CN101899398A - Aspergillusniger strain and application thereof - Google Patents
Aspergillusniger strain and application thereof Download PDFInfo
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Abstract
The invention discloses an aspergillusniger strain for producing beta-glucuroide. Aspergillusniger T2 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on 26th September, 2008 with the preservation number of CGMCC No.2715. The aspergillusniger T2 strain is the aspergillusniger strain which is obtained by performing mutagenesis with ultraviolet rays and nitrous acid and screening by using a Congo red-carboxymethylcellulose (CMC) double-layer flat plate, has high enzymatic activity and is used for producing the beta-glucuroide. The aspergillusniger T2 strain is applied to the production of the beta-glucuroide. The beta-glucuroide produced by performing liquid state fermentation on corn cob, yeast powder and the like serving as raw materials and the aspergillusniger T2 strain serving as the strain has the characteristics of enzymatic activity, simple production method and the like.
Description
Technical field
The present invention relates to a kind of Aspergillus niger strain and application thereof, Aspergillus niger strain and production method and application especially for producing beta-glucosidase belong to the enzyme engineering field.
Technical background
Be hydrolyzed in the process of glucose at natural cellulose, must rely on the synergy of endo-type dextranase, circumscribed-type dextranase and three kinds of enzymes of beta-glucosidase just can finish.Wherein beta-glucosidase is hydrolyzed into glucose molecule with cellobiose or cell-oligosaccharide, is the rate-limiting factor of this process, and its molecular weight is about 76ku.In utilizing the cellulosic process of cellulase hydrolysis, because the deficiency of beta-glucosidase causes the accumulation of cellobiose, and cellobiose can form the intensive feedback inhibition to the katalysis of cellulase.Therefore, strengthening the vigor of beta-glucosidase, is one of key measure that improves cellulose hydrolysis speed and glucose yield.Aspergillus niger (Aspergillus niger) is not only generally acknowledged safe bacterial strain (GRAS), and be one of stronger microorganism of cellulase-producing ability, at present aspect the production beta-glucosidase, a lot of researchists have carried out selection by mutation, medium optimization, liquid state or solid state fermentation conditions optimization (conditions such as temperature, pH value, rotating speed, inoculum size), the separation and purification of enzyme and property research.
Yang Jianhai (food and fermentation industries, 2005 the 6th phases, " seed selection of aspergillus niger beta-glucosidase superior strain Co～(60)-52-23 ") is a starting strain with aspergillus niger (Aspergillus niger), through ultraviolet ray, ethyl sulfate (DES) and Co
60Complex mutation makes beta-glucosidase work obtain bigger raising, has higher genetic stability.He Xiaoxian (food science and technology, o. 11th in 2005, " research of black mold beta-glucanase bacterial strain mutagenic and breeding ") with ultraviolet, ethyl sulfate (DES) mutagenesis aspergillus niger (Aspergillusniger) FH1, adopt the GCN flat band method to screen, enzyme work improves 2.23 times than starting strain.Wang Qin (industrial microorganism, the 25th phase of nineteen ninety-five, " the synthetic regulation and control of aspergillus niger W25 bacterial strain cellulase preliminary study on problems ") is with working as (NH
4)
2SO
4, (NH
2)
2CO and peptone are that nitrogenous source and wheat bran are carbon source when inducing, and beta-glucosidase is lived and is 9IU/ml.Though above-mentioned document has proposed the report of some relevant fermentative production beta-glucosidases, fermenting enzyme is lived generally lower.
Summary of the invention
At the deficiencies in the prior art, the invention provides a kind of easy to operately, cost is low, enzyme live Aspergillus niger strain and the production method and the application of higher production beta-glucosidase.
The invention provides a strain and produce the Aspergillus niger strain of beta-glucosidase, aspergillus niger Aspergillusniger T2, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on September 26th, 2008, its preserving number is CGMCC No.2715.
Aspergillus niger strain Aspergillus niger T2 of the present invention (CGMCC No.2715) has following microbial characteristic:
1, morphological feature:
Aspergillus niger strain Aspergillus niger T2, the biology form is for comprising several parts such as conidium, born of the same parents' stalk, top capsule, product born of the same parents structure.The conidial head sphere is to radiation shape, diameter 150-450 μ m, and conidiophore betides matrix.Born of the same parents obstruct stem 1000-3000 (length) * 12-20 (diameter) μ m, yellow or tawny, and wall is level and smooth; Top capsule sphere or almost spherical, diameter 45-75 μ m, the surface can be educated comprehensively; Produce born of the same parents' structure bilayer, metulae 10-20 (length) * 4.5-7.0 (diameter) μ m, bottle stalk 6-10 (length) * 2.5-3.5 (diameter) μ m, conidium is spherical or subsphaeroidal, diameter 3-4.5 μ m, brown, wall is coarse.
2, cultivate feature:
Bacterial strain is grown on the wort agar substratum rapidly, 25 ℃ of 5 days diameter 75mm; Quality velvet shape or be with cotton-shaped slightly; The conidium structure is a large amount of, brown-black, no transudate; The bacterium colony reverse side is slightly yellow.
3, physiological and biochemical property:
Aspergillus niger strain Aspergillus niger T2 can be at potato, Semen Maydis powder, Zulkovsky starch, and molasses etc. are gone up growth, optimum pH 5-6,30-35 ℃ of growth optimum temperuture, 30-32 ℃ of the suitableeest product enzyme temperature.
Aspergillus niger strain Aspergillus niger T2 of the present invention is by ultraviolet ray, nitrous acid mutagenesis, the strain product beta-glucosidase high Aspergillus niger strain alive that Congo red-CMC double-layer plate screening obtains.
Aspergillus niger strain Aspergillus niger T2 of the present invention is applied to the production of beta-glucosidase.A kind of concrete grammar comprises: with corn cob, yeast powder etc. is raw material, and aspergillus niger Aspergillus niger T2 is a bacterial strain, and beta-glucosidase is produced in liquid state fermentation.
The plain enzyme common application of beta-glucosidase that the present invention produces and commercial fibre is hydrolyzed in steaming quick-fried stalk, and the enzymolysis yield is improved.
The present invention's nitrous acid, ultraviolet mutagenesis aspergillus niger (Aspergillus niger) obtains aspergillus niger Aspergillus niger T2, and easy to operate, cost is low.In order to corn cob is inductor, and adds an amount of yeast powder, KH
2PO
4, MgSO
4, CaCl
2, and trace element, obtaining the beta-glucosidase that high enzyme is lived, enzyme is lived to 2.46U/ml, ferments four times, and output and enzyme are lived stable.To steam quick-fried stalk hydrolysis in the plain enzyme of beta-glucosidase adding commercial fibre that make, the enzymolysis yield is 78.4%, improves 7 percentage points than adding beta-glucosidase enzymolysis yield.
Biomaterial preservation explanation
Aspergillus niger Aspergillus niger T2 bacterial strain provided by the invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; Deposit number: CGMCC No.2715; Preservation date: on 09 26th, 2008.
Description of drawings
Fig. 1 is an aspergillus niger Aspergillus niger T2 strain morphology enlarged photograph of the present invention.
Embodiment
The present invention is by the change of nitrous acid Venezuelan, ultraviolet mutagenesis, and Congo red-CMC double-layer plate screening obtains a strain and produces beta-glucosidase high Aspergillus niger strain alive.
Initial bacterium derives from the active sludge of waste water treatment plant.With the active sludge dilution, be suspended in the sterilized water, add fermention medium, cultivate 72-84h for 30 ℃.Get an amount of nutrient solution, Congo red-CMC double-layer plate is coated in dilution, cultivates 72h for 30 ℃.Select big being inoculated on the potato plug of transparent circle.Wash spore with acetate buffer solution, add NaNO
2After the solution mutagenesis, add Na
2HPO
4Solution is made terminator.Bacterium liquid after the above-mentioned processing is carried out gradient dilution, be coated with Congo red-CMC double-layer plate, place 30 ℃ of incubators, choose the big single bacterium colony of transparent circle behind the cultivation 72h, do the liquid state fermentation experiment, measure the beta-glucosidase ability of producing.Select the 2-3 strain enzyme high repeated experiments of doing of living, select the starting strain of the highest bacterial strain of a strain output as next section mutagenesis.Use the uviolizing phosphoric acid spore suspension of 15w then, dilution back spread plate places 30 ℃ of incubators, and constant temperature culture 72h chooses the big single bacterium colony of transparent circle, does the liquid state fermentation experiment, measures the beta-glucosidase ability of producing.
Full cycle is carried out 3 and is taken turns, and finally obtains the more stable beta-glucosidase superior strain of a strain heritability.
The method that the present invention produces beta-glucosidase with aspergillus niger comprises: with corn cob, yeast powder etc. is raw material, and aspergillus niger Aspergillus niger T2 is a bacterial strain, and beta-glucosidase is produced in liquid state fermentation.Detailed process is as follows:
1, preparation fermention medium: KH
2PO
41-4g/l, MgSO
40.05-0.6g/l, CaCl
20.05-0.6g/l, yeast extract paste 10-25g/l, corn cob 15-35g/l, trace element: FeSO
42-10mg/l, MnSO
40.2-5mg/l, ZnSO
40.3-6mg/l, pH 5-8.
2, preparation seed: above-mentioned substratum is placed triangular flask, 121 ℃ of sterilizations 30 minutes, cooling back is by 10% inoculation aspergillus niger Aspergillus niger T2 of the present invention, 28-33 ℃ of constant temperature culture 24 hours.
3, liquid state fermentation: 121 ℃ of sterilizations 30 minutes, the cooling back was inoculated in the substratum by 10% seed with preparation, 28-33 ℃, cultivates 84-96 hour with above-mentioned substratum.
Steam the beta-glucosidase that adds plain enzyme of commercial fibre and fermentation acquisition in the quick-fried stalk and be hydrolyzed, the enzymolysis yield is improved.
The enzymic hydrolysis Mierocrystalline cellulose: in triangular flask, add to steam respectively quick-fried stalk+citrate buffer solution+distilled water, add the pool, Shandong then and give birth to enzyme and beta-glucosidase, 50 ℃ of shaking baths, final glucose content is surveyed in hydrolysis.
The result: beta-glucosidase is lived and is 2.46U/ml, optimal pH 5-6, and 50 ℃ of optimum temperutures are fermented four times, and output and enzyme are lived stable.
Example 1
The inclined-plane seed culture medium: add glucose 20g by soaking in the juice of making of 200g potato, agar 20g adds tap water and is settled to 1000ml, the pH nature.This inclined-plane is used for actication of culture, preservation and test tube slant seed etc.
Screen double-deck Congo red substratum:
KH
2PO
41g, (NH
4)
2SO
40.7g, MgSO
40.3g, CaCl
20.15g 0.5ml Mandel ' s nutritive medium---constant volume is to 500ml ... be designated as solution (1)
Lower floor is 200ml solution (1)+4g agar
The upper strata is 1g CMC, the 0.1g sodium deoxycholate, and 0.01g is Congo red, is dissolved in the 100ml solution (1), adds 1g agar.
Initial bacterium derives from the active sludge of waste water treatment plant.With the active sludge dilution, be suspended in the sterilized water, add fermention medium, cultivate 72-84h for 30 ℃.Get an amount of nutrient solution, by 10
-4, 10
-5, 10
-6Congo red-CMC double-layer plate is coated in dilution, cultivates 72h for 30 ℃.Select big being inoculated on the potato plug of transparent circle.
Example 2
Nitrous acid mutagenesis: acetate and the 0.2mol/L sodium acetate of 0.2mol/L is mixed with pH4.4 by 63: 37 (V/V) acetate-sodium acetate buffer, with acetate-sodium acetate buffer the aspergillus niger Aspergillus niger T2 spore on the inclined-plane is washed, make the acetate spore suspension.Add 0.1mol/L NaNO again
2Solution 2mL, behind mutagenesis 5min and the 10min, each adds 2mL 0.7mol/L Na
2HPO
4Solution is made terminator.Get 0.05mol/L NaNO with same operation
2Carry out mutagenesis.Bacterium liquid after the above-mentioned processing and blank are carried out gradient dilution, get and be diluted to original content 10
-3, 10
-4, 10
-5Each 0.2mL of solution coating plate, numbering is placed on 30 ℃ of incubators, behind the constant temperature culture 72h, chooses the big single bacterium colony of transparent circle, does triangular flask liquid state fermentation experiment, measures enzymatic productivity, selects the starting strain of the highest bacterial strain of a strain output as next section mutagenesis.
Ultraviolet mutagenesis; The SODIUM PHOSPHATE, MONOBASIC of the Sodium phosphate dibasic of 0.2mol/L and 0.2mol/L is mixed with Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of pH 7.0 by 61: 39 (V/V), with Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution the aspergillus niger Aspergillus niger T2 spore on the inclined-plane is washed, make the phosphoric acid spore suspension.At the UV-lamp 20cm place of distance power 15w, the phosphoric acid spore suspension is shone 2min, 4min, 6min respectively, under red light, dilute 10 of original content respectively
-4, 10
-5, 10
-6Doubly, respectively be coated with 3 plates, each plate meets bacterium liquid 0.2mL, evenly coats the plate substratum.Above-mentioned plate numbering back is good with black paper bag, place 30 ℃ of incubators, constant temperature culture 72h chooses the big single bacterium colony of transparent circle, does triangular flask liquid state fermentation experiment, measures enzymatic productivity, selects the starting strain of the highest bacterial strain of a strain output as next section mutagenesis.
Full cycle is carried out 3 and is taken turns, and finally obtains the more stable beta-glucosidase superior strain of a strain heritability.Be accredited as aspergillus niger Aspergillus niger T2 through microbial strains preservation management committee of the Chinese Academy of Sciences, be preserved in microbial strains preservation management committee of Chinese Academy of Sciences common micro-organisms center, preserving number is CGMCCNo.2715.
Example 3
Preparation fermention medium: dress 4.95g corn cob in the 500ml triangular flask, 3.5g yeast powder, KH
2PO
40.4g, MgSO
40.06g, CaCl
20.06g, trace element: FeSO
41mg, MnSO
40.32mg, ZnSO
40.2mg, adding water to 200ml, pH 5.0.
The preparation seed: with above-mentioned substratum as in the triangular flask (500ml), 121 ℃ of sterilizations 30 minutes, cooling back is by 10% inoculation aspergillus niger Aspergillus niger T2 of the present invention, 28-33 ℃ of constant temperature culture 24 hours.
Liquid state fermentation: 121 ℃ of sterilizations 30 minutes, the cooling back was inoculated in the substratum by 10% seed with preparation with above-mentioned substratum, and 30 ℃, rotating speed 200rpm cultivated 72 hours.The above-mentioned triangular flask test-results that repeats 4 batches shows that the average enzyme work of beta-glycosidase reaches 2.46U/ml.
Example 4
Enzymic hydrolysis Mierocrystalline cellulose: in six 500ml triangular flasks, add respectively (30g steams citrate buffer solution+95ml of the 1mol/l of quick-fried stalk+5mlpH4.5,90ml, distilled water), add the pool, Shandong then and give birth to enzyme 2ml+ beta-glucosidase (0,5,10,15,20,25ml), be numbered 1,2,3,4,5,6,50 ℃ of shaking baths, hydrolysis 63h takes a sample every 24h.
Do not add beta-glucosidase, the enzymolysis yield is 71.9%, and behind the adding beta-glucosidase, the enzymolysis yield is up to 78.4%.
Analytical procedure:
(1) beta-glycosidase enzyme activity determination method
The fermented liquid of cultivating behind the 3d is taken out, and filtrate is centrifugal 5min in the whizzer of 8000r/min at revolution, removes mycelia, get supernatant liquor, suitably dilution, the 0.5% Whitfield's ointment glycosides citric acid that adds 1.5mL is a damping fluid, and adding 1.5mL DNS solution deactivating enzyme is alive, makes blank.Saligenin is a substrate, is incubated 60min in 50 ℃ of water-baths, respectively adds 1.5mL DNS solution after the taking-up immediately to stop enzyme reaction in test tube, shakes up back boiling water bath 5min, takes out the back and is settled to 20mL with distilled water, fully mixing.Surveying absorbancy under the 540nm wavelength. on the glucose typical curve, find corresponding glucose content, be calculated as follows out the beta-glycosidase vigor:
Beta-glycosidase (U/ml)=glucose content (mg)/10.8
(2) the sugared composition in the hydrolyzed solution adopts the analysis of high performance liquid chromatography (HPLC) method
(3) cellulose amount (g) in reducing sugar total amount (g) * 0.9 * 100/ substrate in enzymolysis yield (%)=liquid glucose.
Claims (6)
1. the Aspergillus niger strain of beta-glucosidase is produced in a strain, and aspergillus niger Aspergillus niger T2 is preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on September 26th, 2008, and its preserving number is CGMCC No.2715.
2. according to the described Aspergillus niger strain of claim 1, it is characterized in that Aspergillus niger strain Aspergillusniger T2 morphological feature is: comprise conidium, born of the same parents' stalk, top capsule and produce born of the same parents' structure; The conidial head sphere is to radiation shape, diameter 150-450 μ m, and conidiophore betides matrix; Born of the same parents obstruct stem 1000-3000 * 12-20 μ m, yellow or tawny, and wall is level and smooth; Top capsule sphere or almost spherical, diameter 45-75 μ m, the surface can be educated comprehensively; Produce born of the same parents' structure bilayer, metulae 10-20 * 4.5-7.0 μ m, bottle stalk 6-10 * 2.5-3.5 μ m, conidium is spherical or subsphaeroidal, diameter 3-4.5 μ m, brown, wall is coarse.
3. according to the described Aspergillus niger strain of claim 1, it is characterized in that Aspergillus niger strain Aspergillusniger T2 cultivates and is characterized as: bacterial strain is grown on the wort agar substratum rapidly, 25 ℃ of 5 days diameter 75mm; Quality velvet shape or be with cotton-shaped slightly; The conidium structure is a large amount of, brown-black, no transudate; The bacterium colony reverse side is slightly yellow.
4. according to the described Aspergillus niger strain of claim 1, it is characterized in that Aspergillus niger strain Aspergillusniger T2 physiological and biochemical property is: bacterial strain can be grown on potato, Semen Maydis powder, Zulkovsky starch, molasses, optimum pH 5-6,30-35 ℃ of growth optimum temperuture, 30-32 ℃ of the suitableeest product enzyme temperature.
5. the application of the described Aspergillus niger strain Aspergillus of claim 1 niger T2 in the production of beta-glucosidase.
6. according to the described application of claim 5, it is characterized in that: with corn cob, yeast powder is raw material, and aspergillus niger Aspergillus niger T2 is a bacterial strain, and beta-glucosidase is produced in liquid state fermentation.
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| CN109161482A (en) * | 2018-08-17 | 2019-01-08 | 吉林农业大学 | A kind of microbial bacterial agent and preparation method of efficient degradation cellulose |
| CN113122522A (en) * | 2019-12-31 | 2021-07-16 | 中国石油化工股份有限公司 | Method for promoting biological fermentation to produce cellulase |
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