CN105385609A - Aspergillus niger for high-yield glucose oxidase and application thereof - Google Patents
Aspergillus niger for high-yield glucose oxidase and application thereof Download PDFInfo
- Publication number
- CN105385609A CN105385609A CN201510988872.8A CN201510988872A CN105385609A CN 105385609 A CN105385609 A CN 105385609A CN 201510988872 A CN201510988872 A CN 201510988872A CN 105385609 A CN105385609 A CN 105385609A
- Authority
- CN
- China
- Prior art keywords
- aspergillus niger
- blcc6
- enzyme
- fermentation
- glucose oxidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses aspergillus niger for high-yield glucose oxidase. The aspergillus niger is aspergillus niger BLCC6-0009, and has already been preserved in the China model culture collection center on December 4th, 2015, and the collection serial number of the aspergillus niger is CCTCC NO:M 2015722. The invention further discloses a production method for glucose oxidase. According to the production method, the aspergillus niger BLCC6-0009 described in the claim 1 is fermented in a fermentation medium to obtain glucose oxidase fermentation liquor. The aspergillus niger obtained through screening has the advantage of being high in glucose oxidase yield and can be used for preparing fodder enzyme preparations.
Description
Technical field
The present invention relates to aspergillus niger and the application thereof of a plant height malaga carbohydrate oxidase, belong to fermentable industrial technical field.
Background technology
Glucose oxidase (GlucoseOxidase is called for short GOD) can form redox enzyme system with catalase (HPD).Glucose oxidase can generate gluconic acid and hydrogen peroxide by oxidation of beta-D-Glucose under molecular oxygen existent condition.Glucose oxidase is distributed in animal, plant and microbe widely, and it is fast that microorganism has growth and breeding speed, and the features such as wide material sources make it the main source becoming glucose oxidase.In addition, glucose oxidase allows one of feed enzyme preparation used as country, or a kind of novel zymin.Glucose oxidase can generate gluconic acid and hydrogen peroxide by oxidizing glucose under oxygen molecule exists.Therefore, glucose oxidase may play the effect of souring agent and probiotic bacterium by the product acid in enteron aisle and oxygen consumption.
Glucose oxidase also exists huge application potential at foodstuffs industry, medicine, livestock and poultry breeding industry and biological field, but the zymin purity of China's purifying production is all the time lower, vigor is poor, and glucose oxidase almost all depends on import, and production cost also improves thereupon.Therefore, screening malaga carbohydrate oxidase enzyme higher bacterial strain alive, has great actual application value.
Summary of the invention
For above-mentioned prior art, the object of this invention is to provide aspergillus niger and the application thereof of a plant height malaga carbohydrate oxidase.
For achieving the above object, the present invention adopts following technical proposals:
The Aspergillus niger strain of one plant height malaga carbohydrate oxidase, it is aspergillus niger (Aspergillusniger) BLCC6-0009, be preserved in the China typical culture collection center (CCTCC) of Wuhan University of Wuhan, China city on December 04th, 2015, its deposit number is CCTCCNO:M2015722.
The morphological feature of this bacterial strain is: mycelia is white, produces spore rear surface black, Powdered.The top capsule of conidial head is black, spherical or subsphaeroidal, and stigma is double-deck, and the first layer is thick, and the second layer is short and small, radially arranges, and is covered with whole top capsule.
The application of above-mentioned aspergillus niger (Aspergillusniger) BLCC6-0009 in malaga carbohydrate oxidase fermentative production is also the scope of protection of the invention.
Another object of the present invention is to provide a kind of microbial inoculum, and its activeconstituents is above-mentioned aspergillus niger (Aspergillusniger) BLCC6-0009 or its tunning.
Above-mentioned aspergillus niger (Aspergillusniger) BLCC6-0009 and/or microbial inoculum are also protection scope of the present invention preparing the application in feed enzyme preparation.
The present invention also provides a kind of production method of glucose oxidase, and ferment above-mentioned aspergillus niger (Aspergillusniger) BLCC6-0009 in the fermentation medium, namely obtains glucose oxidase fermented liquid.
Consisting of of described fermention medium: glucose 10%, peptone 0.1%, potassium primary phosphate 0.4%, SODIUMNITRATE 0.4%, magnesium sulfate 0.07%, Repone K 0.05%, calcium carbonate 0.07%, be mass percent, pH nature.
In aforesaid method, the temperature of fermentation is 25-35 DEG C (being preferably 30 DEG C), and the time of fermentation is 96-120h.In aforesaid method, the inoculum size of aspergillus niger (Aspergillusniger) BLCC6-0009 is 3-5%, is preferably 4% (volume fraction).
In aforesaid method, before described fermentation, also comprise the steps:
The inclined-plane seed of aspergillus niger (Aspergillusniger) BLCC6-0009 is inoculated in seed culture medium after sterilization, cultivates 2d at 25-35 DEG C of (being preferably 30 DEG C), 180r/min, obtain liquid seeds liquid.
Consisting of of described seed culture medium:
Sucrose 3%, SODIUMNITRATE 0.2%, dipotassium hydrogen phosphate 0.1%, Repone K 0.05%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, be mass percent, pH nature.
Beneficial effect of the present invention:
(1) the present invention screens the bacterial strain of malaga carbohydrate oxidase from orchard soil, in conjunction with physics and chemomorphosis, obtain the bacterial strain that malaga carbohydrate oxidase performance is higher, the genetic stability of this bacterial strain is better, improves the work of bacterial strain enzyme further by the optimization of substratum and fermentation condition.
(2) Aspergillus niger strain of the present invention has the high advantage of glucose oxidase production of enzyme, and in fermented liquid, the output of glucose oxidase can reach more than 10U/mL; In the lyophilized powder of preparation, the enzyme work of glucose oxidase can reach more than 150U/g.Far above the product enzyme performance (4.6U/mL) screening the Aspergillus niger strain obtained in prior art from occurring in nature.
Accompanying drawing explanation
Fig. 1: the plate screening result of malaga carbohydrate oxidase bacterial strain;
The cultural colony of Fig. 2 a-Fig. 2 c: bacterial strain BLCC6-0009 and microscopy state; Fig. 2 a is bacterium colony picture, and Fig. 2 b is conidium picture, and Fig. 2 c is conidial head picture;
Fig. 3: bacterial strain BLCC6-0009 amplified production agargel electrophoresis figure;
Fig. 4: phylogenetic tree.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1: the separation screening of bacterial strain
1 test materials
1.1 substratum
Plate isolation base: glucose 8%, peptone 0.3%, ammonium sulfate 0.04%, potassium primary phosphate 0.019%, magnesium sulfate 0.016%, calcium carbonate 0.35%, Zulkovsky starch 1%, potassiumiodide 0.17%, Sodium desoxycholate 0.02%, agar powder 1.5%, acetate buffer solution 60mmol/L, pH5.6.
Slant medium: sucrose 3%, SODIUMNITRATE 0.2%, dipotassium hydrogen phosphate 0.1%, Repone K 0.05%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, agar powder 1.5%, pH nature.
Fermention medium: glucose 8%, peptone 0.3%, potassium primary phosphate 0.2%, magnesium sulfate 0.07%, Repone K 0.05%, SODIUMNITRATE 0.4%, calcium carbonate 0.35%, pH nature.
Above-mentionedly be mass percent.
1.2 soil sample
Tai'an periphery and peach garden, Linyi and Cherry Pink And Apple Blossom White adopt multiple spot mixing to gather soil sample.
2 test methods
The collection of 2.1 samples
Multiple spot mixed soil sample method is adopted to take the degree of depth to be the soil sample of 0 ~ 10cm from Tai'an periphery, peach garden, Linyi and Cherry Pink And Apple Blossom White.
The primary dcreening operation of 2.2 bacterial strains
Take 10g to gather the soil sample come and be placed in and sterilizedly 100mL physiological saline be housed and containing the 250mL triangular flask of granulated glass sphere, oscillation treatment 30min.Get 1mL sterile saline respectively after being mixed by soil supernatant and be diluted to 10
-4, 10
-5two extent of dilution, respectively with the pipette, extract 0.1mL of sterilizing coat in advance preparation and on the plate isolation base of sterilizing, to occurring Blue circle cultivate 3d at 28 DEG C after.The inoculation got colors in circle is cultivated to slant medium.
The multiple sieve of 2.3 bacterial strains
Adopt Underbofler volumetry.
(1) crude enzyme liquid preparation: by the above-mentioned inoculation screened in fermention medium, cultivates 5d, shaking speed 180r/min in 30 DEG C of constant-temperature tables.After fermentation, the centrifugal 10min of 5000r/min obtains crude enzyme liquid.
(2) enzyme activity determination: get 250mL triangular flask, add 2% glucose hac buffer 25mL and 1mL crude enzyme liquid, be placed in 30 DEG C of water-baths immediately to vibrate 1 hour, then 0.1mol/LNaOH solution 20mL is added with termination reaction, with the remaining NaOH of 0.1mol/LHCl titration, record the HCl volume A consumed, blank group adds 20mLNaOH solution and need not vibrate before enzyme-added liquid, other operations are identical, record the HCl volume B consumed.
Enzyme work formula: GOD enzyme work=(B-A) × F × N × 1000/60
F: enzyme liquid extension rate
N: concentration of hydrochloric acid
Enzyme unit representation method alive: the enzyme amount of the glucose of per minute catalyzed oxidation 1 μm of ol/L is defined as a Ge Meihuo unit under these experimental conditions, represents, i.e. 1U/mL with 1 μm of ol/min.
The mutagenesis of 2.4 bacterial strains
2.4.1 the preparation of spore suspension
By the appropriate stroke-physiological saline solution wash-out of the spore in slant medium, be placed in the triangular flask of sterilization zone granulated glass sphere in advance, shake 20min on shaking table after, cross the monospore suspension of elimination mycelia to dispersion with the absorbent cotton of sterilizing, count with blood counting chamber, be diluted to 10
8the spore suspension of cfu/mL.
2.4.2 ultraviolet mutagenesis
Spore suspension is laid on equably one in sterilizing 7cm watch-glass, the thin layer of about 1mm processed.Under the ultraviolet lamp of Bechtop, be placed on middle position, by magnetic stirrer, irradiate different time respectively, dilution gradient is 10
-1~ 10
-6.Get 10 respectively
-4~ 10
-63 dilution spore suspension 0.2mL, coat on plate culture medium, and 30 DEG C of lucifuges cultivate 2 ~ 3d.Single colony inoculation on picking flat board, to slant medium, is cultured to spore and produces, then is lived by the enzyme of the glucose oxidase of the bacterial strain on each inclined-plane of liquid culture based assays.Choose enzyme high bacterial strain alive to preserve.
2.4.3 ethyl sulfate (DES) mutagenesis
Getting 5mL spore suspension, to join volume be in the triangular flask of 25mL, then add the DES (volumetric concentration 50%) of 0.2mL, concussion different time, then add Sulfothiorine (volumetric concentration 85%) termination reaction of 0.5mL.Dilution gradient is 10
-1~ 10
-6.Get 10
-4~ 10
-63 each 0.2mL of dilution spore suspension coat on plate culture medium, are inverted in 30 DEG C of cultivation 2 ~ 3d in incubator.Single colony inoculation on picking flat board, on slant medium, is cultured to spore and produces, then produces the performance of GOD by each bacterial strain of fermentation culture based assays, chooses the bacterial strain preservation that enzyme is lived high.
The qualification of 2.5 bacterial strains
5.8S-ITS region sequence mensuration is carried out to the good bacterial strain of product enzyme performance screened and identifies genus, phylogenetic tree construction, specify its classification position.
2.5.1 the extraction of bacterial strain DNA
After test strains activates 3 days on slant medium flat board, picking bacterium block is in fermention medium, shaking culture (30 DEG C, 160r/min) 3d, then by mycelia filtering drying, Sangon Biotech's fungal genomic DNA Rapid extraction test kit is adopted to extract genome DNA.
2.5.2PCR amplification and 5.8SrDNA-ITS region sequence measure
Primer I TS1 and ITS4 is adopted to increase to 5.8SrDNA-ITS region sequence.
Primer sequence is as follows:
ITS1:5’-TCCGTAGGTGAACCTGCGG-3’，SEQIDNO:1；
ITS4:5’-TCCTCCGCTTATTGATATGC-3’，SEQIDNO:2。
Pcr amplification selects 50 μ L reaction systems: Mixture25 μ L is (containing Taq DNA polymerase and dNTP etc., Tian Gen biochemical technology company limited), primer I TS1 and ITS4 (concentration is 25 μm of ol/L) each 1 μ L, template DNA 2 μ L, ultrapure water 21 μ L.Pcr amplification reaction condition is: 94 DEG C of sex change 1min, 60 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 7min.Beijing Bo Shang company PCR primer containing object fragment is sent to check order.
The sequence recorded carries out BLAST compare of analysis in NCBI, utilizes DNAMAN6.0 software to carry out multiple sequence and compares, use MEGA3.1 phylogenetic tree construction.
3 results and analysis
The separation primary dcreening operation of 3.1 malaga carbohydrate oxidase bacterial strains
Adopt multiple spot mixed soil sample acquisition method, the degree of depth is taked to be the orchard soil sample of 0 ~ 10cm degree of depth, and adopt gradient dilution and coating method to carry out the screening of malaga carbohydrate oxidase (GOD) bacterial strain, initial gross separation obtains 60 strain malaga carbohydrate oxidases, and (occur blue reacting ring, bacterial strain as shown in Figure 1) also carries out inclined-plane preservation.Number 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, black No. 1, black No. 2, black No. 3, black No. 1-1, black No. 2-1, black No. 3-1, black No. 3-2, 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, black No. 2-2, black No. 2-3, black No. 3-3.
The multiple sieve of 3.2 bacterial strains
The 15 strain bacterium that the dull and stereotyped blue reacting ring of picking is larger are seeded to fermention medium in batches and carry out liquid fermenting 5d, and adopt each strain enzyme-producing performance of Underbofler titration measuring, result is as shown in table 1,2.
The 8 strain bacterium such as table 1ASP-1 produce enzyme performance measurement result
As can be seen from Table 1, produce enzyme performance better with bacterial strain C-2, in fermentation 5d secondary fermentation liquid, the work of GOD enzyme can reach 8.72U/mL.
Black-1 grade 7 strain bacterium of table 2 produces enzyme performance and measures
Can find out that bacterial strain C-2 produces enzyme performance by table 1,2 more stable, producing enzyme when second time measures can reach 8.9U/mL, maintains the level higher than 8.5U/mL, so selected bacterial strain C-2 carries out follow-up test.
3.3 mutagenesis screening results and the checking of product enzyme performance
3.3.1 chemomorphosis the selection result
10min, 20min, 40minDES chemomorphosis is carried out to C-2 bacterial strain fermentation liquor, then gradient dilution spread plate, 30 DEG C cultivate the picking speed of growth faster 8 strain bacterium preserve to inclined-plane, and this 8 strain bacterium is seeded to liquid fermentation medium respectively, 30 DEG C, ferment 5d result of 180r/min is as shown in table 3.
Table 3 chemomorphosis the selection result
8 strain bacterium enzymes of chemomorphosis screening are lived all lower than original strain (C-2) as can be seen from Table 3.
3.3.2 physical mutagenesis combines with chemomorphosis the selection result
Because pure chemistry Mutagenic Effect is undesirable, mutagenesis is carried out to the method that original strain (C-2) adopts physical mutagenesis to combine with chemomorphosis.After mutagenesis, under choosing pure inoculation to fermention medium 30 DEG C, 180r/min condition, cultivate the work of 5d mensuration enzyme, result is as shown in table 4.
The method the selection result that table 4 physics combines with chemomorphosis
Note: inside parantheses, a represents spore suspension only with DES process 20min, 40min etc., does not carry out physical mutagenesis; B represent spore suspension under ultraviolet lamp after 15s again with DES process 20min, 40min etc.
As can be seen from Table 4, bacterial strain a-40-2 and the b-20-2 two strain bacterium enzyme work of mutagenesis screening, higher than original strain (C-2), is carried out next step and is produced enzyme checking.
3.3.3 mutagenesis screening produces enzyme checking
The strain enzyme-producing performance verification that table 5 screens
Finding to only have strain b--20-2 enzyme after the bacterial strain that GOD better performances are produced in 2 strains of screening as can be seen from Table 5 carries out the checking of products enzyme performance, alive comparatively C-2 is high, and be about 10.5U/mL, other strain stability is bad.Selected strain b--20-2 carries out follow-up test, and by its Uniform Name BLCC6-0009.
3.4 bacterial strain BLCC6-0009 genetic stabilities are on the impact of product enzyme performance
Bacterial strain BLCC6-0009 is carried out genetic stability mensuration, and result is as shown in table 6.
In table 6 bacterial strain BLCC6-00091 ~ 8 generation, produces GOD stability
Can find out that bacterial strain BLCC6-0009 produces enzyme performance within 1 ~ 8 generation by table more stable, maintain about 10U/mL.
The qualification of 3.5 bacterial strain BLCC6-0009
3.5.1 the growth characteristics of bacterial strain BLCC6-0009
By bacterial strain on flat board in 30 DEG C of cultivations, colony growth is fast, and mycelia be white, product spore rear surface black, Powdered.The top capsule of conidial head is black, spherical or subsphaeroidal, and stigma is double-deck, and the first layer is thick, and the second layer is short and small, radially arranges, and is covered with whole top capsule, as shown in Figure 2.
3.5.2PCR amplification
Utilize primer I TS1 and ITS4, bacterial strain BLCC6-0009 increases object clip size at about 600bp, as shown in Figure 3.
By increasing, the PCR primer obtained send Beijing Bo Shang Bioisystech Co., Ltd to check order.Sequencing result is as shown in SEQIDNO.3.
3.5.3 the Phylogenetic Analysis of object bacterial strain
The 5.8SrDNA – ITS region sequence of object bacterial strain is carried out BLAST analysis in NCBI, and downloads representative species in the maximum sequence of similarity and this genus, utilize MEGA3.1 software Neighboring-joining method phylogenetic tree construction (Fig. 4).
The 5.8SrDNA-ITS region sequence recording bacterial strain is carried out BLAST analysis in NCBI, from Genebank, transfers their relevant sequences carry out sequential analysis.The 5.8SrDNA-ITS region sequence of AspergillusnigerJF838357.1, HQ850370.1 etc. derives from GenBank.Hierarchial-cluster analysis finds that it is nearest with the black-koji mould sibship of Aspergillus, similarity reaches 99.6%, and the colonial morphology of comprehensive above bacterial strain, growth characteristics, microscopy form and 5.8SrDNA-ITS region sequence phylogenetic analysis proof bacterial strain BLCC6-0009 are aspergillus niger (Aspergillusniger).
Embodiment 2: the optimization of strain fermentation condition
1 material
1.1 basal fermentation medium:
A fills a prescription: glucose 8%, peptone 0.3%, potassium primary phosphate 0.2%, magnesium sulfate 0.07%, Repone K 0.05%, SODIUMNITRATE 0.4%, calcium carbonate 0.35%, pH nature.
B fills a prescription: glucose 6%, peptone 0.3%, potassium primary phosphate 0.2%, magnesium sulfate 0.07%, Repone K 0.05%, SODIUMNITRATE 0.4%, calcium carbonate 1%, pH nature.
1.2 seed culture mediums:
Sucrose 3%, SODIUMNITRATE 0.2%, dipotassium hydrogen phosphate 0.1%, Repone K 0.05%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, pH nature.
Above-mentionedly be mass percent.
2 methods
2.1 fermention mediums and fermentation time are determined
Select A, B to fill a prescription at 30 DEG C, shaking culture 2d, 5d under 180r/min condition respectively, in sampling and measuring fermented liquid, GOD enzyme is lived, and chooses GOD enzyme and to live the highest formula and fermentation time.
The preparation of 2.2 seed liquor
The bacterial classification recovered in inclined-plane is chosen in seed culture medium, 30 DEG C, namely shaking culture 2d obtains liquid seeds under 180r/min condition.
2.3 different vaccination amounts are on the impact of object strain enzyme-producing performance
The different vaccination amount choosing 2%, 4%, 6%, 8% (volume fraction) 4 levels 30 DEG C, shaking culture 5d carries out product enzyme performance and measures under 180r/min condition, research inoculum size is on the impact of object strain enzyme-producing performance.
The optimization of 2.4 fermentation conditions
2.4.1 the preparation of spore suspension
By the appropriate stroke-physiological saline solution wash-out of the spore in slant medium, be placed in the triangular flask of sterilization zone granulated glass sphere in advance, shake 20min on shaking table after, cross the monospore suspension of elimination mycelia to dispersion with the absorbent cotton of sterilizing, count with blood counting chamber, be diluted to 10
8the spore suspension of cfu/mL.
2.4.2 Orthogonal Method is adopted to be optimized fermention medium
With the orthogonal test of fermention medium 4 composition designs 4 factors 3 levels filtered out, carry out fermentation culture to the object bacterial strain of screening, sampling and measuring produces enzyme performance, obtains an Optimal compositions of fermentation medium formula.
2.4.3 lyophilized powder preparation and enzyme activity determination
Preparation seed liquor, is seeded to fermentor tank (10L) with optimum inoculum size, 30 DEG C, shaking culture 5d under 180r/min condition, the centrifugal and lyophilize by fermented liquid, is pulverized by the bacterium powder after lyophilize to get part and add sterile purified water back dissolving and measure enzyme and live.
3 results and analysis
3.1 adopt the product enzyme performance of two kinds of substratum checking bacterial strains, result is as shown in table 7,8.
Table 7A formula (glucose content is 8%) strain enzyme-producing result
Table 8B formula (glucose content is 6%) strain enzyme-producing result
From table 7,8, carry out product enzyme performance measure with the substratum that glucose content is different, it is alive that result shows two kinds of formula, fermenting 5d enzymes enzyme all higher than fermentation 2d alive, and the enzyme of A formula, fermenting 5d is lived as 9.6U/mL, higher than the 6.39U/mL that B fills a prescription.So selected A formula, fermentation 5d carries out follow-up test.
3.2 different vaccination amounts are to strain enzyme-producing performance impact
The different vaccination amount choosing 2%, 4%, 6%, 8% (volume fraction) 4 levels carries out product enzyme performance mensuration, and result is as shown in table 9.
Table 9 different vaccination amount is to strain enzyme-producing performance impact
As can be seen from Table 9, inoculum size produces GOD performance impact difference not significantly to bacterial strain BLCC6-0006, considers inoculum size relatively good with 4%.
3.3 the optimization of fermentative medium formula
4 factors using kind of the component of 4 in the fermention medium filtered out as orthogonal test, design the orthogonal test of 4 factor 3 levels, to produce enzyme performance for testing index, and the addition of further Optimal Medium each component.Orthogonal test level of factor table is in table 10.
Table 10 orthogonal test level of factor table
Orthogonal experiments is in table 11.
Table 11 produces enzyme performance fermentation condition optimization analysis of results table
As can be seen from Table 11, Different factor horizontal combination, fermentation broth enzyme is lived different, through to fermentation condition 9 kinds of orthogonals, and carries out analysis to result and can find out, the addition of 4 factors is KH on the order of producing enzyme impact
2pO
4>NaNO
3> glucose > peptone, can obtain each component addition by range analysis table and be respectively glucose 8%, peptone 0.3%, KH
2pO
40.4%, NaNO
30.4%.
Carry out the checking of product enzyme performance by the good prescription of product enzyme performance 5,7 that draws orthogonal table analysis and theoretical optimum prescription (A), result is as shown in table 12.
The formula of table 12 orthogonal carries out the checking of product enzyme performance
As can be seen from Table 12, the work of BLCC6-0009 fermentation 5d enzyme can reach 10.91U/mL.The optimal medium formula filtered out is: glucose: 10%, peptone 0.1%, potassium primary phosphate 0.4%, SODIUMNITRATE 0.4%, magnesium sulfate 0.07%, Repone K 0.05%, calcium carbonate 0.07%, pH nature.
To sum up, through optimizing the fermentation condition of the bacterial strain BLCC6-0009 obtained be: the inoculum size of bacterial strain BLCC6-0009 is 4%, fermentation time is 5d, consisting of of fermention medium: glucose: 10%, peptone 0.1%, potassium primary phosphate 0.4%, SODIUMNITRATE 0.4%, magnesium sulfate 0.07%, Repone K 0.05%, calcium carbonate 0.07%, pH nature.
The large cultivation and fermentation liquid of 3.4 bacterial strain BLCC6-0009 and lyophilized powder enzyme activity determination
Under the fermentation condition optimized, cultivate greatly (100mL/500mL) to bacterial strain BLCC6-0009 measure fermented liquid and the work of lyophilized powder enzyme, result is as shown in table 13,14.
Table 13 bacterial strain BLCC6-0009 fermentation broth enzyme is lived
Table 14 bacterial strain BLCC6-0009 lyophilized powder enzyme is lived
Note: BLCC6-0009-1, BLCC6-0009-2 are two repetitions of bacterial strain BLCC6-0009.
Can find out that the large cultivation of BLCC6-0009 (100mL/500mL) fermented liquid and lyophilized powder enzyme activity determination result show the product enzyme performance of this bacterial strain by table 13 and 14 more stable, fermented liquid and lyophilized powder enzyme are lived and are respectively 10.225U/mL and 150U/g.
The bacterial strain of existing malaga carbohydrate oxidase, because yield of enzyme is low, needs further to be separated fermented liquid, purifying obtains glucose oxidase; And adopting the lyophilized powder that Aspergillus niger strain BLCC6-0009 of the present invention is prepared after enlarged culturing, enzyme work, up to 150U/g, can directly use as feed enzyme preparation, without the need to further separation and purification, has good industrial application value.
Claims (10)
1. the aspergillus niger of a plant height malaga carbohydrate oxidase, it is aspergillus niger (Aspergillusniger) BLCC6-0009, and be preserved in China typical culture collection center on December 04th, 2015, its deposit number is CCTCCNO:M2015722.
2. the application of aspergillus niger BLCC6-0009 according to claim 1 in glucose oxidase fermentative production.
3. a microbial inoculum, is characterized in that, its activeconstituents is aspergillus niger BLCC6-0009 described in claim 1 or its tunning.
4. the application in feed enzyme preparation prepared by aspergillus niger BLCC6-0009 according to claim 1 and/or microbial inoculum according to claim 3.
5. a production method for glucose oxidase, is characterized in that, step is: ferment aspergillus niger BLCC6-0009 according to claim 1 in the fermentation medium, namely obtains glucose oxidase fermented liquid.
6. production method according to claim 5, it is characterized in that, consisting of of described fermention medium: glucose 10%, peptone 0.1%, potassium primary phosphate 0.4%, SODIUMNITRATE 0.4%, magnesium sulfate 0.07%, Repone K 0.05%, calcium carbonate 0.07%, be mass percent, pH nature.
7. production method according to claim 5, is characterized in that, the temperature of fermentation is 25-35 DEG C, and the time of fermentation is 96-120h.
8. production method according to claim 5, is characterized in that, the inoculum size of aspergillus niger BLCC6-0009 is 3-5%.
9. production method according to claim 5, is characterized in that, before described fermentation, also comprises the steps:
By in the inclined-plane seed of aspergillus niger BLCC6-0009 inoculation seed culture medium after sterilization, 25-35 DEG C, 180r/min cultivates 2d, obtains liquid seeds liquid.
10. production method according to claim 9, is characterized in that, consisting of of described seed culture medium:
Sucrose 3%, SODIUMNITRATE 0.2%, dipotassium hydrogen phosphate 0.1%, Repone K 0.05%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, be mass percent, pH nature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510988872.8A CN105385609B (en) | 2015-12-24 | 2015-12-24 | The aspergillus niger of one plant height malaga carbohydrate oxidase and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510988872.8A CN105385609B (en) | 2015-12-24 | 2015-12-24 | The aspergillus niger of one plant height malaga carbohydrate oxidase and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105385609A true CN105385609A (en) | 2016-03-09 |
| CN105385609B CN105385609B (en) | 2018-10-26 |
Family
ID=55418394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510988872.8A Active CN105385609B (en) | 2015-12-24 | 2015-12-24 | The aspergillus niger of one plant height malaga carbohydrate oxidase and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105385609B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106071143A (en) * | 2016-06-21 | 2016-11-09 | 山东宝来利来生物工程股份有限公司 | A kind of granular pattern microbial ecological agent improving ruminant production performance and preparation method thereof |
| CN106434393A (en) * | 2016-09-09 | 2017-02-22 | 南京百斯杰生物工程有限公司 | Recombinant Aspergillus niger expression strain |
| CN106520575A (en) * | 2016-11-25 | 2017-03-22 | 山东隆科特酶制剂有限公司 | Aspergillus niger strain capable of realizing high yield of catalase and application thereof |
| CN106591249A (en) * | 2016-11-30 | 2017-04-26 | 温县兴发生物科技有限公司 | Glucose oxidase preparation technology |
| CN107904224A (en) * | 2017-12-27 | 2018-04-13 | 济南诺能生物工程有限公司 | A kind of liquid glucose oxidizing ferment for the stabilizer of liquid glucose oxidizing ferment and comprising the stabilizer and preparation method thereof |
| CN108795892A (en) * | 2018-06-19 | 2018-11-13 | 张宝华 | A method of preparing, detach and purify 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5866406A (en) * | 1990-02-02 | 1999-02-02 | The Board Of Regents Of The University Of Nebraska | Oxidase-producing aspergillus niger |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN103642764A (en) * | 2013-11-21 | 2014-03-19 | 周懂懂 | Process for fermenting glucose oxidase |
-
2015
- 2015-12-24 CN CN201510988872.8A patent/CN105385609B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5866406A (en) * | 1990-02-02 | 1999-02-02 | The Board Of Regents Of The University Of Nebraska | Oxidase-producing aspergillus niger |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN103642764A (en) * | 2013-11-21 | 2014-03-19 | 周懂懂 | Process for fermenting glucose oxidase |
Non-Patent Citations (13)
| Title |
|---|
| A. A. KHATTAB ET AL: "Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| MUHAMMAD RAMZAN ET AL: "Enhanced production of glucose oxidase from UV-mutant of Aspergillus niger", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》 * |
| 何国庆 等: "《食品发酵与酿造工艺学》", 31 August 2003, 中国农业出版社 * |
| 居乃琥 等: "《酶工程手册》", 31 August 2011, 中国轻工业出版社 * |
| 张婷: "Aspergillus niger Z-25产葡萄糖氧化酶发酵条件优化及酶的分离纯化研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 张学洪 等: "《甘蔗糖蜜及其酒精废液资源化处理技术研究》", 31 January 2012, 中国环境科学出版社 * |
| 张德华等: "《蛋白质与酶工程》", 30 September 2015 * |
| 朱运平等: "产胞外葡萄糖氧化酶菌株的分离鉴定及其诱变育种研究", 《中国食品学报》 * |
| 朱运平等: "微生物葡萄糖氧化酶的生产及其在食品工业中应用的研究进展", 《中国食品添加剂》 * |
| 李艳等: "葡萄糖氧化酶及其应用", 《食品工程》 * |
| 温辉梁 等: "《饲料添加剂生产技术与配方》", 31 December 2009, 江西科学技术出版社 * |
| 范新蕾: "产葡萄糖氧化酶菌株的诱变选育及发酵条件优化", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 辛国芹等: "高产葡萄糖氧化酶菌株的分离鉴定及发酵条件优化", 《饲料广角》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106071143A (en) * | 2016-06-21 | 2016-11-09 | 山东宝来利来生物工程股份有限公司 | A kind of granular pattern microbial ecological agent improving ruminant production performance and preparation method thereof |
| 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 |
| CN106434393A (en) * | 2016-09-09 | 2017-02-22 | 南京百斯杰生物工程有限公司 | Recombinant Aspergillus niger expression strain |
| CN106520575A (en) * | 2016-11-25 | 2017-03-22 | 山东隆科特酶制剂有限公司 | Aspergillus niger strain capable of realizing high yield of catalase and application thereof |
| 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 |
| CN107904224A (en) * | 2017-12-27 | 2018-04-13 | 济南诺能生物工程有限公司 | A kind of liquid glucose oxidizing ferment for the stabilizer of liquid glucose oxidizing ferment and comprising the stabilizer and preparation method thereof |
| CN108795892A (en) * | 2018-06-19 | 2018-11-13 | 张宝华 | A method of preparing, detach and purify glucose oxidase |
| 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 |
| 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105385609B (en) | 2018-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105385609A (en) | Aspergillus niger for high-yield glucose oxidase and application thereof | |
| TW200914610A (en) | Isopropyl alcohol-producing bacteria and method for producing isopropyl alcohol using the same | |
| CN103898004B (en) | The method of Selective medium and fermentative production U-32070E thereof | |
| CN105039171B (en) | Bolt bacterium and its application | |
| CN106191173A (en) | A kind of method improving Cordyceps militaris fermenting and producing yield of Cordycepin | |
| CN109182147B (en) | Penicillium and method for producing fumagillin by using same | |
| CN105274086A (en) | High-throughput screening method for bacillus circulans | |
| CN101855973B (en) | Fungus strain irpex iacteus for producing laccase, and culturing method and application thereof | |
| CN1730653A (en) | Aspergillus niger lipase and its preparation method | |
| CN103937691B (en) | One plant production β fructosidases aspergillus oryzae strain and its cultural method and application | |
| CN101215533B (en) | Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid | |
| CN101597627B (en) | Production method of high molecular poly (gamma-glutamic acid) | |
| CN108977367A (en) | One plant of Aspergillus niger strain and its degradation cake of camellia oleifera seeds in Tea Saponin application | |
| CN103275950A (en) | Culture medium and method for producing lipase by aschersonia placenta fermentation | |
| CN101921723B (en) | Chryseobacterium rhizosphaerae and application thereof | |
| CN105483171B (en) | A kind of raising ubiquinone10The production method of industrial output | |
| CN104212820A (en) | Enzyme with catalase activity and encoding gene of enzyme | |
| CN105039386A (en) | Method for constructing monascus strain capable of achieving high yield of acid protease | |
| CN103275898A (en) | Lipase high-producing strain, screening method thereof and method for producing lipase by using strain through fermentation | |
| CN104818220B (en) | One plant is screened the Rhizopus oryzae bacterial strain JHSW01 obtained from rotten stalk | |
| CN105018410A (en) | Method for inducing Blakeslea trispora aging strain to rapidly produce a large number of spores | |
| CN116083307B (en) | Lactococcus garvieae for producing esterase and ester compounds and application thereof | |
| CN106282146B (en) | A kind of process for solid state fermentation of adenylic acid deaminase | |
| CN117384799B (en) | Method for producing beta-glucosidase by using Lu Gesi bacillus A78.1 and microbial inoculum and application | |
| Petlamul et al. | Medium optimization for production of Beauveriabassiana BNBCRC spores from biohydrogen effluent of palm oil mill using taguchi design |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Aspergillus niger with high glucose oxidase production and its application Effective date of registration: 20220527 Granted publication date: 20181026 Pledgee: Taian Taishan Rural Commercial Bank Co.,Ltd. Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2022980006448 |