CN109749967A - The marine bacteria of one plant of malaga carbohydrate oxidase and its application - Google Patents
The marine bacteria of one plant of malaga carbohydrate oxidase and its application Download PDFInfo
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- 101710128063 Carbohydrate oxidase Proteins 0.000 title claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 55
- 108090000790 Enzymes Proteins 0.000 claims abstract description 54
- 229940088598 enzyme Drugs 0.000 claims abstract description 53
- 230000001580 bacterial effect Effects 0.000 claims abstract description 31
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 27
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 26
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 26
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 26
- 244000005700 microbiome Species 0.000 claims abstract description 13
- 241000873310 Citrobacter sp. Species 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims description 34
- 238000012216 screening Methods 0.000 claims description 18
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Abstract
The present invention provides one plant of bacterium for being able to produce glucose oxidase, belongs to microorganisms technical field.The bacterial strain is a kind of III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8-, it is isolated from the Bohai Offshore ooze of Dalian Area, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 24th, 2019, and biological deposits number is CGMCC NO.17228.Bacterial strain of the invention belongs to bacterium, is a kind of new glucose oxidase production bacterial strain.Furthermore the produced GOD of the bacterial strain belongs to cold-adapted enzyme scope, can make up existing GOD low temperature field using blank, therefore have extraordinary Prospect of R & D.
Description
Technical field
The invention belongs to microorganisms technical fields, are able to produce the thin of glucose oxidase in particular to a kind of
Bacterium.
Background technique
Glucose oxidase (Glucose oxidase, GOD) can be catalyzed β-D-Glucose and air to high specificity
In oxygen reaction, so that glucose is oxidized into gluconic acid and hydrogen peroxide, have the effects that glucose, deoxidation, sterilization, and
And it is safe and free of toxic and side effects, the processing and antistaling of food, blood sugar test and in terms of on have and widely answer
With.Existing GOD bacterium source includes aspergillus niger, mould, and the operative temperature that produced GOD is adapted to is at 35 DEG C -50 DEG C, in low temperature
There is apply drawback in terms of detection, food fresh keeping and feed anticorrosion.Traditional source GOD terrestrial animal, plant and soil, it is uneven
There is low temperature attribute.
The bacillus of GOD is produced in pertinent literature report Shi Shuyu et al. screening from ooze, and Xu Defeng et al. is from ooze
The bacterium of GOD is produced in screening in sample, these prior arts all absolutely prove that there is GOD genes in marine microorganism, and
Since marine organisms are because of its special habitat, the enzyme generated have significant specificity (such as pressure-resistant, alkaline-resisting, salt tolerant and
It is cold-resistant etc.), more meet the application requirement of modern biotechnology and different secondary industries.
Summary of the invention
The purpose of the present invention is to provide a kind of low temperature glucose oxidase bacterial strain, bacterial strain institute malaga carbohydrate oxidase
Has cold-adapted enzyme characteristic.
To achieve the above object, the invention adopts a technical scheme as:
A kind of marine bacteria, a kind of III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8-, the bacterium
Strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 24th, 2019, address: Beijing
No. 3 Institute of Microorganism, Academia Sinica, institute of city, North Star West Road, Chaoyang District 1, postcode: 100101, biological deposits number are
CGMCC NO.17228。
The bacterial strain is subjected to 16sRNA identification, as shown in SEQ ID NO.1, No. GenBanK is MK050005, determines bacterial strain
Belong to for citric acid bacillus (Citrobacter sp.).
III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8- can prepare grape glycosyloxy
Change and is applied in enzyme.
The screening technique of the malaga carbohydrate oxidase marine bacteria the following steps are included:
Step 1, the ooze sample for taking Dalian Area Bohai Offshore under aseptic technique are added and train equipped with sterile enrichment
It in the triangular flask for supporting base and bead, sufficiently shakes up, is put into shaking table culture case 20-25 DEG C, 160-200r/min cultivates 12-
For 24 hours, sample bacterium solution is obtained.
Enriched medium: yeast extract 0.5-1%, peptone 1-3%, sodium chloride 1-2%.
Step 2, by each sample bacterium solution after enrichment, be diluted to different gradients with antiseptic sea water, each gradient takes 100 μ L to apply
It is distributed on solid screening and culturing medium plate, is placed at 20-25 DEG C, be inverted culture 48-72h, parallel laboratory test is set.Choose periphery
There is the bacterium colony of blue colouration reaction in culture medium, continues plate streaking purifying.
Solid screening and culturing medium: glucose 4.5-6%, peptone 0.27-0.45%, KH2PO40.02-0.04%,
(NH4)2HPO40.035-0.05%, CaCO30.3-0.5%, soluble starch 0.5-1%, MgSO40.015-0.02% takes off
Oxycholic acid sodium 0.02-0.03%, chloramphenicol 0.01-0.03%, Polyoxin 0.01-0.03%, horseradish peroxidase 0.02-
0.04%, dianisidine 0.1-0.2%, agar powder 2-2.5%.
The purifying strain inoculated that step 3, picking are obtained by step 1 and 2 primary dcreening operations is in fermentation medium, and 20-25 DEG C, 160-
200r/min cultivates 48-72h.Then bacterium solution is centrifuged 15min in 8000r/min, takes supernatant that enzyme activity reaction system is added,
Its OD value is measured at 460nm.Primary at interval of observation in one minute, the continuous rising of OD value presentation, which represents the produced enzyme of the bacterial strain, to be existed
Enzyme activity.
Fermentation medium: glucose 4-6%, peptone 0.3-0.5%, potassium dihydrogen phosphate 0.2-0.4%, magnesium sulfate
0.05-1%, potassium chloride 0.05-1%, sodium nitrate 4-6%, pH7.0.
Step 4, the bacterium after being centrifuged in step 3 is observed on LB solid medium secondary screening obtain the color of bacterium colony, shape,
The modal feature such as gloss, transparency；Utilize the form of Electronic Speculum observation microorganism；It is carried out after 20-25 DEG C of culture 48-72h
16sRNA identification, as shown in SEQ ID NO.1, determines that bacterial strain belongs to for citric acid bacillus.
LB solid medium: yeast extract 0.5-1%, peptone 0.5-1%, sodium chloride 0.5-1%, agar 2-
2.5%.
The preparation glucose oxidase method is as follows:
Preservation strain is inoculated in LB liquid medium with 2-3% ratio by step 1, and 20-25 DEG C, 160-200rpm is cultivated
48-72h.LB liquid medium: 0.5-1% sodium chloride, 0.5-1% yeast extract, 0.5-1% peptone；
Step 2 takes the cultured 200 μ L of bacterium solution of step 1 to be inoculated in 100mL fermentation medium, and 20-25 DEG C, 160-
200rpm cultivates 48-72h.
Step 2 gained bacterium solution is mixed with saturated ammonium sulfate solution ammonium sulfate saturation degree is made to reach 75%, 0-4 DEG C by step 3
Stand 12-16h.
Step 3 gained bacterium solution 8000rpm is centrifuged 15min by step 4, abandons supernatant, and it is heavy to be resuspended with the PBS of 10mLpH7.4
It forms sediment.
Re-suspension liquid ultrasound is cracked 15min (opening 3S, be spaced 5S, power 200W) by step 5.By lysate 10000r/min,
4 DEG C, it is centrifuged 20min, supernatant is crude enzyme liquid.
Step 6, crude protein are through G-100 and Ni2+- NTA column (Novagen) carries out affinitive layer purification and obtains Portugal after purification
Grape carbohydrate oxidase, 4 DEG C of protein liquid preservations of elution.
Enzyme solution obtained in step 6 after purification is placed in freeze dryer and is freeze-dried by step 7, and it is glycoxidative to obtain grape
Enzyme enzyme powder.
Finally test to the physiological and biochemical property of institute's malaga carbohydrate oxidase.
The invention has the benefit that the bacterial strain filtered out in the present invention belongs to bacterium, the malaga glycosyloxy being previously reported
Changing enzyme bacterial strain is almost fungi.Bacterium is smaller compared to fungal gene group, is easier to be studied and be transformed from gene level.The Portugal
Grape carbohydrate oxidase optimum temperature is and higher in 0-20 DEG C of opposite enzyme activity at 15 DEG C, has cold-adapted enzyme characteristic, is other fungies
Not available, in low temperature field research potential with higher.
Detailed description of the invention
Fig. 1 is that malaga carbohydrate oxidase marine bacteria bacterium colony screens colour developing figure；
Fig. 2 is the electron microscopic morphology figure of III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8-；
Fig. 3 is glucose oxidase optimum temperature curve graph；
Fig. 4 glucose oxidase thermal stability curve graph；
Fig. 5 glucose oxidase optimal pH curve graph；
Fig. 6 glucose oxidase ph stability curve graph；
The influence schematic diagram of Fig. 7 metal ion and chelating agent to enzyme activity.
Specific embodiment
Following embodiment will the present invention is further illustrated in conjunction with attached drawing.
One, the screening of object bacteria
Step 1, the ooze sample 4g for taking Dalian Area Bohai Offshore under aseptic technique, be added equipped with 100mL without
It in the triangular flask of bacterium enriched medium and appropriate bead, sufficiently shakes up, is put into shaking table culture case 20 DEG C, 160r/min culture
12h。
Enriched medium: yeast extract 0.5%, peptone 1%, sodium chloride 1%.
Step 2, will enrichment after each sample bacterium solution, be diluted to 10 with antiseptic sea water-6、10-7、10-8Three gradients, Mei Geti
Degree take 100 μ L be coated on containing horseradish peroxidase, dianisidine be color development system solid screening and culturing medium plate on,
Chloramphenicol and Polyoxin inhibit fungi growth in the primary dcreening operation plate.25 DEG C of constant temperature are inverted culture 72h, and 3 groups of setting is real in parallel
It tests.After colony diameter reaches 1mm-4mm, as relayed kind of object.It chooses periphery culture medium and blue colouration reaction occurs
Bacterium colony continue plate streaking purifying.
Screening and culturing medium: glucose 4.5%, peptone 0.27%, KH2PO40.02%, (NH4)2HPO40.042%,
CaCO30.3%, soluble starch 0.8%, MgSO40.016%, NaTDC 0.02%, chloramphenicol 0.01%, polyoxy is mould
Element 0.01%, horseradish peroxidase 0.02%, dianisidine 0.12%, agar powder 2%.
The purifying strain inoculated that step 3, picking primary dcreening operation obtain is in fermentation medium, and 25 DEG C, 160r/min cultivates 48h.So
Bacterium solution is centrifuged 15min in 8000r/min afterwards, takes supernatant that enzyme activity reaction system is added, its OD value is measured at 460nm.Every
Primary every observation in one minute, the continuous rising of OD value presentation represents the produced enzyme of the bacterial strain, and there are enzyme activity.
Fermentation medium: glucose 6%, peptone 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.07%, potassium chloride
0.05%, sodium nitrate 4%, pH7.0.
Enzyme activity determination method is the continuous spectrophotometry of dianisidine: it is molten that 5% glucose is sequentially added in 96 orifice plates
Liquid 150uL, dianisidine solution 150uL and horseradish peroxidase solution 10uL, and by the reaction system as 25 DEG C of perseverances
Temperature stands 10min, and 10uL crude enzyme liquid is then added.Microplate reader wavelength 460nm is set, measures absorbance A at interval of 1min, continuously
5min is measured, rising representative is presented in OD value, and there are enzyme activity.
Step 4, observation secondary screening obtains the forms such as color, shape, gloss, the transparency of bacterium colony on LB solid medium
On feature；Utilize the form of Electronic Speculum observation microorganism.It was found that the fast growing on LB culture medium, 25 DEG C of culture 72h diameter 5-
8mm, bacterium colony are presented milky (as shown in Figure 1), there is slight tart flavour.Bacterial strain analytic type scanning electron microscopic observation, the bacterial strain are nothing
The medium sized straight-bar bacterium of gemma, size are 2.63 μ ms (0.276-0.561) μm.Thallus both ends blunt circle, it is more to exist in pairs,
Also it is dispersed in (as shown in Figure 2).
Step 5, reference " common bacteria system identification handbook " carry out glucose fermentation test to each bacterial strain, methyl red tries
It tests, V-P experiment, gelatin liquefaction experiment, Starch Hydrolysis experiment, gelatin liquefaction experiment, urea test, hydrogen peroxide test, Twee
The test such as n80.As shown in table 1.
1 bacterial strain part biochemical character of table
Step 6, by strain inoculated on LB plate, be sent to after 25 DEG C of culture 48h Beijing source Nuo Hezhi company progress
16sRNA identification, as shown in SEQ ID NO.1, No. GenBanK is MK050005, determines that bacterial strain belongs to for citric acid bacillus
(Citrobacter sp.)。
A kind of III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8-, the bacterial strain is in 2019 1
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 24th, address: the Chaoyang District, Beijing City North Star
No. 3 Institute of Microorganism, Academia Sinica, institute of West Road 1, postcode: 100101, biological deposits number are CGMCC NO.17228.
Two, glucose oxidase the preparation method is as follows:
Preservation strain is inoculated in LB liquid medium with 2% ratio by step 1, and 20 DEG C, 160rpm cultivates 48h.
LB culture medium: 1% sodium chloride, 0.5% yeast extract, 1% peptone；
Step 2 takes the cultured 200 μ L of bacterium solution of step 1 to be inoculated in 100mL fermentation medium, and 20 DEG C, 160rpm is cultivated
48h。
Fermentation medium: 6% glucose, 0.3% peptone, 0.2% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.05%
Potassium chloride, 0.4% sodium nitrate pH6.5.
Step 2 gained bacterium solution is mixed with saturated ammonium sulfate solution ammonium sulfate saturation degree is made to reach 75% by step 3, and 4 DEG C quiet
Set 12h.
Step 3 gained bacterium solution 8000rpm is centrifuged 15min by step 4, abandons supernatant, and it is heavy to be resuspended with the PBS of 10mLpH7.4
It forms sediment.
Re-suspension liquid ultrasound is cracked 15min (opening 3S, be spaced 5S, power 200W) by step 5.By lysate 10000r/min,
4 DEG C, it is centrifuged 20min, supernatant is crude enzyme liquid.
Step 6, crude protein are through G-100 and Ni2+- NTA column (Novagen) carries out affinitive layer purification and obtains Portugal after purification
Grape carbohydrate oxidase, 4 DEG C of protein liquid preservations of elution.
Enzyme solution obtained in step 6 after purification is placed in freeze dryer and is freeze-dried by step 7, and it is glycoxidative to obtain grape
Enzyme enzyme powder.
Step 8 tests to the physiological and biochemical property of institute's malaga carbohydrate oxidase
Glucose oxidase optimum temperature: will appropriate diluted enzyme solution respectively 0,5,10,15,20,25,30,35,
40, it is reacted under 45,50,55 and 60, and carries out enzyme activity determination according to the continuous spectrophotometry of dianisidine.Most with enzyme activity
Gao Zhewei 100% calculates opposite enzyme activity, so that it is determined that the optimum temperature of the enzyme, as shown in figure 3, in the activity of 0-40 DEG C of enzyme
Relatively high, optimum temperature is 15 DEG C.
The thermal stability of glucose oxidase: appropriate diluted enzyme solution is put into 0 respectively, 5,10,15,20,25,30,35,
40,45,50,55 and 60 lower heat preservation 1h, samples every 30min, places the continuous spectrophotometric of dianisidine after 5min on ice
Method carries out enzyme activity determination.It is the opposite enzyme activity of 100% calculating with the enzyme activity for the enzyme solution not being heat-treated, recombinates glucose to determine
The thermal stability of oxidizing ferment, as shown in figure 4, glucose oxidase is relatively stable at 0-45 DEG C, it is steady to reach highest at 15 DEG C
Definite value.
Glucose oxidase Optimun pH: prepare pH be respectively 4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,
8.0,8.5,9.0,9.5,10.0 and 10.5 PBS buffer system is reacted after diluting enzyme solution with these buffers respectively,
Then enzyme activity determination is carried out according to the continuous spectrophotometry of dianisidine.It is the opposite enzyme activity of 100% calculating with enzyme activity soprano,
So that it is determined that the most suitable action pH of glucose oxidase, as shown in figure 5, the activity of enzyme is relatively high when pH value is 5-8.5,
Its optimum pH is 6.5.
The pH stability of glucose oxidase: respectively by enzyme solution pH be 4.0,4.5,5.0,5.5,6.0,6.5,7.0,
7.5,30min (temperature is 25 DEG C) is kept the temperature in 8.0,8.5,9.0,9.5,10.0 and 10.5 PBS buffer solution, and joins fennel according to neighbour
The fragrant continuous spectrophotometry of amine carries out enzyme activity determination.It is 100% calculating residual enzyme activity with the enzyme activity without isothermal holding, thus
The pH stability of glucose oxidase is determined, as shown in fig. 6, glucose oxidase is relatively stable, in pH when pH value is 4-10
Value reaches highest stabilizing value when being 6.5.
Metal ion influences the enzyme activity of glucose oxidase: in enzyme activity reaction system, Na is added+、K+、Ca2+、Ni2+、
Fe2+、Cu2+、Mg2+、Fe3+、Zn2+、Ag+And ethylenediamine tetra-acetic acid (ethylenediaminetetraacetic acid, EDTA),
Make its final concentration of 0.35mmol/L, then carries out enzyme activity determination according to the continuous spectrophotometry of dianisidine.Gold is not added
The enzyme activity for belonging to the reaction system of ion is defined as 100%, calculates the opposite enzyme activity under different metal ions or compound, such as Fig. 7
It is shown, K+、Ni2+Effect is obviously promoted to the activity of glucose oxidase；Na+、Zn2+、Fe3+、Cu2+、Ag+、Ca2+、Fe2+、
Mg2+There is inhibiting effect to glucose oxidase activity with EDTA, wherein Cu2+、Ag+、Ca2+、Fe2+And Mg2+It is serious to inhibit glucose
Oxidase active.Speculate that this may be and then enzyme to be caused to inactivate since these ions are in conjunction with the functional group at enzyme center.
Sequence table
<110>University Of Dalian
The marine bacteria of<120>one plants of malaga carbohydrate oxidases and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1348
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cttcttttgc aacccactcc catggtgtga cgggcggtgt gtacaaggcc cgggaacgta 60
ttcaccgtgg cattctgatc cacgattact agcgattccg acttcatgga gtcgagttgc 120
agactccaat ccggactacg acatacttta tgaggtccgc ttgctctcgc gaggtcgctt 180
ctctttgtat atgccattgt agcacgtgtg tagccctact cgtaagggcc atgatgactt 240
gacgtcatcc ccaccttcct ccagtttatc actggcagtc tcctttgagt tcccggccga 300
accgctggca acaaaggata agggttgcgc tcgttgcggg acttaaccca acatttcaca 360
acacgagctg acgacagcca tgcagcacct gtctcagagt tcccgaaggc accaaagcat 420
ctctgctaag ttctctggat gtcaagagta ggtaaggttc ttcgcgttgc atcgaattaa 480
accacatgct ccaccgcttg tgcgggcccc cgtcaattca tttgagtttt aaccttgcgg 540
ccgtactccc caggcggtcg acttaacgcg ttagctccgg aagccacgcc tcaagggcac 600
aacctccaag tcgacatcgt ttacggcgtg gactaccagg gtatctaatc ctgtttgctc 660
cccacgcttt cgcacctgag cgtcagtctt tgtccagggg gccgccttcg ccaccggtat 720
tcctccagat ctctacgcat ttcaccgcta cacctggaat tctacccccc tctacaagac 780
tctagcctgc cagtttcgga tgcagttccc aggttgagcc cggggatttc acatccgact 840
tgacagaccg cctgcgtgcg ctttacgccc agtaattccg attaacgctt gcaccctccg 900
tattaccgcg gctgctggca cggagttagc cggtgcttct tctgcgagta acgtcaatcg 960
ttgcggttat taaccacaac gccttcctcc tcgctgaaag tactttacaa cccgaaggcc 1020
ttcttcatac acgcggcatg gctgcatcag gcttgcgccc attgtgcaat attccccact 1080
gctgcctccc gtaggagtct ggaccgtgtc tcagttccag tgtggctggt catcctctca 1140
gaccagctag ggatcgtcgc ctaggtgagc cgttacccca cctactagct aatcccatct 1200
gggcacatcc gatggcaaga ggcccgaagg tccccctctt tggtcttgcg acgttatgcg 1260
gtattagcta ccgtttccag tagttatccc cctccatcgg gcagtttccc agacattact 1320
cacccgtccg ccactcgtca cccaagga 1348
Claims (8)
1. a kind of III bacterial strain of malaga carbohydrate oxidase marine bacteria (Citrobacter sp.) 8-, the bacterial strain is in January, 2019
It was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 24th, address: Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica, institute of road 1, postcode: 100101, biological deposits number are CGMCC NO.17228.
2. a kind of malaga carbohydrate oxidase marine bacteria according to claim 1, which is characterized in that screening technique include with
Lower step:
Step 1, the ooze sample for taking Dalian Area Bohai Offshore under aseptic technique are added and sterile enriched medium are housed
It in the triangular flask of bead, sufficiently shakes up, is put into shaking table culture case 20-25 DEG C, 160-200r/min cultivates 12-24h, obtains
To sample bacterium solution；
Step 2, by each sample bacterium solution after enrichment, be diluted to different gradients with antiseptic sea water, each gradient takes 100 μ L to be coated on
It on solid screening and culturing medium plate, is placed at 20-25 DEG C, is inverted culture 48-72h, parallel laboratory test is set；Choose periphery culture
There is the bacterium colony of blue colouration reaction in base, continues plate streaking purifying；
The purifying strain inoculated that step 3, picking are obtained by step 1 and 2 primary dcreening operations is in fermentation medium, and 20-25 DEG C, 160-
200r/min cultivates 48-72h；Then bacterium solution is centrifuged 15min in 8000r/min, takes supernatant that enzyme activity reaction system is added,
Its OD value is measured at 460nm；Primary at interval of observation in one minute, the continuous rising of OD value presentation, which represents the produced enzyme of the bacterial strain, to be existed
Enzyme activity；
Bacterium after being centrifuged in step 3 is observed secondary screening on LB solid medium and obtains the color, shape, light of bacterium colony by step 4
The modal feature such as pool, transparency；Utilize the form of Electronic Speculum observation microorganism；It is carried out after 20-25 DEG C of culture 48-72h
16sRNA identification, as shown in SEQ ID NO.1, No. GenBanK is MK050005, determines that bacterial strain belongs to for citric acid bacillus
(Citrobacter sp.)。
3. screening technique according to claim 2, which is characterized in that the enrichment culture based formulas are as follows: yeast extracts
Object 0.5-1%, peptone 1-3%, sodium chloride 1-2%.
4. screening technique according to claim 2, which is characterized in that the solid screening and culturing based formulas are as follows: grape
Sugared 4.5-6%, peptone 0.27-0.45%, KH2PO40.02-0.04%, (NH4)2HPO40.035-0.05%, CaCO3
0.3-0.5%, soluble starch 0.5-1%, MgSO40.015-0.02%, NaTDC 0.02-0.03%, chloramphenicol
0.01-0.03%, Polyoxin 0.01-0.03%, horseradish peroxidase 0.02-0.04%, dianisidine 0.1-
0.2%, agar powder 2-2.5%.
5. screening technique according to claim 2, which is characterized in that the fermentative medium formula are as follows: glucose 4-
6%, peptone 0.3-0.5%, potassium dihydrogen phosphate 0.2-0.4%, magnesium sulfate 0.05-1%, potassium chloride 0.05-1%, sodium nitrate
4-6%, pH7.0.
6. screening technique according to claim 2, which is characterized in that the LB solid culture based formulas are as follows: yeast mentions
Take object 0.5-1%, peptone 0.5-1%, sodium chloride 0.5-1%, agar 2-2.5%.
7. III bacterial strain of marine bacteria (Citrobacter sp.) 8- of malaga carbohydrate oxidase is answered in preparing glucose oxidase
With.
8. the use as claimed in claim 7, which is characterized in that steps are as follows:
III bacterial strain of 8- is inoculated in LB liquid medium with 2-3% ratio by step 1, and 20-25 DEG C, 160-200rpm cultivates 48-
72h；LB liquid medium: 0.5-1% sodium chloride, 0.5-1% yeast extract, 0.5-1% peptone；
Step 2 takes the cultured 200 μ L of bacterium solution of step 1 to be inoculated in 100mL fermentation medium, and 20-25 DEG C, 160-200rpm is trained
Support 48-72h；
Step 2 gained bacterium solution is mixed with saturated ammonium sulfate solution ammonium sulfate saturation degree is made to reach 75%, 0-4 DEG C of standing by step 3
12-16h；
Step 3 gained bacterium solution 8000rpm is centrifuged 15min by step 4, abandons supernatant, and precipitating is resuspended with the PBS of 10mLpH7.4；
Re-suspension liquid ultrasound is cracked 15min by step 5, is opened 3S, is spaced 5S, power 200W；By lysate 10000r/min, 4 DEG C,
It is centrifuged 20min, supernatant is crude enzyme liquid；
Step 6, crude protein are through G-100 and Ni2+- NTA column carries out affinitive layer purification and obtains glucose oxidase after purification, washes
4 DEG C of protein liquid de- preservations；
Enzyme solution obtained in step 6 after purification is placed in freeze dryer and is freeze-dried by step 7, obtains glucose oxidase enzyme
Powder.
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