic acid and hydrogen peroxide, and the glucose oxidase has wide application in the fields of food, medicine, biology and the like.

Because glucose oxidase can catalyze glucose to consume oxygen and generate gluconic acid and hydrogen peroxide, the glucose oxidase is widely applied in the food industry, and is mainly expressed in the following four aspects: removing residual glucose in food, deoxidizing, sterilizing, and measuring glucose content in food.

Glucose oxidase, as a novel green enzyme preparation, was listed by the nation in 1999 as one of 12 permitted feed additives. The action mechanism of the glucose oxidase for promoting the growth is as follows: the glucose oxidase added into the animal feed has an antioxidant function, and can remove a large amount of free radicals generated in intestinal epithelial cells under the stress state of livestock to protect the integrity of the intestinal epithelial cells.

The principle of the medical urine glucose test paper is that hydrogen peroxide is generated according to the enzymatic reaction of glucose oxidase, the hydrogen peroxide is further decomposed by catalase to generate water and oxygen, the oxygen oxidizes reduced and colorless o-tolidine dye on the test paper into a blue substance, the generation amount of the blue substance is proportional to the concentration of glucose, and the color shade of the test paper is compared with a standard color plate to judge the content of the glucose in the urine. The glucose oxidase oxygen rate method for determining glucose has the advantages of high accuracy, wide linear range, strong reaction specificity and good repeatability.

There are several dozen enzyme sensors that have been developed internationally, and Updike and Hicks have first covered a GOD film on a platinum electrode in 1967 to produce an enzyme sensor for quantitatively detecting the content of glucose in serum, and have successfully produced the first glucose biosensor.

Glucose Oxidase (GOD) is widely distributed in animals, plants and microorganisms. However, the method has a small content in animals and plants and has a certain limitation in extraction, so the method is less adopted.

In microorganisms, glucose oxidase is produced mainly by a mold fermentation method, and aspergillus and penicillium are generally used as production strains, such as aspergillus niger, aspergillus oryzae, penicillium, kluyveromyces lactis and kluyveromyces fragilis, and are common zymogens for industrial production of GOD. At present, glucose oxidase is produced at home and abroad by adopting a microbial fermentation method. Because of the strong ability of mold to produce glucose oxidase under certain conditions, aspergillus niger and penicillium (penicillium innotation) are mainly used for the industrial production of glucose oxidase. However, glucose oxidase is almost completely imported, and the production cost is increased. Therefore, the screening of the glucose oxidase with excellent performance has great practical application value.

The invention content is as follows:

in order to solve the technical problems, the invention provides oxidation-resistant low-temperature glucose oxidase and a production method and application thereof.

The glucose oxidase is produced from aspergillus niger, the aspergillus niger is a high-yield low-temperature glucose oxidase strain with improved oxidation resistance obtained by physical mutagenesis and chemical compound mutagenesis in a laboratory, and the strain is identified to belong to aspergillus niger, in particular to aspergillus niger CH870, and the strain is preserved in the general microbiological culture collection center of China microbiological culture collection management committee at 7-13.2017, with the address: the microbial research institute of Chinese academy of sciences, No. 3 Xilu-Beijing, Chaozhou, Chao code No.1, facing Yang, has a preservation number of CGMCC No. 14138.

The invention also provides a liquid microbial fermentation production method of the glucose oxidase, which has the advantages of high fermentation enzyme activity, high extraction yield and low manufacturing cost, and the method specifically comprises the following steps:

aspergillus niger CH870 as producing strain;

(1) seed tank culture:

the pressure of the tank is 0.05-0.08MPa, the culture temperature is 30 ℃, and the ventilation rate is 30m³H, stirring speed is 180r/min, and pH is controlled to be 7.0; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing;

seeding tank culture medium: glucose 20%, peptone 25%, (NH)₄)₂ SO ₄5％，K₂HPO₄1％，MgSO₄·7H₂O0.5％，pH 7.0；

(2) Cultivation in fermenter

The pressure of the tank is 0.05-0.08Mpa, the culture temperature is 30 ℃, the stirring speed is 300r/min, and the pH is controlled to be 6.5-7.0; ventilation quantity: 0-40h is 30m³40-58h is 45m³Per, 58 h-can discharge is 40m³H; when the pH value rises to 7.0, feeding is started, and the pH value is controlled to be 6.5-7.0;

fermentation medium: 12.30% of sucrose, 0.41% of peptone and NaNO₃0.6％、KH₂PO₄0.2％、KCl0.05％、MgSO₄·7H₂O 0.07％、CaCO ₃1％，pH 7.0；

A supplemented medium: 20% of sucrose, 30% of corn steep liquor, 0.5% of calcium chloride and 7.0 of pH value.

(3) Can for placing food

Culturing in a fermentation tank for 96-110h, slowly increasing enzyme activity, and placing the thallus into the tank when the thallus begins to autolyze partially;

through determination, the enzyme activity of the fermentation liquid of the glucose oxidase after fermentation is over 2300U/ml;

(4) extraction of glucose oxidase

Fermentation liquor-pretreatment-filter pressing-ultrafiltration-standardization-fine filtration-blending-filling-detection-finished product.

The glucose oxidase of the present invention is characterized in that:

(1) pH: is stable in the pH range of 2.0-8.0;

(2) temperature: the reaction is stable below 40 ℃, and the optimal reaction temperature is 20 ℃; the activity can still be kept by more than 90% after the temperature is kept for 1h at 35 ℃, when the temperature is increased to 40 ℃, the enzyme activity is reduced to about 70% along with the prolonging of the heat preservation time, and the thermal stability is obviously weakened at 45 ℃;

(3) oxidation resistance: under the condition of 30mmol of hydrogen peroxide, the residual enzyme activity is 100 percent; under the condition of 200mmol of hydrogen peroxide, the residual enzyme activity is 90.8 percent.

Has the advantages that:

the invention provides oxidation-resistant low-temperature glucose oxidase which is stable in the pH range of 2.0-8.0, the optimal reaction temperature is 20 ℃, the stability is good, the glucose oxidase has oxidation resistance, and the residual enzyme activity is 100% under the condition of 30mmol of hydrogen peroxide; under the condition of 200mmol of hydrogen peroxide, the residual enzyme activity is 90.8 percent, and the application is wide.

Description of the drawings:

FIG. 1 is a graph showing the relative enzyme activity of glucose oxidase at different temperatures;

FIG. 2 is a graph showing the relative enzyme activity of glucose oxidase at different pH values;

FIG. 3 is a thermal stability curve for glucose oxidase;

FIG. 4 is a pH stability curve of glucose oxidase.

The specific implementation mode is as follows:

the invention is illustrated in more detail below by means of specific examples:

EXAMPLE 1 mutagenic Breeding of strains

Preparation of spore suspension: eluting spore on the original strain slant with appropriate amount of sterile normal saline, placing in a sterilized triangular flask with glass beads, shaking on a shaking table for 20min, filtering with sterilized absorbent cotton to remove mycelium to obtain dispersed monospore suspension, and counting with blood counting plate. Diluting to 10⁸Spore suspension per mL.

Microwave mutagenesis: placing the test tube containing 5mL spore suspension in a beaker containing ice, irradiating the test tube one by one at different time by using a microwave oven with 2450MHz frequency and 700W output power, and diluting with gradient of 10^-1～10^-6. Taking 10 of each dose^-4～10^-6And (3) coating 0.2mL of spore suspension with 3 dilutions on a plate culture medium, culturing at 30 ℃ for 2-3 d, calculating the colony number, and drawing a lethality curve. Selecting screening plate (screening plate culture medium: bottom layer culture medium: potato 20%, glucose 2%, NaNO)₃0.2％、K₂HPO₄0.1％、KCl 0.05％、MgSO₄0.05 percent of agar and 1.5 to 2.0 percent of agar; upper medium: 2% of glucose, 1% of soluble starch, 0.17% of KI, 0.1mol/L of phosphate buffer, 1.5-2.0% of agar and pH 5.5. ) And inoculating the single colony with the larger upper blue color circle on the inclined plane, culturing until spores are produced, and measuring the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium. Selecting strains with higher yield of glucose oxidase, which can stably inherit more than 3 generations of strains, preserving the strains as inclined planes, and using the strains as starting strains for further diethyl sulfate mutagenesis.

Diethyl sulfate (DES) mutagenesis: 5mL of spore suspension was added to a 25mL Erlenmeyer flask, followed by addition of 0.2mL of DES (50% by volume), shaking and quenching with 0.5mL of sodium thiosulfate (85%) for various periods of time. Dilution gradient of 10^-1～10^-6. Taking 10 of each dose^-4～10^-6And (3) coating 0.2mL of spore suspension with gradient on a plate culture medium, inverting the plate culture medium, culturing in an incubator, and culturing for 2-3 days at 30 ℃. The number of colonies was counted and a lethality curve was plotted. And selecting a single colony with a large blue circle on the screening plate, inoculating the single colony on the inclined plane, culturing until spores are produced, measuring the glucose oxidase enzyme activity of the strain on each inclined plane through a liquid culture medium, and selecting the strain with high enzyme activity for storage. Repeating the steps for mutagenesis and screening to screen out a high-yield low-temperature glucose oxidase strain CH870 with improved oxidation resistance, wherein the strain has the advantages of higher growth speed, less spore production, improved oxidation resistance, lower fermentation enzyme production temperature, higher enzyme activity and capability of stabilizing the heritageThe shake flask fermentation enzyme activity is improved by 5.6 times compared with the original strain.

Stable passage experiment of glucose oxidase high-producing strain CH870

Inoculating the fresh slant cultured by high-yield strain CH870 containing glucose oxidase to a fermentation shake flask (fermentation medium of shake flask comprising glucose 6%, peptone 0.3%, and NaNO)₃0.4％、KH₂PO₄0.2％、KCl 0.05％、MgSO₄·7H₂O 0.07％、CaCO ₃1 percent, natural pH, sterilization at 115 ℃ for 20min), culture at 30 ℃ and 220r/min for 96h, and determining the enzyme activity. The shake flask results for 10 serial passages of this strain are shown in table 1:

TABLE 1 results of stability test of strain CH870

The mutant strain is subcultured for 10 generations, and the experimental result can be seen from table 2, so that the mutant strain has good genetic stability.

Example 2 production of glucose oxidase by liquid fermentation of strain CH870 and extraction thereof

(1) Seed tank culture:

the pressure of the tank is 0.05-0.08MPa, the culture temperature is 30 ℃, and the ventilation rate is 30m³H, stirring speed is 180r/min, and pH is controlled to be 7.0; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing to obtain a seed solution;

seeding tank culture medium: glucose 20%, peptone 25%, (NH)₄)₂SO₄5％，K₂HPO₄1％，MgSO₄·7H₂O0.5％，pH 7.0；

(2) Cultivation in fermenter

Inoculating the seed solution into a fermentation tank at 5% inoculation amount under 0.05-0.08Mpa at 30 deg.C, stirring at 300r/min, and controlling pH to 6.5-7.0; ventilation quantity: 0-40h is 30m³40-58h is 45m³Per, 58 h-can discharge is 40m³H; when the pH value rises to 7.0, feeding is started, and the pH value is controlled to be 6.5-7.0;

fermentation medium: 12.30% of sucrose, 0.41% of peptone and NaNO₃0.6％、KH₂PO₄0.2％、KCl0.05％、MgSO₄·7H₂O 0.07％、CaCO ₃1％，pH 7.0。

A supplemented medium: 20% of sucrose, 30% of corn steep liquor, 0.5% of calcium chloride and 7.0 of pH value.

(3) Can for placing food

Culturing in a fermentation tank for 100h, slowly increasing enzyme activity, and putting the thallus into the tank after partial autolysis;

(4) extraction of glucose oxidase

Fermentation liquor-pretreatment-filter pressing-ultrafiltration-standardization-fine filtration-blending-filling-detection-finished product.

a. Pretreatment: after the fermentation broth is placed in a tank, measuring the volume of the fermentation broth, adjusting the pH to about 4.0, sequentially adding 2% of sodium benzoate, 3% of bentonite and 4% of perlite according to the volume of the fermentation broth, and finally adding 1 volume time of water of the fermentation broth;

b. and (3) filter pressing: after the batching is finished, starting valves to feed, and controlling the feeding speed not too fast properly at the beginning to enable the feeding pressure to rise slowly, and simultaneously observing the definition of the press filtrate, and putting the press filtrate into a clear liquid tank after the press filtrate is clear;

c. and (3) ultrafiltration: before filtration, after alkaline water for maintenance in the roll-type membrane is discharged, the membrane is replaced by clear water, and then residual water in the membrane is discharged. Starting ultrafiltration, controlling inlet pressure at about 0.4MPa, outlet pressure at about 0.3MPa, and temperature below 25 deg.C. Ultrafiltering to obtain the final product with activity, and adding into blending tank.

d. Fine filtering: and (3) finely filtering, adding 1 per mill of sodium benzoate, 0.5 per mill of potassium sorbate and various ingredients required by a finished product according to the volume of the ultrafiltrate, adjusting the pH, stirring for 1 hour, feeding and finely filtering.

e. Blending and filling: blending enzyme activity according to the requirement of product specification, and filling.

Table 2 shows the fermentation period and the fermentation broth enzyme activity for 6 batches of fermentations, the average fermentation broth enzyme activity being: 2454U/mL.

TABLE 2.3L results of fermentation experiments in small pots

Batches of	Fermentation period (h)	Ferment enzyme activity (U/mL)
			1	100	2389
2	100	2476
			3	100	2467
4	100	2567
			5	100	2436
6	100	2387

As can be seen from Table 2, the fermentation level of the mutagenic strain CH870 is relatively stable, and the fermentation enzyme activity reaches more than 2300U/ml.

Example 3 method for measuring enzyme Activity of glucose oxidase

Substrate system: 2mL of a 0.07g/L o-dianisidine solution and 1mL of a 5% glucose solution were pipetted into a graduated tube using a 1mL pipette. 0.1mL of 0.1g/L horseradish peroxidase solution was pipetted into the same graduated tube using a 0.5mL pipette. The substrate system is placed in a constant temperature water bath kettle and is kept warm for 10min at 30 ℃.

And (3) enzyme activity determination: 0.1mL of the diluted enzyme solution was pipetted into the substrate and shaken well, and the absorbance at 460nm was measured rapidly using a visible spectrophotometer with a blank tube as a control. Read initial absorbance value as A₀And timing, recording the absorbance value A every 1min_nThe total time was measured for 5 min.

From the measured absorbance values, enzyme activity of the enzyme solution was calculated according to the following formula: x1(U/mL)

ΔA_n+1＝A_n+1-A_n(n＝0,1,2,3,4)

ΔA_Average＝(ΔA₁+ΔA₂+ΔA₃+ΔA₄+ΔA₅)/5

X1＝ΔA_Average×f/(11.3×t×V1/V2)

In the formula: 11.3-extinction coefficient;

t-reaction time, min;

v1-volume of enzyme solution, mL;

v2-total volume of reaction solution, mL;

f-dilution factor of enzyme solution.

EXAMPLE 4 optimum reaction temperature

Taking the finished product of the glucose oxidase prepared in the embodiment 2, dissolving the finished product of the glucose oxidase to prepare an enzyme solution according to the enzyme activity determination method described in the embodiment 3, and determining the activity of the glucose oxidase at the conditions of 10, 15, 20, 25, 30, 35, 40, 45 and 50 ℃ respectively when the pH value is 6.0, and calculating the relative enzyme activity. As shown in FIG. 1, the optimum reaction temperature was 20 ℃ and the enzyme activity was high at 10 ℃. The experimental result shows that the optimum reaction temperature of the glucose oxidase produced by the mutant strain is obviously lower than that of glucose oxidase from other sources, and the low-temperature glucose oxidase can be widely used in the production of low-temperature fresh-keeping, medicines and foods, has great market application value and has wide application value in industrial production.

Example 5 optimum reaction pH

Taking the finished product of the glucose oxidase prepared in the example 2, dissolving the finished product to prepare an enzyme solution, and then measuring the activity of the glucose oxidase under the conditions of pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 respectively at the temperature of 20 ℃ according to the enzyme activity measuring method described in the example 3, and calculating the relative enzyme activity. The measurement result is shown in fig. 2, and the enzyme activity of the glucose oxidase is highest when the pH is 5.5.

Example 6 thermal stability

Taking a finished product of the glucose oxidase prepared in the embodiment 2, dissolving the finished product according to the enzyme activity determination method described in the embodiment 3, respectively placing enzyme solutions at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ℃ for heat preservation treatment for 60min, and determining the relative enzyme activity (the relative enzyme activity is the ratio of the enzyme activity after heat preservation treatment at different temperatures to the initial enzyme activity, and the initial enzyme activity is defined as 100%) after heat preservation is finished, wherein the experimental result is shown in fig. 3. The activity can still be kept more than 90% after the temperature is kept for 1h at 35 ℃, when the temperature is increased to 40 ℃, the enzyme activity is reduced to about 70% along with the prolonging of the heat preservation time, and the thermal stability is obviously weakened at 45 ℃.

The enzyme activity can be lost by mild heat treatment of the low-temperature glucose oxidase generated by the mutagenic strain CH870, and the quality of the product cannot be influenced by the low-temperature treatment, so that the quality of the product can be effectively improved.

Example 7 acid and alkali resistance

Taking the finished product of glucose oxidase prepared in example 2, according to the enzyme activity determination method described in example 3, adjusting the pH of the enzyme solution to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 with 0.1M NaOH or 0.1M HCl, standing at room temperature for 24 hours, and determining the relative enzyme activity (the relative enzyme activity is the ratio of the enzyme activity after treatment under different pH conditions to the initial enzyme activity, and the initial enzyme activity is defined as 100%) at 20 ℃ and pH 5.5. As shown in FIG. 4, the relative enzyme activity was maintained at 80% or more after 24 hours at pH 2.0-8.0.

Example 8 comparison of the Oxidation resistance of glucose oxidase produced by the starting Strain and the mutagenized Strain CH870

According to the method of the embodiment 2, the starting strain and the mutagenic strain CH870 are respectively used as production strains to produce glucose oxidase by fermentation, and after the fermentation is finished, the fermentation liquor is centrifuged to obtain supernatant liquid to obtain crude enzyme liquid. Treating glucose oxidase produced by original strain and glucose oxidase produced by mutant strain CH870 with hydrogen peroxide of different concentrations at 20 deg.C for 2 hr, wherein the buffer solution is phosphate buffer solution with pH of 5.5. After the treatment, a certain amount of catalase is added to remove unreacted hydrogen peroxide, the reaction is stopped, and then the relative enzyme activity of each group of glucose oxidase is determined (the relative enzyme activity is the ratio of the enzyme activity after the hydrogen oxide treatment to the initial enzyme activity, and the initial enzyme activity is defined as 100%), and the relative enzyme activity is shown in table 3:

TABLE 3 comparison of Oxidation resistance of original and mutagenized strains CH870 glucose oxidase

The glucose oxidase can generate hydrogen peroxide in the reaction process, so the enzyme has the antibacterial and bactericidal effects, but the activity of the glucose oxidase can be inhibited by the accumulation of the hydrogen peroxide, and researches show that the activity of the glucose oxidase can be inhibited when the concentration of the hydrogen peroxide reaches 30mmol/L, so that the method has very important significance in breeding the oxidation-resistant glucose oxidase high-yield strain. As can be seen from the experimental results of Table 3, when the concentration of hydrogen peroxide reaches 30mmol/L, the glucose oxidase activity of the original strain is reduced to 90.7%, while the enzyme activity of the mutagenized strain is almost unchanged. After the treatment of hydrogen peroxide with the same concentration, the oxidation resistance of the mutagenic strain is obviously higher than that of a control strain, so that the mutagenic strain CH870 has higher value in practical application.

Claims (3)

1. The oxidation-resistant low-temperature glucose oxidase is characterized in that the glucose oxidase is produced from aspergillus niger, in particular aspergillus niger CH870, and the preservation number is CGMCC No. 14138;

the preparation method of the glucose oxidase specifically comprises the following steps:

aspergillus niger CH870 as producing strain;

(1) seed tank culture:

the pressure of the tank is 0.05-0.08MPa, the culture temperature is 30 ℃, and the ventilation rate is 30m³H, stirring speed is 180r/min, and pH is controlled to be 7.0; culturing until the thallus is deeply dyed and stout and has no mixed bacteria, and finishing culturing;

(2) cultivation in fermenter

The pressure of the tank is 0.05-0.08Mpa, the culture temperature is 30 ℃, the stirring speed is 300r/min, and the pH is controlled to be 6.5-7.0; ventilation quantity: 0-40h is 30m³40-58h is 45m³Per, 58 h-can discharge is 40m³H; when the pH value rises to 7.0, feeding is started, and the pH value is controlled to be 6.5-7.0;

(3) can for placing food

Culturing in a fermentation tank for 96-110h, slowly increasing enzyme activity, and placing the thallus into the tank when the thallus begins to autolyze partially;

(4) extraction of glucose oxidase

Fermentation liquor-pretreatment-filter pressing-ultrafiltration-standardization-fine filtration-blending-filling-detection-finished product;

after the pretreatment, the pH of the fermentation liquor is adjusted to about 4.0 after the fermentation liquor is placed in a tank, 2% of sodium benzoate, 3% of bentonite and 4% of perlite are sequentially added according to the volume of the fermentation liquor, and finally, 1 volume time of water of the fermentation liquor is added;

the blending refers to blending of enzyme activity according to the requirements of product specifications.

2. The method of glucose oxidase of claim 1,

cultivation in seeding tankAnd (3) nutrient medium: glucose 20%, peptone 25%, (NH)₄)₂SO₄5％，K₂HPO₄1％，MgSO₄·7H₂O0.5％，pH 7.0；

Fermentation medium: 12.30% of sucrose, 0.41% of peptone and NaNO₃0.6％、KH₂PO₄0.2％、KCl 0.05％、MgSO₄·7H₂O 0.07％、CaCO₃1％，pH 7.0；

A supplemented medium: 20% of sucrose, 30% of corn steep liquor, 0.5% of calcium chloride and 7.0 of pH value.

3. Use of the glucose oxidase of claim 1 for non-therapeutic purposes in the medical, food, feed fields.

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