CN105385609B - The aspergillus niger of one plant height malaga carbohydrate oxidase and its application - Google Patents
The aspergillus niger of one plant height malaga carbohydrate oxidase and its application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/685—Aspergillus niger
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Abstract
The invention discloses the Aspergillus niger strains of a plant height malaga carbohydrate oxidase, it is aspergillus niger (Aspergillus niger) BLCC6-0009, China typical culture collection center is preserved on December 04th, 2015, deposit number is CCTCC NO:M 2015722.The invention also discloses a kind of production method of glucose oxidase, the aspergillus niger BLCC6-0009 described in claim 1 that ferments in the fermentation medium is to get to glucose oxidase zymotic fluid.The Aspergillus niger strain that the present invention screens can be used for preparing feed enzyme preparation with the high advantage of glucose oxidase yield.
Description
Technical field
The present invention relates to the aspergillus niger of a plant height malaga carbohydrate oxidase and its applications, belong to microbial fermentation industrial technology
Field.
Background technology
Glucose oxidase (Glucose Oxidase, abbreviation GOD) can form oxidation with catalase (HPD) also
Protoenzyme system.It can oxidation of beta-D-Glucose generation gluconic acid and peroxidating under the conditions of glucose oxidase is existing for molecular oxygen
Hydrogen.Glucose oxidase is widely distributed in animal, plant and microbial body, and microorganism has growth and breeding speed fast, is come
The features such as source is extensive makes the main source of glucose oxidase.In addition, glucose oxidase allows to use as country
One of feed enzyme preparation or a kind of novel enzyme preparation.Glucose oxidase can oxidizing glucose in the presence of oxygen molecule
Generate gluconic acid and hydrogen peroxide.Therefore, glucose oxidase may play acidification by production acid in enteron aisle and oxygen consumption
The effect of agent and probiotics.
Glucose oxidase in food industry, medicine, livestock and poultry breeding industry and biological field there is huge application potential,
But all the time China purifying production enzyme preparation purity it is relatively low, vigor is poor, glucose oxidase almost all depend on into
Mouthful, production cost also improves therewith.Therefore, the higher bacterial strain of malaga carbohydrate oxidase enzyme activity is screened, there is great reality to answer
With value.
Invention content
For the above-mentioned prior art, it the object of the present invention is to provide the aspergillus niger of a plant height malaga carbohydrate oxidase and its answers
With.
To achieve the above object, the present invention uses following technical proposals：
The Aspergillus niger strain of one plant height malaga carbohydrate oxidase is aspergillus niger (Aspergillus niger) BLCC6-
0009, in the China typical culture collection center for being preserved in Wuhan University of Wuhan, China city on December 04th, 2015
(CCTCC), deposit number is CCTCC NO:M 2015722.
The morphological feature of the bacterial strain is：Mycelia is white, produces spore rear surface black, powdered.The top of conidial head
Capsule is in black, spherical or subsphaeroidal, and stigma is double-deck, and first layer is coarse, and the second layer is short and small, is arranged radially, and is covered with entire top
Capsule.
Above-mentioned aspergillus niger (Aspergillus niger) BLCC6-0009 answering in malaga carbohydrate oxidase fermenting and producing
With being also the scope of protection of the invention.
It is a further object of the present invention to provide a kind of microbial inoculum, active constituent is above-mentioned aspergillus niger (Aspergillus
Niger) BLCC6-0009 or its tunning.
Above-mentioned aspergillus niger (Aspergillus niger) BLCC6-0009 and/or microbial inoculum are in preparing feed enzyme preparation
Using being also protection scope of the present invention.
The present invention also provides a kind of production methods of glucose oxidase, and ferment above-mentioned aspergillus niger in the fermentation medium
(Aspergillus niger) BLCC6-0009 is to get to glucose oxidase zymotic fluid.
The group of the fermentation medium becomes：Glucose 10%, peptone 0.1%, potassium dihydrogen phosphate 0.4%, sodium nitrate
0.4%, magnesium sulfate 0.07%, potassium chloride 0.05%, calcium carbonate 0.07%, are mass percent, and pH is natural.
In the above method, the temperature of fermentation is 25-35 DEG C (preferably 30 DEG C), and the time of fermentation is 96-120h.Above-mentioned side
In method, the inoculum concentration of aspergillus niger (Aspergillus niger) BLCC6-0009 is 3-5%, preferably 4% (volume fraction).
Further include following steps before the fermentation in the above method：
By the seed culture of the inclined-plane seed inoculation of aspergillus niger (Aspergillus niger) BLCC6-0009 after sterilization
In base, 2d is cultivated in 25-35 DEG C of (preferably 30 DEG C), 180r/min, obtains liquid seeds liquid.
The group of the seed culture medium becomes：
Sucrose 3%, sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, potassium chloride 0.05%, magnesium sulfate 0.05%, sulfuric acid are sub-
Iron 0.001%, is mass percent, and pH is natural.
Beneficial effects of the present invention：
(1) present invention screens the bacterial strain of malaga carbohydrate oxidase from orchard soil, in conjunction with physics and mutagenesis, obtains
The higher bacterial strain of malaga carbohydrate oxidase performance, the genetic stability of the bacterial strain is preferable, passes through the excellent of culture medium and fermentation condition
Change further increases bacterial strain enzyme activity.
(2) Aspergillus niger strain of the invention has the advantages that glucose oxidase yield is high, and grape is glycoxidative in zymotic fluid
The yield of enzyme is up to 10U/mL or more；The enzyme activity of glucose oxidase is up to 150U/g or more in the freeze-dried powder of preparation.It is far above
The producing enzyme performance (4.6U/mL) of the Aspergillus niger strain screened from nature in the prior art.
Description of the drawings
Fig. 1：The plate screening result of malaga carbohydrate oxidase bacterial strain；
Fig. 2 a- Fig. 2 c：The cultural colony and microscopy state of bacterial strain BLCC6-0009；Fig. 2 a are bacterium colony picture, and Fig. 2 b are point
Raw spore picture, Fig. 2 c are conidial head picture；
Fig. 3：Bacterial strain BLCC6-0009 amplified production agargel electrophoresis figures；
Fig. 4：Phylogenetic tree.
Specific implementation mode
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, following the description is merely to explain this
Invention, is not defined its content.
Embodiment 1：The separation screening of bacterial strain
1 test material
1.1 culture medium
Plate isolation base：Glucose 8%, peptone 0.3%, ammonium sulfate 0.04%, potassium dihydrogen phosphate 0.019%,
Magnesium sulfate 0.016%, calcium carbonate 0.35%, soluble starch 1%, potassium iodide 0.17%, NaTDC 0.02%, agar powder
1.5%, acetate buffer solution 60mmol/L, pH5.6.
Slant medium：Sucrose 3%, sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, potassium chloride 0.05%, magnesium sulfate
0.05%, ferrous sulfate 0.001%, agar powder 1.5%, pH are natural.
Fermentation medium：Glucose 8%, peptone 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.07%, potassium chloride
0.05%, sodium nitrate 0.4%, calcium carbonate 0.35%, pH are natural.
Above-mentioned is mass percent.
1.2 soil sample
Tai'an periphery and Linyi peach garden and Cherry Pink And Apple Blossom White are using multiple spot mixing acquisition soil sample.
2 test methods
The acquisition of 2.1 samples
Multiple spot mixed soil sample method is used to take depth for 0~10cm's from Tai'an periphery, Linyi peach garden and Cherry Pink And Apple Blossom White
Soil sample.
The primary dcreening operation of 2.2 bacterial strains
Weigh 10g acquisition come soil sample be placed in it is sterilized be equipped with 100mL physiological saline and the 250mL tri- containing bead
In the bottle of angle, oscillation treatment 30min.1mL will be taken to be diluted to 10 with sterile saline respectively after soil supernatant mixing-4、10-5
Two dilutions are coated on the plate isolation base prepared and sterilized in advance with the pipette, extract 0.1mL of sterilizing respectively
On, it is cultivated at 28 DEG C after 3d to there is Blue circle.It is cultivated in the inoculation to slant medium in enclosing that gets colors.
The secondary screening of 2.3 bacterial strains
Using Underbofler titrations.
(1) prepared by crude enzyme liquid：By the above-mentioned inoculation screened in fermentation medium, in 30 DEG C of constant-temperature table cultures
5d, shaking speed 180r/min.5000r/min centrifuges 10min and obtains crude enzyme liquid after fermentation.
(2) enzyme activity determination：250mL triangular flasks are taken, 2% glucose hac buffer 25mL and 1mL crude enzyme liquid is added, are stood
It is placed in 30 DEG C of water-baths to vibrate 1 hour, 0.1mol/L NaOH solutions 20mL is then added to terminate reaction, uses 0.1mol/L
HCl titrates remaining NaOH, and the consumed HCl volume A of record, 20mL NaOH solutions are added before adding enzyme solution in blank group need not
Oscillation, other operations are identical, the consumed HCl volumes B of record.
Enzyme activity calculating formula：GOD enzyme activity=(B-A) × F × N × 1000/60
F：Enzyme solution extension rate
N：Concentration of hydrochloric acid
Enzyme-activity unit representation method：The enzyme amount of the glucose of 1 μm of ol/L of catalysis oxidation per minute under these experimental conditions
It is defined as an enzyme-activity unit, is indicated with 1 μm of ol/min, i.e. 1U/mL.
The mutagenesis of 2.4 bacterial strains
2.4.1 the preparation of spore suspension
The suitable sterile saline of spore in slant medium is eluted, the three of advance sterilization zone bead are placed in
In the bottle of angle, after shaking 20min on shaking table, mycelia is filtered off to the monospore suspension of dispersion with the absorbent cotton of sterilizing, uses blood cell
Tally is counted, and is diluted to 108The spore suspension of cfu/mL.
2.4.2 ultraviolet mutagenesis
Spore suspension is equably laid in the 7cm surface plate that sterilized, the thin layer of 1mm processed or so.In ultra-clean work
Under the ultraviolet lamp of platform, it is placed on middle position, with magnetic stirrer, irradiates different time, dilution gradient 10 respectively-1~
10-6.10 are taken respectively-4~10-6The spore suspension 0.2mL of 3 dilutions, is coated on plating medium, and 30 DEG C are protected from light culture 2
~3d.Single bacterium colony on picking tablet is inoculated into slant medium, and culture to spore generates, then every by Liquid Culture based assays
The enzyme activity of the glucose oxidase of the bacterial strain on a inclined-plane.The high bacterial strain of enzyme activity is chosen to be preserved.
2.4.3 dithyl sulfate (DES) mutagenesis
It takes 5mL spore suspensions to be added in the triangular flask that volume is 25mL, adds the DES (volumetric concentrations of 0.2mL
50%) different time, is shaken, the sodium thiosulfate (volumetric concentration 85%) for adding 0.5mL terminates reaction.Dilution gradient is
10-1~10-6.Take 10-4~10-6Each 0.2mL of spore suspension of 3 dilutions is coated on plating medium, is inverted in incubator
In 30 DEG C culture 2~3d.Single bacterium colony on picking tablet is inoculated on slant medium, and culture to spore generates, then passes through hair
The performance of each bacterial strain production GOD of ferment culture based assays, chooses the high bacterial strain of enzyme activity and preserves.
The identification of 2.5 bacterial strains
5.8S-ITS region sequences measurement is carried out to the bacterial strain of the producing enzyme better performances screened and identifies category, structure system hair
Tree is educated, its classification position is specified.
2.5.1 the extraction of bacterial strain DNA
After strain to be tested activates 3 days on slant medium tablet, in picking fungus block to fermentation medium, shaken cultivation
(30 DEG C, 160r/min) 3d, then by mycelia filtering drying, using Sangon Biotech's fungi base
Because group DNA Rapid extraction kits extract genome DNA.
2.5.2PCR amplification and 5.8S rDNA-ITS region sequences measure
5.8S rDNA-ITS region sequences are expanded using primer I TS1 and ITS4.
Primer sequence is as follows：
ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ', SEQ ID NO:1；
ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ', SEQ ID NO:2.
PCR amplification selects 50 μ L reaction systems：(archaeal dna polymerase containing Taq and dNTP etc., Tiangeng are biochemical by 25 μ L of Mixture
Science and Technology Ltd.), primer I TS1 and ITS4 (a concentration of 25 μm of ol/L) each 1 μ L, 2 μ L of template DNA, 21 μ L of ultra-pure water.PCR
Amplification reaction condition is：94 DEG C of denaturation 1min, 60 DEG C of annealing 1min, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 7min.
Beijing Bo Shang companies are sent to be sequenced the PCR product containing target fragment.
The sequence measured carries out BLAST in NCBI and compares analysis, and multiple sequence ratio is carried out using DNAMAN6.0 softwares
Compared with MEGA3.1 phylogenetic tree constructions.
3 results and analysis
The separation primary dcreening operation of 3.1 malaga carbohydrate oxidase bacterial strains
Using multiple spot mixed soil sample acquisition method, it is the orchard soil sample of 0~10cm depth to take depth, and dilute using gradient
It releases and the screening of rubbing method progress malaga carbohydrate oxidase (GOD) bacterial strain, initial gross separation obtains 60 plants of malaga carbohydrate oxidases and (go out
Bacterial strain existing blue reacting ring, as shown in Figure 1) simultaneously carries out inclined-plane preservation.Number is as follows：
78b2、ASP-1、ASP-2、ASP-3、ASP-4、ASP-5、C-1、C-2、C-3、C-4、C-5、C-6、C-X、C-X-1、
C-X-2, C-X-4, C-X-5, C-X-6, C-X-7, No. 1 black, No. 2 black, No. 3 black, No. 1-1 black, No. 2-1 black, No. 3-1 black, black 3-2
Number, peach soil -1, mould -1, mould -2, peach soil -2, peach soil -3, peach soil -4, mould -5, black -1, black -2, poplar-z-1, poplar-z-2,
Poplar-z-3, peach -1, peach -2, peach -3, cherry soil -1, cherry soil -2, cherry soil -3, cherry soil -4, cherry soil -5, it is No. 2-2 black, black
No. 2-3, it is No. 3-3 black.
The secondary screening of 3.2 bacterial strains
15 plants of larger bacterium of picking tablet blue reacting ring are seeded to fermentation medium and carry out liquid fermentation 5d in batches, use
The each strain enzyme-producing performance of Underbofler titration measurings, as a result as shown in table 1,2.
8 plants of bacterium producing enzyme performance measurement results such as table 1ASP-1
As can be seen from Table 1, with bacterial strain C-2 producing enzyme better performances, after the 5d that ferments in zymotic fluid GOD enzyme activity up to 8.72U/
mL。
7 plants of bacterium producing enzyme performance measurements such as table 2 black -1
By table 1,2 it can be seen that bacterial strain C-2 producing enzymes performance is relatively stablized, producing enzyme can reach 8.9U/mL when measuring for the second time, tie up
It holds in the level higher than 8.5U/mL, so selected bacterial strain C-2 carries out follow-up test.
3.3 mutagenesis screening results and producing enzyme performance verification
3.3.1 mutagenesis the selection result
10min, 20min, 40min DES mutagenesis are carried out to C-2 bacterial strain fermentation liquors, then gradient dilution coating is flat
Plate, 30 DEG C of culture faster 8 plants of bacterium of the picking speed of growth to inclined-plane preserve, and this 8 plants of bacterium are seeded to liquid fermentation training respectively
Support base, 30 DEG C, 180r/min fermentation 5d the results are shown in Table 3.
3 mutagenesis the selection result of table
8 plants of bacterium enzyme activity of mutagenesis screening are all less than original strain (C-2) as can be seen from Table 3.
3.3.2 physical mutagenesis is combined the selection result with mutagenesis
Since pure chemistry Mutagenic Effect is undesirable, original strain (C-2) is combined using physical mutagenesis with mutagenesis
Method carry out mutagenesis.After mutagenesis, culture 5d is surveyed under the conditions of choosing pure inoculation to 30 DEG C of fermentation medium, 180r/min
Determine enzyme activity, the results are shown in Table 4.
The method the selection result that 4 physics of table is combined with mutagenesis
Note：A represents spore suspension and only handles 20min, 40min etc. with DES inside parantheses, without physical mutagenesis；B generations
Table spore suspension handles 20min, 40min etc. after 15s with DES again in the UV lamp.
As can be seen from Table 4, two plants of bacterium enzyme activity of bacterial strain a-40-2 and b-20-2 of mutagenesis screening are higher than original strain (C-
2) next step producing enzyme verification, is carried out.
3.3.3 mutagenesis screening producing enzyme is verified
The strain enzyme-producing performance verification that table 5 screens
The bacterial strain for the 2 plants of production GOD better performances screened as can be seen from Table 5 finds there was only bacterium after carrying out producing enzyme performance verification
Strain b-20-2 enzyme activity is 10.5U/mL or so, other strain stabilities are bad compared with C-2 high.Selected strain b- -20-2 carries out follow-up
Experiment, and by its Uniform Name BLCC6-0009.
3.4 bacterial strain BLCC6-0009 genetic stabilities influence producing enzyme performance
Bacterial strain BLCC6-0009 is subjected to genetic stability measurement, the results are shown in Table 6.
In 6 generations of bacterial strain BLCC6-00091~8 of table, produce GOD stability
By table it can be seen that bacterial strain BLCC6-0009 producing enzyme performances within 1~8 generation are relatively stablized, the left sides 10U/mL are maintained
It is right.
The identification of 3.5 bacterial strain BLCC6-0009
3.5.1 the growth characteristics of bacterial strain BLCC6-0009
By bacterial strain in 30 DEG C of cultures on tablet, bacterium colony growth is fast, and mycelia is white, produces spore rear surface black, powdered.
The top capsule of conidial head is in black, spherical or subsphaeroidal, and stigma is double-deck, and first layer is coarse, and the second layer is short and small, radial row
Row, are covered with entire top capsule, as shown in Figure 2.
3.5.2PCR amplification
Using primer I TS1 and ITS4, bacterial strain BLCC6-0009 is expanded to target fragment size in 600bp or so, such as Fig. 3
It is shown.
Beijing Bo Shang Bioisystech Co., Ltd is sent to be sequenced the PCR product that amplification obtains.Sequencing result such as SEQ
Shown in ID NO.3.
3.5.3 the Phylogenetic Analysis of the bacterial strain of mesh
5.8SrDNA-ITS the region sequences of purpose bacterial strain are subjected to BLAST analyses in NCBI, and download similitude maximum
Sequence and the category in representative species, utilize MEGA3.1 software Neighboring-joining methods phylogenetic tree construction (figure
4)。
The 5.8S rDNA-ITS region sequences for measuring bacterial strain are subjected to BLAST analyses in NCBI, are transferred from Genebank
Their relevant sequences carry out sequence analysis.The 5.8S of Aspergillus niger JF838357.1, HQ850370.1 etc.
RDNA-ITS region sequences derive from GenBank.Hierarchial-cluster analysis finds that the black-koji mould affiliation of it and aspergillus is nearest,
Similarity is up to 99.6%, the in summary colonial morphology of bacterial strain, growth characteristics, microscopy form and 5.8S rDNA-ITS region sequences
Phylogenetic analysis proves that bacterial strain BLCC6-0009 is aspergillus niger (Aspergillus niger).
Embodiment 2：The optimization of strain fermentation condition
1 material
1.1 basal fermentation medium：
A is formulated:Glucose 8%, peptone 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.07%, potassium chloride
0.05%, sodium nitrate 0.4%, calcium carbonate 0.35%, pH are natural.
B is formulated：Glucose 6%, peptone 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.07%, potassium chloride
0.05%, sodium nitrate 0.4%, calcium carbonate 1%, pH are natural.
1.2 seed culture medium：
Sucrose 3%, sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, potassium chloride 0.05%, magnesium sulfate 0.05%, sulfuric acid are sub-
Iron 0.001%, pH are natural.
Above-mentioned is mass percent.
2 methods
2.1 fermentation mediums and fermentation time determine
A, B formula shaken cultivation 2d, 5d under the conditions of 30 DEG C, 180r/min are selected respectively, and GOD in zymotic fluid is measured by sampling
Enzyme activity chooses the highest formula of GOD enzyme activity and fermentation time.
The preparation of 2.2 seed liquors
The strain that inclined-plane is recovered is chosen into seed culture medium, 30 DEG C, under the conditions of 180r/min shaken cultivation 2d up to liquid
Body seed.
Influence of the 2.3 different vaccination amounts to purpose strain enzyme-producing performance
2%, 4%, 6%, 8% (volume fraction) 4 horizontal different vaccination amounts of selection are under the conditions of 30 DEG C, 180r/min
Shaken cultivation 5d carries out producing enzyme performance measurement, influence of the research inoculum concentration to purpose strain enzyme-producing performance.
The optimization of 2.4 fermentation conditions
2.4.1 the preparation of spore suspension
The suitable sterile saline of spore in slant medium is eluted, the three of advance sterilization zone bead are placed in
In the bottle of angle, after shaking 20min on shaking table, mycelia is filtered off to the monospore suspension of dispersion with the absorbent cotton of sterilizing, uses blood cell
Tally is counted, and is diluted to 108The spore suspension of cfu/mL.
2.4.2 fermentation medium is optimized using Orthogonal Method
With 4 factor, the 3 horizontal orthogonal tests of composition design one of fermentation medium 4 filtered out, to the mesh of screening
Bacterial strain carry out fermented and cultured, be measured by sampling producing enzyme performance, obtain an Optimal compositions of fermentation medium formula.
2.4.3 freeze-dried powder preparation and enzyme activity determination
Seed liquor is prepared, fermentation tank (10L) is seeded to optimal inoculum concentration, 30 DEG C, shaken cultivation under the conditions of 180r/min
Zymotic fluid is centrifuged and is freeze-dried by 5d, takes part plus sterile purified water back dissolving to measure enzyme the bacterium powder crushing after freeze-drying
It is living.
3 results and analysis
3.1 verify the producing enzyme performance of bacterial strain using two kinds of culture mediums, as a result as shown in table 7,8.
Table 7A is formulated (glucose content 8%) strain enzyme-producing result
Table 8B is formulated (glucose content 6%) strain enzyme-producing result
By table 7,8 it is found that carrying out producing enzyme performance measurement with the different culture medium of glucose content, two kinds of formulas are as a result shown
5d enzyme activity of fermenting all is higher than the enzyme activity of fermentation 2d, and the enzyme activity of A formula, fermentings 5d is 9.6U/mL, higher than the 6.39U/ of B formulas
mL.So selected A formulas, fermentation 5d carry out follow-up test.
3.2 different vaccination amounts influence strain enzyme-producing performance
It chooses 2%, 4%, 6%, 8% (volume fraction) 4 horizontal different vaccination amounts and carries out producing enzyme performance measurement, as a result
As shown in table 9.
9 different vaccination amount of table influences strain enzyme-producing performance
As can be seen from Table 9, inoculum concentration is not notable on bacterial strain BLCC6-0006 production GOD performances influence differences, considers
Inoculum concentration is relatively good with 4%.
The optimization of 3.3 fermentative medium formulas
Using 4 kinds of components in the fermentation medium filtered out as 4 factors of orthogonal test, 4 factor, 3 water is designed
Flat orthogonal test advanced optimizes the additive amount of culture medium each component using producing enzyme performance as testing index.Orthogonal test factor
Water-glass is shown in Table 10.
10 orthogonal test factor level table of table
Orthogonal experiments are shown in Table 11.
11 producing enzyme performance fermentation condition optimization analysis of results table of table
As can be seen from Table 11, different factor level combinations, zymotic fluid enzyme activity is different, through to 9 kinds of orthogonal sieves of fermentation condition
Choosing, and analysis is carried out on result as can be seen that the additive amount of 4 factors is KH on the sequence that producing enzyme influences2PO4>NaNO3>Grape
$ >Peptone, it is respectively glucose 8%, peptone 0.3%, KH that can obtain each component additive amount by range analysis table2PO40.4%,
NaNO30.4%.
It is produced by the prescription 5,7 and theoretical optimal prescription (A) of the producing enzyme better performances obtained to orthogonal table analysis
Enzyme performance is verified, as a result as shown in table 12.
The formula of 12 orthogonal of table carries out producing enzyme performance verification
As can be seen from Table 12, BLCC6-0009 ferments 5d enzyme activity up to 10.91U/mL.The optimal medium filtered out is matched
Fang Wei：Glucose：10%, peptone 0.1%, potassium dihydrogen phosphate 0.4%, sodium nitrate 0.4%, magnesium sulfate 0.07%, potassium chloride
0.05%, calcium carbonate 0.07%, pH are natural.
To sum up, the fermentation condition of optimized obtained bacterial strain BLCC6-0009 is：The inoculum concentration of bacterial strain BLCC6-0009 is
4%, the group of fermentation time 5d, fermentation medium become：Glucose：10%, peptone 0.1%, potassium dihydrogen phosphate 0.4%,
Sodium nitrate 0.4%, magnesium sulfate 0.07%, potassium chloride 0.05%, calcium carbonate 0.07%, pH are natural.
The big cultivation and fermentation liquid of 3.4 bacterial strain BLCC6-0009 and freeze-dried powder enzyme activity determination
Under the fermentation condition of optimization to bacterial strain BLCC6-0009 carry out culture (100mL/500mL) greatly measure zymotic fluid and
Freeze-dried powder enzyme activity, as a result as shown in table 13,14.
13 bacterial strain BLCC6-0009 zymotic fluid enzyme activity of table
14 bacterial strain BLCC6-0009 freeze-dried powder enzyme activity of table
Note：BLCC6-0009-1, BLCC6-0009-2 are two repetitions of bacterial strain BLCC6-0009.
By table 13 and 14 it can be seen that big culture (100mL/500mL) zymotic fluids of BLCC6-0009 and freeze-dried powder enzyme activity determination
As a result show that the producing enzyme performance of the bacterial strain is relatively stablized, zymotic fluid and freeze-dried powder enzyme activity are respectively 10.225U/mL and 150U/g.
The bacterial strain of existing malaga carbohydrate oxidase is needed further to detach zymotic fluid, be purified since yield of enzyme is low
Obtain glucose oxidase；And the freeze-dried powder that Aspergillus niger strain BLCC6-0009 using the present invention is prepared after expanding and cultivating,
Enzyme activity is up to 150U/g, can be used directly as feed enzyme preparation, without further isolating and purifying, has preferable work
Industry application value.
Claims (5)
1. the aspergillus niger of a plant height malaga carbohydrate oxidase is aspergillus niger (Aspergillus niger) BLCC6-0009,
China typical culture collection center is preserved on December 04th, 2015, deposit number is CCTCC NO:M 2015722.
2. applications of the aspergillus niger BLCC6-0009 described in claim 1 in glucose oxidase fermenting and producing, it is characterized in that：
The group of fermentation medium becomes：Glucose 10%, peptone 0.1%, potassium dihydrogen phosphate 0.4%, sodium nitrate 0.4%, magnesium sulfate
0.07%, potassium chloride 0.05%, calcium carbonate 0.07%, are mass percent, and pH is natural；
The temperature of the fermentation is 25-35 DEG C, and the time of fermentation is 96-120h；
The inoculum concentration of the aspergillus niger BLCC6-0009 is 3-5%.
3. a kind of microbial inoculum, which is characterized in that its active constituent is aspergillus niger BLCC6-0009 described in claim 1 or its fermentation production
Object, the microbial inoculum are freeze-dried powder, and preparation method includes：Prepare aspergillus niger (Aspergillus niger) BLCC6-0009 kinds
Sub- liquid, is seeded to fermentation tank, and zymotic fluid is centrifuged and is freeze-dried to get jelly by 30 DEG C, shaken cultivation 5d under the conditions of 180r/min
Dry powder.
4. the microbial inoculum described in aspergillus niger BLCC6-0009 described in claim 1 and/or claim 3 is preparing feed enzyme preparation
In application.
5. a kind of production method of glucose oxidase, which is characterized in that step is：It ferments in the fermentation medium claim
Aspergillus niger BLCC6-0009 described in 1 is to get to glucose oxidase zymotic fluid；
The group of the fermentation medium becomes：Glucose 10%, peptone 0.1%, potassium dihydrogen phosphate 0.4%, sodium nitrate
0.4%, magnesium sulfate 0.07%, potassium chloride 0.05%, calcium carbonate 0.07%, are mass percent, and pH is natural；The fermentation
Temperature is 25-35 DEG C, and the time of fermentation is 96-120h；
The inoculum concentration of the aspergillus niger BLCC6-0009 is 3-5%.
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| CN106434393B (en) * | 2016-09-09 | 2019-06-14 | 南京百斯杰生物工程有限公司 | Recombinant aspergillus niger expresses bacterial strain |
| CN106520575B (en) * | 2016-11-25 | 2019-11-15 | 山东隆科特酶制剂有限公司 | One plant height produces Aspergillus niger strain and its application of catalase |
| CN106591249A (en) * | 2016-11-30 | 2017-04-26 | 温县兴发生物科技有限公司 | Glucose oxidase preparation technology |
| CN107904224B (en) * | 2017-12-27 | 2021-08-31 | 济南百斯杰生物工程有限公司 | Stabilizer for liquid glucose oxidase, liquid glucose oxidase containing stabilizer and preparation method of stabilizer |
| CN108795892B (en) * | 2018-06-19 | 2021-01-12 | 北京天一辉远生物科技有限公司 | Method for preparing, separating and purifying glucose oxidase |
| CN109430537A (en) * | 2018-12-29 | 2019-03-08 | 北京盛拓达生物技术有限公司 | Application of the glucose oxidase in feed |
| CN109628331A (en) * | 2019-02-25 | 2019-04-16 | 大连大学 | The basidiomycetes of one plant of production low temperature glucose oxidase |
| 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 |
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