CN107488640A - A kind of resistance to oxidation low temperature glucose oxidase and its production method and application - Google Patents
A kind of resistance to oxidation low temperature glucose oxidase and its production method and application Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, more particularly to a kind of low temperature glucose oxidase and its production method and application.The glucose oxidase originates from aspergillus niger (Aspergillu niger) CH870, and deposit number is CGMCC No.14138.The zymotic fluid enzyme activity of the glucose oxidase of strain fermentation production reaches more than 2300U/ml, and the glucose oxidase produced is stable in the range of pH2.0 8.0,20 DEG C of optimal reactive temperature, stability is good, and there is oxidative resistance, under 30mmol Hydrogen Peroxides, remaining enzyme activity is 100%；Under 200mmol Hydrogen Peroxides, remaining enzyme activity is 90.8%, is widely used.
Description
Technical field：
The invention belongs to technical field of bioengineering, more particularly to a kind of low temperature glucose oxidase of oxidative resistance raising
And its production method and application.
Background technology：
β-D-Glucose exclusively can be oxidized to gluconic acid and hydrogen peroxide, grape by glucose oxidase (GOD)
Carbohydrate oxidase suffers from being widely applied in fields such as food, medicine and biologies.
Because glucose oxidase can be catalyzed glucose consumption oxygen, gluconic acid and hydrogen peroxide are produced, so it
Applied widely in food service industry, in terms of being mainly manifested in following four：Remove food in remain glucose, deoxidation,
Sterilization, measure Determination of Glucose in Food content.
Glucose oxidase was classified as 12 kinds of feedings being allowed to use in 1999 as new green enzyme preparation by country
One of feed additives.The somatotrophic mechanism of action of glucose oxidase is：The glucose oxidase energy added in animal feed
With oxidation resistant function, can dispose under livestock stress situation in a large amount of free radicals caused by intestinal epithelial cell, to protect
Protect the integrality of intestinal epithelial cell.
Medically Tes-Tape is used for determining the glucose content in diabetes patient's urine, and principle is glycoxidative according to grape
Enzyme enzymatic reaction produces hydrogen peroxide, and hydrogen peroxide is further decomposed by catalase can produce water and oxygen, and oxygen is by test paper
On reduced form and colourless o-tolidine dye oxidation be blue material, blue material growing amount and concentration of glucose into than
Example, by the depth of test paper color with Standard colour board compared with, judge urinate in glucose content how much.Glucose oxidase oxygen speed
The degree of accuracy height of method measure glucose, the range of linearity is wide, atopic is strong, repeatability is good.
The enzyme sensor succeeded in developing in the world has tens kinds at present, and Updike and Hicks are in 1967 first by GOD films
It is covered in so as to which enzyme sensor be made on platinum electrode, it is used for the content of quantitative detection glucose in serum, and successfully
First glucose biological sensor has been made.
Glucose oxidase (GOD) is distributed widely in animal, plant and microbial body.But due to being included in animal and plant body
Measure less, and extract and have certain limitation, therefore less use.
In microorganism, the production of glucose oxidase mainly uses mold fermentation method, is typically done using aspergillus and mould
It is industrial production GOD such as aspergillus niger, aspergillus oryzae, mould, Kluyveromyces lactis and Kluyveromyces fragilis to produce strain
Universal proenzyme.At present, Production by Microorganism Fermentation glucose oxidase is used both at home and abroad.Due to mould under certain condition
The ability of generation glucose oxidase is strong, and therefore, the industrialized production of glucose oxidase mainly uses aspergillus niger
(Aspergillu niger) and Penicillium notatum (Penicilliunnotation).But glucose oxidase almost all depends on
Import, production cost also improve therewith.Therefore, the superior glucose oxidase enzyme of screenability, there is great practical application
Value.
The content of the invention：
In order to solve the above-mentioned technical problem, the present invention will provide a kind of resistance to oxidation low temperature glucose oxidase and its producer
Method and application.
The glucose oxidase originates from one plant of aspergillus niger, and the aspergillus niger is that this laboratory is answered through physical mutagenesis and chemistry
The high-yield of low-temperature glucose oxidase bacterial strain that one plant of oxidative resistance improves is obtained after closing mutagenesis, identified its belongs to aspergillus niger, has
Body is aspergillus niger (Aspergillu niger) CH870, and the bacterial strain is preserved in Chinese microorganism strain on July 13rd, 2017
Preservation administration committee common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Thing research institute, postcode 100101, deposit number are CGMCC No.14138.
The present invention also provides the liquid of the low glucose oxidase of a kind of high enzyme activity that ferments, extract yield height, manufacturing cost
State microbial fermentation production method, it is specific as follows：
Using aspergillus niger (Aspergillu niger) CH870 as production strain；
(1) seed tank culture：
Tank presses 0.05-0.08MPa, 30 DEG C of cultivation temperature, ventilation 30m3/ h, speed of agitator 180r/min, pH control
7.0；Culture to thalline dyes deep, sturdy, no miscellaneous bacteria, terminates culture；
Seed tank culture base：Glucose 20%, peptone 25%, (NH4)2SO45%, K2HPO41%, MgSO4·7H2O
0.5%, pH 7.0；
(2) fermentation tank culture
Tank presses 0.05-0.08Mpa, 30 DEG C of cultivation temperature, speed of agitator 300r/min, pH control 6.5-7.0；Ventilation：
0-40h is 30m3/ h, 40-58h 45m3It is 40m that/h, 58h-, which put tank,3/h；When pH rises to 7.0, start feed supplement, control pH exists
6.5-7.0；
Fermentation medium：Sucrose 12.30%, peptone 0.41%, NaNO30.6%th, KH2PO40.2%th, KCl
0.05%th, MgSO4·7H2O 0.07%, CaCO31%, pH 7.0；
Supplemented medium：Sucrose 20%, corn steep liquor 30%, calcium chloride 0.5%, pH 7.0.
(3) tank is put
Fermentation tank culture to 96-110h, enzyme activity increasess slowly, thalline beginning self-dissolving, you can put tank；
After measured, the zymotic fluid enzyme activity of glucose oxidase reaches more than 2300U/ml after the completion of fermentation；
(4) extraction of glucose oxidase
Zymotic fluid-pretreatment-press filtration-ultrafiltration-standardization-refined filtration-allotment-filling-detection-finished product.
The glucose oxidase of the present invention, it is characterised in that：
(1)pH：It is stable in the range of pH 2.0-8.0；
(2) temperature：It is relatively stable below 40 DEG C, 20 DEG C of optimal reactive temperature；1h is incubated under the conditions of 35 DEG C to remain to protect
More than 90% activity is held, when temperature rises to 40 DEG C, enzyme activity drops to 70% or so, 45 with the extension enzyme activity of soaking time
DEG C when its heat endurance substantially weaken；
(3) oxidative resistance：Under 30mmol Hydrogen Peroxides, remaining enzyme activity is 100%；200mmol Hydrogen Peroxides
Under, remaining enzyme activity is 90.8%.
Beneficial effect：
The invention provides a kind of resistance to oxidation low temperature glucose oxidase, the glucose oxidase is in pH2.0-8.0 models
Interior stabilization is enclosed, 20 DEG C of optimal reactive temperature, stability is good, and has oxidative resistance, under 30mmol Hydrogen Peroxides, residual enzyme
Living is 100%；Under 200mmol Hydrogen Peroxides, remaining enzyme activity is 90.8%, is widely used.
Brief description of the drawings：
Fig. 1 is the relative enzyme activity curve of glucose oxidase under different temperatures；
Fig. 2 is the relative enzyme activity curve of glucose oxidase under different pH；
Fig. 3 is the heat endurance curve of glucose oxidase；
Fig. 4 is the pH stability curves of glucose oxidase.
Embodiment：
More detailed description is done to the present invention below by way of specific embodiment：
The mutagenic and breeding of the bacterial strain of embodiment 1
The preparation of spore suspension：Spore on original strain inclined-plane is eluted with appropriate sterile saline, is placed in pre-
In the triangular flask of first sterilization zone bead, after 20min is vibrated on shaking table, mycelia is filtered off to scattered with the absorbent cotton of sterilizing
Monospore suspension, counted with blood counting chamber.It is diluted to 108Individual/mL spore suspension.
Microwave irradiation：Test tube equipped with 5mL spore suspensions is placed in the beaker containing ice cube, frequency of use 2450MHz,
Power output is 700W micro-wave oven, test tube is irradiated one by one by the different time, dilution gradient 10-1~10-6.Take
The 10 of each dosage-4~10-6The spore suspension 0.2mL of 3 dilution factors, is coated on plating medium, 30 DEG C of 2~3d of culture,
Clump count is calculated, draws fatal rate curve.Picking screening flat board (screening flat board culture medium：Bottom culture medium：Potato 20%,
Glucose 2%, NaNO30.2%th, K2HPO40.1%th, KCl 0.05%, MgSO40.05%th, agar 1.5%~2.0%；On
Layer culture medium：Glucose 2%, soluble starch 1%, KI 0.17%, phosphate buffer 0.1mol/L, agar 1.5-2.0%,
pH5.5.) the larger single bacterium colony of blue color ring is inoculated on inclined-plane, cultivated to spore and produced, then pass through fluid nutrient medium
Determine the glucose oxidase enzyme activity of the bacterial strain on each inclined-plane.Select glucose oxidase yield higher, and can stablize and lose
3 bacterial strains more than generation are passed, make slant preservation, and as the starting strain of further dithyl sulfate mutagenesis.
Dithyl sulfate (DES) mutagenesis：Take 5mL spore suspensions to be added in the triangular flask that volume is 25mL, add
0.2mL DES (volumetric concentration 50%), different time is vibrated, add 0.5mL sodium thiosulfate (85%) terminating reaction.
Dilution gradient is 10-1~10-6.Take the 10 of each dosage-4~10-6The spore suspension 0.2mL of 3 gradients, is coated on flat board culture
On base, incubator culture, 30 DEG C of 2~3d of culture are upside down in.Clump count is calculated, draws fatal rate curve.In picking screening flat board
Blueness is enclosed larger single bacterium colony and is inoculated on inclined-plane, is cultivated to spore and produced, then each oblique by Liquid Culture based assays
The glucose oxidase enzyme activity of the bacterial strain in face, choose the high bacterial strain of enzyme activity and preserved.Repeat the above steps carry out mutagenesis and
Screening, filters out the high-yield of low-temperature glucose oxidase bacterial strain CH870 that one plant of oxidative resistance improves, the strain growth speed compared with
It hurry up, production spore amount is few, and antioxygenic property improves, enzymatic production temperature is relatively low, and enzyme activity is higher and can be stable hereditary, shake flask fermentation enzyme
It is living to improve 5.6 times than starting strain.
Glucose oxidase superior strain CH870 stability passage assays
By the cultured fresh inclined-planes of glucose oxidase superior strain CH870, an oese lawn is taken with sterile spades
(hair shaking flask ferment culture medium is inoculated into fermentation shake flask：Glucose 6%, peptone 0.3%, NaNO30.4%th, KH2PO40.2%th,
KCl 0.05%, MgSO4·7H2O 0.07%, CaCO31%th, pH is naturally, 115 DEG C of sterilizing 20min), 30 DEG C, 220r/min trainings
96h is supported, determines enzyme activity.The bacterial strain continuous passage shake flask results of 10 times are as shown in table 1：
The bacterial strain CH870 Detection of Stability results of table 1.
By the generation of mutant strain Secondary Culture 10, experimental result as can be seen from Table 2, the genetic stability of the mutant strain
It is good.
The bacterial strain CH870 liquid fermentations of embodiment 2 produce glucose oxidase and its extraction
(1) seed tank culture：
Tank presses 0.05-0.08MPa, 30 DEG C of cultivation temperature, ventilation 30m3/ h, speed of agitator 180r/min, pH control
7.0；Culture to thalline dyes deep, sturdy, no miscellaneous bacteria, and seed liquor is cultivated to obtain in end；
Seed tank culture base：Glucose 20%, peptone 25%, (NH4)2SO45%, K2HPO41%, MgSO4·7H2O
0.5%, pH 7.0；
(2) fermentation tank culture
Seed liquor is seeded to fermentation tank, tank pressure 0.05-0.08Mpa, 30 DEG C of cultivation temperature, stirring turn by 5% inoculum concentration
Fast 300r/min, pH control 6.5-7.0；Ventilation：0-40h is 30m3/ h, 40-58h 45m3It is 40m that/h, 58h-, which put tank,3/h；
When pH rises to 7.0, start feed supplement, pH is in 6.5-7.0 for control；
Fermentation medium：Sucrose 12.30%, peptone 0.41%, NaNO30.6%th, KH2PO40.2%th, KCl
0.05%th, MgSO4·7H2O 0.07%, CaCO31%, pH 7.0.
Supplemented medium：Sucrose 20%, corn steep liquor 30%, calcium chloride 0.5%, pH 7.0.
(3) tank is put
Fermentation tank culture to 100h, enzyme activity increasess slowly, thalline beginning self-dissolving, you can put tank；
(4) extraction of glucose oxidase
Zymotic fluid-pretreatment-press filtration-ultrafiltration-standardization-refined filtration-allotment-filling-detection-finished product.
A. pre-process：After putting tank, measure fermentating liquid volume, then adjust pH to 4.0 or so, according to zymotic fluid volume successively
Add 2% sodium benzoate, 3% bentonite, 4% perlite, be eventually adding the water of 1 times of volume of zymotic fluid；
B. press filtration：After dispensing, the charging of each valve is opened, charging rate want suitable control not make too soon during beginning
Feed pressure slowly rises, while observes the definition of pressing filtering liquid, and Clear liquid tank is put into after pressing filtering liquid is clear；
C. ultrafiltration：Before playing filter, after bleeding off the maintenance buck in rolled film, replaced with clear water, then bleed off residual water in film.Open
Beginning ultrafiltration, inlet pressure control are controlled in 0.3Mpa or so in 0.4Mpa or so, outlet pressure, and temperature control is below 25 DEG C.
When ultrafiltration to finished product vigor requires, squeeze into and dissolve tank.
D. refined filtration：Refined filtration adds 1 ‰ sodium benzoates, 0.5 ‰ potassium sorbates and finished product requirement according to the volume of ultrafiltrate
Various dispensings, adjust pH, stir 1h rear feeding refined filtrations.
E. allocate, be filling：By the requirement of product specification, the allotment of enzyme activity is carried out, it is filling.
Table 2 is the fermentation period and fermentation broth enzyme vigor for carrying out 6 wholesale ferment, and average fermentation liquid enzyme activity is：2454U/mL.
The 3L canister fermenting experiment results of table 2.
| Batch | 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, mutagenic strain CH870 fermentation levels are relatively stable, fermentation enzyme activity reached 2300U/ml with
On.
The glucose oxidase enzyme activity determination method of embodiment 3
Substrate system：2mL 0.07g/L dianisidine solution, the glucose solutions of 1mL 5% are pipetted with 1mL liquid-transfering gun
In scale test tube.0.1mL0.1g/L horseradish peroxidases solution is pipetted in identical graduation test tube with 0.5mL pipette
In.The substrate system is placed in 30 DEG C of insulation 10min in thermostat water bath.
Enzyme activity determination：Enzyme liquid after drawing 0.1mL dilutions with liquid-transfering gun shakes up in substrate, fast using blank tube as control
Speed visible spectrophotometer measure light absorption value at 460nm.It is A to read initial absorbance value0And timing, recorded every 1min
Absorbance An, 5min is determined altogether.
By the absorbance measured, enzyme liquid enzyme activity is calculated as follows：X1(U/mL)
ΔAn+1=An+1-An(n=0,1,2,3,4)
ΔAIt is average=(Δ A1+ΔA2+ΔA3+ΔA4+ΔA5)/5
X1=Δs AIt is average×f/(11.3×t×V1/V2)
In formula：11.3-extinction coefficient；
T- reaction time, min；
V1- enzyme liquid volumes, mL；
V2- reaction solution cumulative volumes, mL；
F- enzyme liquid extension rates.
The optimal reactive temperature of embodiment 4
Glucose oxidase finished product prepared by Example 2, the enzyme activity determination method according to embodiment 3, dissolving system
After into enzyme liquid in pH6.0, glucose oxidase is determined under the conditions of 10,15,20,25,30,35,40,45,50 DEG C respectively and is lived
Power, calculate enzyme activity.As a result as shown in figure 1, optimal reactive temperature is 20 DEG C, also there is higher enzyme activity at 10 DEG C.
From experimental result, the optimal reactive temperature of the mutant strain institute malaga carbohydrate oxidase is significantly lower than the grape in other sources
Carbohydrate oxidase, and low temperature glucose oxidase can be widely applied in the production of preservation by low temperature, medicine and food, have very big
Market application value, is with a wide range of applications in the industrial production.
The optimal reaction pH of embodiment 5
Glucose oxidase finished product prepared by Example 2, the enzyme activity that dissolving is made after enzyme liquid according to embodiment 3 are surveyed
Determine method, under the conditions of temperature is 20 DEG C, determine respectively in pH value at 4.0,4.5,5.0,5.5,6.0,6.5,7.0 and 7.5
Glucose oxidase vigor under part, calculate enzyme activity.Measurement result as shown in Fig. 2 the enzyme activity of glucose oxidase in pH
For 5.5 when enzyme activity highest.
The heat endurance of embodiment 6
Glucose oxidase finished product prepared by Example 2, the enzyme activity determination method according to embodiment 3, after dissolving
Isothermal holding 60min under the conditions of enzyme liquid is respectively placed in into 20,25,30,35,40,45,50,55,60,65,70 DEG C, insulation terminate
Determining it afterwards, (with respect to the ratio that enzyme activity is the enzyme activity after different temperatures isothermal holding and initial enzyme activity, definition is just with respect to enzyme activity
Beginning enzyme activity is 100%), its experimental result is as shown in Figure 3.The activity that 1h remains to keep more than 90% is incubated under the conditions of 35 DEG C,
When temperature rises to 40 DEG C, enzyme activity drops to 70% or so with the extension enzyme activity of soaking time, and its heat endurance is bright at 45 DEG C
It is aobvious to weaken.
Low temperature glucose oxidase caused by mutagenic strain CH870 can just lose enzyme activity by gentle heat treatment
Lose, and relatively low gentle Temperature Treatment does not interfere with the quality of product, therefore, can effectively improve the matter of product
Amount.
The resistance to acids and bases of embodiment 7
Glucose oxidase finished product prepared by Example 2, the enzyme activity determination method according to embodiment 3, is used respectively
The pH of enzyme liquid is adjusted to 1.0 by 0.1m NaOH or 0.1M HCl, 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,
10.0th, 11.0, it is respectively placed in after standing 24h under room temperature condition, is 20 DEG C, determines it with respect to enzyme activity under the conditions of pH5.5 in temperature
Power (with respect to the ratio that enzyme activity is the enzyme activity after condition of different pH is handled and initial enzyme activity, defines initial enzyme activity as 100%).Survey
Result is determined as shown in figure 4, after 24h under conditions of pH2.0-8.0, and enzyme activity stills remain in more than 80%.
The starting strain of embodiment 8 is compared with the experiment of mutagenic strain CH870 institutes malaga carbohydrate oxidase oxidative resistances
According to the method for embodiment 2, grape is produced as production strain fermentation using starting strain and mutagenic strain CH870 respectively
Carbohydrate oxidase, zymotic fluid centrifuging and taking supernatant is obtained into crude enzyme liquid after fermentation ends.By glucose oxidase caused by original strain with
The hydrogen peroxide treatment through various concentrations, treatment temperature are 20 DEG C to glucose oxidase caused by mutagenic strain CH870 respectively, place
The reason time is 2h, and buffer solution is pH5.5 phosphate buffer.Processing adds a certain amount of catalase removing after terminating not anti-
The hydrogen peroxide answered, terminating reaction, the relative enzyme activity for then determining each group glucose oxidase (is through hydrogen peroxide with respect to enzyme activity
The ratio of enzyme activity and initial enzyme activity after processing, initial enzyme activity is defined as 100%), it is shown in Table 3 with respect to enzyme activity：
The original strain of table 3. is compared with the oxidative resistance of mutagenic strain CH870 glucose oxidases
Glucose oxidase can produce hydrogen peroxide during the course of the reaction, and therefore, the enzyme has the function that antibiotic and sterilizing, but
It is that the accumulation of hydrogen peroxide can also suppress the activity of glucose oxidase, research is found, when the concentration of hydrogen peroxide reaches
During 30mmol inhibitory action, therefore, the glucose oxidase of seed selection oxidative resistance will be produced to the activity of glucose oxidase
Superior strain is of great significance.Concentration when hydrogen peroxide it can be seen from the experimental result of table 3 reaches 30mmol
When, the glucose oxidase activity of original strain is reduced to original 90.7%, and mutagenic strain enzyme activity does not almost become.Through identical dense
After the hydrogen peroxide treatment of degree, the oxidation resistent susceptibility of mutagenic strain is apparently higher than control strain, and therefore, mutagenic strain CH870 exists
There is higher value in practical application.
Claims (4)
1. one plant of resistance to oxidation low temperature glucose oxidase, it is characterised in that the glucose oxidase originates from aspergillus niger, is specially
Aspergillus niger (Aspergillu niger) CH870, deposit number are CGMCC No.14138.
2. the method for glucose oxidase described in claim 1, it is characterised in that specific as follows：
Using aspergillus niger (Aspergillu niger) CH870 as production strain；
(1) seed tank culture：
Tank presses 0.05-0.08MPa, 30 DEG C of cultivation temperature, ventilation 30m3/ h, speed of agitator 180r/min, pH control 7.0；Culture
Deep, sturdy, no miscellaneous bacteria is dyed to thalline, terminates culture；
(2) fermentation tank culture
Tank presses 0.05-0.08Mpa, 30 DEG C of cultivation temperature, speed of agitator 300r/min, pH control 6.5-7.0；Ventilation：0-40h
For 30m3/ h, 40-58h 45m3It is 40m that/h, 58h-, which put tank,3/h；When pH rises to 7.0, start feed supplement, pH is in 6.5- for control
7.0；
(3) tank is put
Fermentation tank culture to 96-110h, enzyme activity increasess slowly, thalline beginning self-dissolving, you can put tank；
(4) extraction of glucose oxidase
Zymotic fluid-pretreatment-press filtration-ultrafiltration-standardization-refined filtration-allotment-filling-detection-finished product.
3. the method for glucose oxidase as claimed in claim 2, it is characterised in that
Seed tank culture base：Glucose 20%, peptone 25%, (NH4)2SO45%, K2HPO41%, MgSO4·7H2O
0.5%, pH 7.0；
Fermentation medium：Sucrose 12.30%, peptone 0.41%, NaNO30.6%th, KH2PO40.2%th, KCl 0.05%,
MgSO4·7H2O 0.07%, CaCO31%, pH 7.0；
Supplemented medium：Sucrose 20%, corn steep liquor 30%, calcium chloride 0.5%, pH 7.0.
4. the application of glucose oxidase described in claim 1.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107488600A (en) * | 2017-09-18 | 2017-12-19 | 山东隆科特酶制剂有限公司 | One plant height produces the aspergillus niger of resistance to oxidation low temperature glucose oxidase |
| CN108384766A (en) * | 2018-06-11 | 2018-08-10 | 张宝华 | A method of preparing glucose oxidase using microbial fermentation |
| CN109652390A (en) * | 2019-02-25 | 2019-04-19 | 大连大学 | A kind of marine low temperature glucose oxidase and its application |
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| CN109864188A (en) * | 2019-02-28 | 2019-06-11 | 大连大学 | A kind of feed addictive of the glucose oxidase containing low temperature and application thereof |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488600A (en) * | 2017-09-18 | 2017-12-19 | 山东隆科特酶制剂有限公司 | One plant height produces the aspergillus niger of resistance to oxidation low temperature glucose oxidase |
| CN107488600B (en) * | 2017-09-18 | 2020-09-25 | 山东隆科特酶制剂有限公司 | Aspergillus niger capable of producing oxidation-resistant low-temperature glucose oxidase with high yield |
| CN108384766A (en) * | 2018-06-11 | 2018-08-10 | 张宝华 | A method of preparing glucose oxidase using microbial fermentation |
| CN108384766B (en) * | 2018-06-11 | 2020-12-08 | 宁波天皓生物科技有限公司 | Method for preparing glucose oxidase by utilizing microbial fermentation |
| CN109652390A (en) * | 2019-02-25 | 2019-04-19 | 大连大学 | A kind of marine low temperature glucose oxidase and its application |
| CN109749967A (en) * | 2019-02-28 | 2019-05-14 | 大连大学 | The marine bacteria of one plant of malaga carbohydrate oxidase and its application |
| CN109864188A (en) * | 2019-02-28 | 2019-06-11 | 大连大学 | A kind of feed addictive of the glucose oxidase containing low temperature and application thereof |
| CN109880809A (en) * | 2019-02-28 | 2019-06-14 | 大连大学 | A kind of genetic engineering bacterium and preparation method thereof producing low temperature glucose oxidase |
| CN111500473A (en) * | 2020-06-05 | 2020-08-07 | 宏葵生物（中国）股份有限公司 | Method for producing low-temperature glucose oxidase by microbial fermentation |
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