CN102586133A - Cellulose-producing strain, cellulose and method for producing and fermenting cellulose - Google Patents
Cellulose-producing strain, cellulose and method for producing and fermenting cellulose Download PDFInfo
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- CN102586133A CN102586133A CN2011104355659A CN201110435565A CN102586133A CN 102586133 A CN102586133 A CN 102586133A CN 2011104355659 A CN2011104355659 A CN 2011104355659A CN 201110435565 A CN201110435565 A CN 201110435565A CN 102586133 A CN102586133 A CN 102586133A
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Abstract
The invention discloses a bacillus sp. strain for producing cellulose and a production method for utilizing the strain to produce cellulose. By optimizing and screening culture media and optimizing and controlling fermentation process conditions, the stable fermentation method is established, and the produced cellulase preparation has good enzymatic activity. The invention can be widely used in flue-cured tobacco aging, food processing, feed processing and other industries, and is significant.
Description
Invention field
The present invention relates to mikrobe and microbial fermentation field.Specifically, the present invention relates to a kind of Bacillus strain of production of cellulose enzyme, cellulase and working method.The preserving number of bacillus (Bacillus sp.) bacterial strain is: CGMCC No.5311; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on September 29th, 2011.
Background technology
Mierocrystalline cellulose is the staple of cell walls, is to pass through the wire high polymer that β-(1,4) glycosidic link is formed by connecting by glucose.
Cellulase is a kind of biology that when decomposition of cellulose, rises
KatalysisEnzyme.Cellulase extensively is present in the natural organism.Can both produce cellulase in the bacterium, fungi, animal body etc.Its 26S Proteasome Structure and Function of the cellulase of different sources differs greatly.
Cellulase can be divided into according to the difference of its catalyzed reaction function
VISOSEInscribe
Enzyme(1,4-β-D-glucan glucanohydrolase or endo-1,4-β-D-glucanase; EC3.2.1.4), and the VISOSE excision enzyme (1,4-β-D-glucan cellobilhydrolase or exo-1; 4-β-D-glucannase; EC.3.2.1.91) and beta-glucan glycosides enzyme (β-1,4-glucosidase, EC.3.2.1.21).NCE5 is the inner unformed area of the plain polysaccharide chain of cutting fibre at random, produces the oligosaccharides of different lengths and the end of new chain.Exoglucanase acts on the end of the fibrination sugar chain of these reductibilities and irreducibility, discharges glucose or cellobiose.Beta-glucosidase hydrolysis fiber disaccharides produces bimolecular glucose.
The cellulase reaction is different with general enzyme reaction, and its major different is that cellulase is a polycomponent enzyme system, and substrate structure is extremely complicated.Because the ES mixture process that the water-insoluble of substrate, REACTION OF ADSORPTION OF CELLULASE have replaced enzyme-to-substrate to form.Cellulase is adsorbed on the substrate Mierocrystalline cellulose earlier specifically, under the synergy of several kinds of components, Mierocrystalline cellulose is resolved into glucose then.
Influence the factor of yield of enzyme and vigor: the factor that influences yield of cellulase and vigor is a lot, except that bacterial classification, also has culture temperature, pH, moisture, matrix, incubation time etc.These factors are not isolated, but connect each other.
At present, both at home and abroad the cellulase-producing bacterial strain requirement condition of degenerating easily and ferment is harsh, the production cost height, can not large-scale application to industrial production.
Summary of the invention
The object of the invention is to overcome above-mentioned prior art shortcoming, and a kind of genus bacillus (Bacillus sp.) bacterial strain and the working method of utilizing this bacterial strain production of cellulose enzyme that produces cellulase is provided.Through medium optimization screening and optimization of fermentation condition control, established stable fermentation process, the cellulase preparation of production has good enzymic activity.
The present invention seeks to accomplish like this: a kind of genus bacillus (Bacillus sp), its preserving number is: CGMCCNo.5311, this bacterial strain has the ability of high cellulase-producing.
The cellulase that can obtain effectively.
Utilize the fermentation culture method of the described genus bacillus of claim 1 (Bacillus sp) bacterial classification production of cellulose enzyme, it is characterized in that this bacterial classification inoculation is fit in its substratum in LB substratum or other, be 10～35 ℃ in temperature and cultivated 12 hours;
Separation and purification bacterial strain to be measured is transferred to the single bacterium colony on the Mastplate flat board, inoculates on the cellulase strain screening culture medium flat board, place 37 ℃ of constant incubator 10h;
When treating the about 3～6mm of colony diameter size; Congo red solution-dyed 2h with 0.2%; With 5%NaCl solution decolouring 1h, measure the size of hydrolysis circle and bacterial strain diameter then then, calculate the ratio of hydrolysis circle and bacterial strain diameter; The experimental strain that utilizes activation to filter out carries out the fermentation culture experiment, obtains cellulase preparation of the present invention;
Said LB medium component is following: yeast powder 5g, peptone 10g, sodium-chlor 10g.
In the fermentation culture process, can be in seed culture medium with the bacterial classification direct inoculation, be inoculated in 1～10% inoculum size then and carry out fermentation culture in the fermention medium.
Selecting culture temperature is 10 ℃～40 ℃, and incubation time is 30～75 hours, and the pH value of cultivation can be in 4.5～9.5 production range.
Following condition culture temperature is preferably 15～40 ℃, and pH value is more suitable for the production in cellulase of the present invention in 5～8 scopes, the production of cellulose zymin, and through above-mentioned,
Preferably carboxymethyl cellulose sodium is as carbon source, and consumption is 0.3～1.8%; Optimization protein peptone, yeast extract paste are as the nitrogenous source of the best, and consumption is 0.1～1.5%.
Obtain cellulase preparation by following method: utilize filtration method or centrifuging separating thallus and substratum material from nutrient solution, obtain supernatant or filtrating, then methods such as filtrating ultrafiltration and concentration are made cellulase preparation at last.
The bacterial strain of cellulase-producing of the present invention can be widely used in industries such as flue-cured tobacco alcoholization, food-processing, feed processing, and significant.
Description of drawings
Below in conjunction with accompanying drawing, further specify content of the present invention, but do not limit the present invention with this.
Fig. 1: bacterial strain 48-1 Phylogenetic Analysis.
Fig. 2: the influence that different carbon sources are lived to bacterial strain 48-1 cellulase-producing.
Fig. 3: the influence that different nitrogen sources is lived to bacterial strain 48-1 cellulase-producing.
Embodiment
The present invention is through the flue-cured tobacco surface isolation in Yuxi, and purifying and screening obtain the microorganism strains of a collection of cellulase-producing, therefrom select the bacterial strain that a strain is numbered 48-1, have the good character of high cellulase-producing.Identify and the Physiology and biochemistry evaluation that through 16S rDNA belong to the bacterial strain of bacillus (Bacillus sp.), its preserving number is: CGMCC No.5311.
The present invention provides the working method of this bacterial strain cellulase on the basis that obtains bacillus (Bacillus sp.) 48-1 bacterial strain, obtain through the concrete fermentation process that the present invention confirms through utilizing bacterial strain of the present invention.
Cellulase-producing bacterial strain 48-1 of the present invention can adopt following method it is screened and shake flask fermentation to obtain cellulase preparation of the present invention.
Bacterial classification inoculation of the present invention is fit in its substratum in LB substratum or other, cultivated 12 hours for 10～35 ℃.Its LB medium component is following: yeast powder 5g, peptone 10g, sodium-chlor 10g.Separation and purification bacterial strain to be measured is transferred to the single bacterium colony on the Mastplate flat board, inoculates on the cellulase strain screening culture medium flat board, place 37 ℃ of constant incubator 10h.When treating the about 3～6mm of colony diameter size, the Congo red solution-dyed 2h with 0.2% then with the 5%NaCl solution 1h that decolours, measures the size of hydrolysis circle and bacterial strain diameter, the ratio of calculating hydrolysis circle and bacterial strain diameter then.Generally speaking, the size of ratio is relevant with strains for degrading Mierocrystalline cellulose ability size.The ratio of hydrolysis circle and bacterial strain diameter is big more, and the cellulosic ability of this strains for degrading is strong more, and the ability of the plain enzyme of eccrine fiber is also strong more, so ratio calculated is the better foundation of further screening bacterial strain.The Congo red dull and stereotyped transparent circle sieve method of Mierocrystalline cellulose is used for the qualitative test of cellulose-decomposing bacterium enzyme activity size, and the size of transparent circle has directly reflected the height of enzyme concn.This method is determined speed conveniently, is suitable for a large amount of bacterial strain screenings.
The experimental strain of the present invention that utilizes activation to filter out carries out the fermentation culture experiment, obtains cellulase preparation of the present invention.
In the fermentation culture process, can be in seed culture medium with the bacterial classification direct inoculation, be inoculated in 1～10% inoculum size then and carry out fermentation culture in the fermention medium.
As the nutrition source of substratum, the nutrition source that can be widely used and be generally used for cultivating.So long as can be used as the carbon cpd of carbon assimilation or contain this carbon cpd, microbial strains is utilizable, is suitable for the carbon source that fermentation culture produces cellulase and gets final product, and for example, can use glucose, wort, sucrose, available polysaccharide etc.Preferably carboxymethyl cellulose sodium is as carbon source among the present invention, and consumption is 0.3～1.8%.
As nitrogenous source; So long as can be used as the nitrogen compound of nitrogenous source assimilation or contain getting final product of this nitrogen compound; For example, available ammonium salt, nitrate salt, soyflour, yeast extract paste etc., the selection of its nitrogenous source and consumption; Can be according to the composition of other nutrition sources and consumption different and different, as long as be fit to the cultivation of cellulase producing bacteria of the present invention and the production of enzyme.In the present invention, optimization protein peptone, yeast extract paste are as the nitrogenous source of the best, and consumption is 0.1～1.5%.
For inorganic salts, can suitably add phosphoric acid hydrogen ammonia, salts such as phosphoric acid salt such as potassium primary phosphate, magnesium salts, calcium salt, manganese salt, culture condition changes according to the composition difference of substratum, as long as suitable thalline produces cellulase of the present invention.
Usually, can select following condition to cultivate, culture temperature is 10 ℃～40 ℃, is preferably 15～40 ℃, and incubation time is 30～75 hours, as long as when yield of cellulase of the present invention reaches the highest, finish fermentation culture.The pH value of cultivating can particularly be more suitable for the production in cellulase of the present invention in 4.5～9.5 production range in 5～8 scopes.Through like above-mentioned fermentation culture method, produce cellulase preparation of the present invention.
From the nutrient solution of as above gained, extract cellulase, can be according to the method that is generally used for the collection of cells exoenzyme, like ammonium sulfate precipitation, ultrafiltration and concentration, lyophilize, chromatographic separation etc., separation and purification forms.The present invention can obtain cellulase preparation by following method: utilize filtration method or centrifuging separating thallus and substratum material from nutrient solution, obtain supernatant or filtrating, then methods such as filtrating ultrafiltration and concentration are made cellulase preparation at last.
Bacillus of the present invention (Bacillus sp.) bacterial strain can be produced cellulase of the present invention effectively.Embodiment 1:
Cellulase-producing bacterial strain 48-1 bacterial strain physiological and biochemical property
The flue-cured tobacco surface isolation from Yuxi, purifying and screening obtain the bacterial isolates that a strain is numbered the high cellulase-producing of 48-1.This bacterial strain is in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.5311.
The bacterium colony of bacterial strain 48-1 is bacterium colony circle, white, convexity, and bacterium colony size 5.0-6.0mm, glossy, bacterium colony is opaque, the quality toughness.This bacterial strain can be at 10～35 ℃, and the pH scope is all can grow in 4.5～9.5 scopes.The 16S rDNA sequence of bacterium is carried out homologous sequence comparison search (BLAST) through submitting ncbi database in GenBank, and downloads the 16S rDNA sequence of relevant monoid mikrobe type strain.Utilize ClustalX software that the 16SrDNA sequence of 16S rDNA sequence and type strain is compared; Made up systematic evolution tree with MEGA4.0 software according to the Neighbor-Joinin method then; And carrying out the Bootstrap statistical test 1000 times, its systematic evolution tree is seen Fig. 1.
The physiological and biochemical property of high yield cellulase strain 48-1 of the present invention is as shown in table 1.Its physiological and biochemical property uses the bacterium trace biochemical reaction pipe of sky, Hangzhou and microorganism reagent ltd to analyze the physiological and biochemical property of urea, semi-lactosi, hydrogen sulfide reduction, pectinose, gelatin, nitrate reduction, glucose, wood sugar, cellobiose and the N.F,USP MANNITOL of bacterium.
In conjunction with physiological and biochemical property, be the bacterial strain of bacillus (Bacillus sp.) with the 48-1 identification of strains.
Embodiment 2:
Screening and the fermentation condition of cellulase-producing bacterial strain 48-1
On the LB substratum that contains 1% Xylo-Mucine (CMC), its composition is: yeast powder 5g, peptone 10g with the inoculation that is separated to; Sodium-chlor 10g cultivated 12 hours for 0～35 ℃, when treating the about 3～6mm of colony diameter size; Congo red solution-dyed 2h with 0.2%; With 5%NaCl solution decolouring 1h, measure the size of hydrolysis circle and bacterial strain diameter then then, calculate the ratio of hydrolysis circle and bacterial strain diameter.Generally speaking, the size of ratio is relevant with strains for degrading Mierocrystalline cellulose ability size.The ratio of hydrolysis circle and bacterial strain diameter is big more, and the cellulosic ability of this strains for degrading is strong more, and the ability of the plain enzyme of eccrine fiber is also strong more,
The measuring method that the multiple sieve of cellulase-producing bacterial strain adopts enzyme to live carries out, and the mensuration of enzyme activity is with reference to the DNS method of Miller G.L, and this method can know waits to screen bacterial strain cellulase-producing unit of activity.The definition of enzyme activity unit: under 7.0,37 ℃ of pH, it is enzyme unit alive that interior catalyse cellulose of unit time generates the required enzyme amount of 1 μ g glucose.Primary dcreening operation there is the bacterium colony of degraded cellulose vigor be transferred in liquid LB substratum and liquid (CMC+LB) substratum with inoculating needle; Put under 37 ℃, 150rpm condition and cultivate 10h; By 1% inoculum size nutrient solution is inserted in the fresh liquid LB substratum then, cultivate 60h under 37 ℃, 150rpm condition.Adopt the DNS method to detect the cellulase activity in the bacterial strain solution of alcoholized, and difference same strains for degrading Mierocrystalline cellulose ability in liquid LB substratum and liquid (CMC+LB) substratum have indifference.The result shows that the vigor of bacterial strain 48-1 cellulase-producing is the highest.
Embodiment 3:
High yield cellulase strain 48-1 condition of enzyme production is optimized
Carbon source is to producing the influence of enzyme:
Basic medium A: yeast powder 0.2g/L, Repone K 0.5g/L, potassium hydrogenphosphate 1g/L, SODIUMNITRATE 1g/L, sal epsom 0.4g/L, ferrous sulfate 0.01g/L.Add glucose, sucrose, SANMALT-S, starch, glycerine, fructose and CMC-Na (final concentration 1g/L) respectively, pH 7.0.Provoke a little lawn from 48-1 bacterial strain inclined-plane with inoculating needle and insert the liquid LB substratum 37 ℃ of shaking culture 2～3d respectively.Insert among the basic medium A 37 ℃ of shaking culture 2d again by 1% inoculum size.Adopt the DNS method to detect the cellulase activity in each fermented liquid.The result shows: in various carbon sources, dextrin is lived the highest, and SANMALT-S takes second place, sucrose, starch and glycerine yield of enzyme relatively low (Fig. 2).
Nitrogenous source is to producing the influence of enzyme:
Basic medium B: glucose 1g/L, yeast powder 0.2g/L, potassium hydrogenphosphate 1g/L, sal epsom 0.4g/L, Repone K 0.5g/L, ferrous sulfate 0.01g/L.Add ammonium sulfate, SODIUMNITRATE, peptone, yeast powder, urea, ammonium citrate, ammonium chloride (final concentration 1g/L) respectively, pH 7.0.48-1 is inserted in the liquid LB substratum 37 ℃ of shaking culture 2～3d with inoculating needle.Inoculum size by 1% inserts among the basic medium B, 37 ℃ of shaking culture 2d.Adopt the DNS method to detect the cellulase activity in each fermented liquid.The result shows: in 7 kinds of nitrogenous sources of participating in the experiment, inorganic nitrogen-sourced effect is superior to organic nitrogen source, and in inorganic nitrogen-sourced with ammonium sulfate best results (Fig. 3).
Through carbon source, nitrogenous source, pH, temperature, time-optimized, show that the optimization condition of enzyme production of 48-1 bacterial strain is: substratum is formed: yeast powder 0.2g/L, glucose 1g/L; Ammonium sulfate 2g/L, potassium hydrogenphosphate 1g/L, sal epsom 0.4g/L; Repone K 0.5g/L, ferrous sulfate 0.01g/L, pH 9.0.Culture temperature: 25 ℃.Incubation time: 48h.The enzyme activity of 48-1 bacterial strain has improved 4.7 times before optimizing under optimal conditions.In optimizing process, find that the carbon source (greater than 0.1%) of high density is unfavorable for the 48-1 strain enzyme-producing, this possibly receive glucose feedback inhibition and bacterial strain long term growth in the poor relatively environment of carbon source nutrition with cellulase, and it is relevant to form unique growth metabolism type.
Table 1: the physiological and biochemical property of bacterial strain 48-1
Table 1
Annotate :+expression positive reaction-expression negative reaction.
Claims (8)
1. cellulase-producing bacterial strain, its bacterial strain is genus bacillus (Bacillus sp), preserving number is: CGMCCNo.5311, this bacterial strain has the ability of cellulase-producing.
2. cellulase-producing bacterial strain according to claim 1 is characterized in that genus bacillus (Bacillus sp) can obtain the cellulase of high yield effectively.
3. utilize the fermentation culture method of the described genus bacillus of claim 1 (Bacillus sp) bacterial classification production of cellulose enzyme, it is characterized in that this bacterial classification inoculation is fit in its substratum in LB substratum or other, be 10～35 ℃ in temperature and cultivated 12 hours;
Separation and purification bacterial strain to be measured is transferred to the single bacterium colony on the Mastplate flat board, inoculates on the cellulase strain screening culture medium flat board, place 37 ℃ of constant incubator 10h;
When treating the about 3～6mm of colony diameter size; Congo red solution-dyed 2h with 0.2%; With 5%NaCl solution decolouring 1h, measure the size of hydrolysis circle and bacterial strain diameter then then, calculate the ratio of hydrolysis circle and bacterial strain diameter; The experimental strain that utilizes activation to filter out carries out the fermentation culture experiment, obtains cellulase preparation of the present invention;
Said LB medium component is following: yeast powder 5g, peptone 10g, sodium-chlor 10g.
4. the fermentation culture method of genus bacillus according to claim 3 (Bacillus sp); It is characterized in that in the fermentation culture process; Can be in seed culture medium with the bacterial classification direct inoculation, be inoculated in 1～10% inoculum size then and carry out fermentation culture in the fermention medium.
5. the fermentation culture method of bacillus according to claim 3 (Bacillus sp), it is characterized in that selecting culture temperature is 10 ℃～40 ℃, and incubation time is 30～75 hours, and the pH value of cultivation can be in 4.5～9.5 production range.
6. according to the fermentation culture methods of claim 3 or 5 described bacillus (Bacillus sp); It is characterized in that following condition culture temperature is preferably 15～40 ℃; PH value is more suitable for the production in cellulase of the present invention in 5～8 scopes, the production of cellulose zymin is through above-mentioned.
7. the fermentation culture method of bacillus according to claim 3 (Bacillus sp) is characterized in that preferably carboxymethyl cellulose sodium as carbon source, and consumption is 0.3～1.8%; Optimization protein peptone, yeast extract paste are as the nitrogenous source of the best, and consumption is 0.1～1.5%.
8. the fermentation culture method of bacillus according to claim 3 (Bacillus sp); It is characterized in that obtaining cellulase preparation: utilize filtration method or centrifuging separating thallus and substratum material from nutrient solution by following method; Obtain supernatant or filtrating, then methods such as filtrating ultrafiltration and concentration are made cellulase preparation at last.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048331A (en) * | 2017-12-15 | 2018-05-18 | 福建省金燕海洋生物科技股份有限公司 | Seaweed slag fermentation method prepares the production technology of production microalgae culture medium |
| CN109182304A (en) * | 2018-09-18 | 2019-01-11 | 云南中烟工业有限责任公司 | A kind of alpha-amylase gene and its application |
| CN109439574A (en) * | 2018-11-08 | 2019-03-08 | 唐山师范学院 | Cellulase producing bacteria and its application |
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| CN1110708A (en) * | 1994-04-21 | 1995-10-25 | 长濑生化学工业株式会社 | Bacterial preparation for agricultural use no priority right |
| CN1214082A (en) * | 1996-03-12 | 1999-04-14 | 金克克国际有限公司 | Alkaline cellulase and method of producing same |
| CN101643708A (en) * | 2008-08-29 | 2010-02-10 | 南京工业大学 | Constitutive acidic incision cellulase high-yield strain |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1110708A (en) * | 1994-04-21 | 1995-10-25 | 长濑生化学工业株式会社 | Bacterial preparation for agricultural use no priority right |
| CN1214082A (en) * | 1996-03-12 | 1999-04-14 | 金克克国际有限公司 | Alkaline cellulase and method of producing same |
| CN101643708A (en) * | 2008-08-29 | 2010-02-10 | 南京工业大学 | Constitutive acidic incision cellulase high-yield strain |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108048331A (en) * | 2017-12-15 | 2018-05-18 | 福建省金燕海洋生物科技股份有限公司 | Seaweed slag fermentation method prepares the production technology of production microalgae culture medium |
| CN109182304A (en) * | 2018-09-18 | 2019-01-11 | 云南中烟工业有限责任公司 | A kind of alpha-amylase gene and its application |
| CN109182304B (en) * | 2018-09-18 | 2021-05-25 | 云南中烟工业有限责任公司 | Alpha-amylase gene and application thereof |
| CN109439574A (en) * | 2018-11-08 | 2019-03-08 | 唐山师范学院 | Cellulase producing bacteria and its application |
| CN109439574B (en) * | 2018-11-08 | 2022-09-02 | 唐山师范学院 | Cellulase producing strain and application thereof |
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