CN103937732A - Bacterial strain for producing neutral cellulose, breeding method and method for producing cellulase - Google Patents
Bacterial strain for producing neutral cellulose, breeding method and method for producing cellulase Download PDFInfo
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Abstract
The invention discloses a bacterial strain for producing neutral cellulose. The bacterial strain is characterized by being preserved at the 'China Center for Type Culture Collection' in Wuhan University of China on March 3, 2014, wherein the preservation number is CCTCCNO: M2014056, the bacterial strain is named (I) Streptomycesparvus S10A10 in class, and the bacterial strain is derived from insect guts. The invention provides an rDNA sequence for identified strain culture. The invention also provides enzyme production medium and culture condition of the bacterial strain, enzymatic property of the cellulose produced by the bacterial strain, and a production method. The neutral cellulose produced by the invention is high in activity, low in production cost and short in fermentation cycle. The obtained neutral cellulose is applicable to be used as a food and a feed additive, the food quality is improved, animal growth is promoted, the feed coefficient is reduced, and the neutral cellulose can be applied to textile industry.
Description
Technical field
The present invention relates to the fields such as microbiology, enzyme engineering, fermentation engineering, food engineering and livestock-raising, be specifically related to a kind of method of producing neutral cellulase bacterial strain and selection and its production of cellulose enzyme.The cellulase that the present invention produces is mainly used in the industries such as food-processing, fodder additives and weaving.
Background technology
Cellulase is Mierocrystalline cellulose and derivative thereof to be hydrolyzed into the general name of one group of enzyme of glucose.One group of complete cellulase system is comprised of cellulolytic three fermentoids of mutual concerted catalysis conventionally: endo cellulase (CMCase), circumscribed cellulase and glucuroide.Optimal pH by catalyzed reaction, can be divided into endo cellulase: acidic incision cellulase (optimal pH 3～5), neutral endo cellulase (optimal pH 6～8), alkaline endo cellulase (optimal pH 8～10).Current domestic commercialization cellulase mostly is acid high temperature enzyme, and the neutral cellulase of domestic use is nearly all external manufacturer production.Technical monopoly causes its commercialization neutral cellulase expensive, has restricted its marketing.Therefore, this area is in the urgent need to developing the bacterial strain of the high vigor neutral cellulase of new production.
If can produce microbial host fungi and the part bacterium of endo cellulase.Fungi institute cellulase-producing mostly is acid extracellular enzyme and product enzyme efficiency is high, and enzyme architecture is complete, but fungi institute cellulase-producing is all prozyme, and enzyme architecture is complicated, molecular weight is large, is difficult for separation and Extraction, and its product enzyme process need to add cellulose substances to induce conventionally, and fermentation period is long, therefore aspect industrial enzymes exploitation, demonstrating many deficiencies.Although bacterium institute cellulase-producing composition single (conventionally only containing a fibrid element enzyme), can simplify extraction step, but bacteria cellulose enzyme mostly is neutral or alkaline intracellular enzyme, be adsorbed in bacterium wall more, and its enzyme is lived generally lower, in addition, most of bacterial strain produces induction type endo cellulase, need to add inductor, be not suitable with for industrial mass production.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to, a kind of method of producing neutral cellulase bacterial strain and selection and its production of cellulose enzyme is provided, and cellulase pH good stability, catalytic capability that this bacterial strain produces are strong, are applicable to higher enzyme reaction temperature.
For solving the problems of the technologies described above, the technical solution used in the present invention is: the streptomyces bacterial strain of neutral cellulase is produced in a strain, it is characterized in that: it is preserved in Wuhan, China university on March 3rd, 2014, and " Chinese Typical Representative culture collection " center "; deposit number is CCTCCNO:M2014056, Classification And Nomenclature is StreptomycesparvusS10A10.
Neutral cellulase bacterial strain is produced in an aforesaid strain, it is characterized in that: this bacterial strain is actinomycetes, can effectively obtain the neutral cellulase of high yield.
A selection for product neutral cellulase bacterial strain as claimed in claim 1, is characterized in that, said method comprising the steps of:
(a) get the aseptic grinding of insect gut and add in liquid seed culture medium after enrichment culture 1d, the bacterium liquid after enrichment culture is diluted to 10
-4, get diluent, on primary dcreening operation substratum, be coated with dull and stereotyped cultivation;
(b) cultivate after 7d, with inoculating needle, choose the actinomycetes bacterium colony of different shape on same flat board, then be transferred to that on flat board, to carry out single bacterium colony separated, finally obtain multi-strain bacteria;
(c) every strain bacterium is inoculated in respectively in liquid seed culture medium and enzymatic production substratum, in 28 ℃ of constant incubators, cultivate 36h, get fermented liquid and carry out the rapid screening of Congo red flat board, Congo red solution-dyed 30min through 1mg/mL, after the NaCl solution decolouring 30min of 1mol/L, measure dull and stereotyped upper bacterium colony hydrolysis circle and colony diameter, calculate the ratio of hydrolysis circle and bacterial strain diameter, filter out the bacterial strain that ratio is greater than 5, through experiment screening repeatedly, obtain a strain and in two kinds of cultivations, all can produce the bacterial strain of obvious transparent circle, by its called after StreptomycesparvusS10A10.
The selection of aforesaid product neutral cellulase bacterial strain, is characterized in that, in above-mentioned steps (a) and step (c), and the consisting of of described liquid seed culture medium: CMC-Na, 10g; KH
2pO
4, 4.0g; MgSO
4, 0.03g; Distilled water, 1000mL, pH6.0～6.5.
The selection of aforesaid product neutral cellulase bacterial strain, is characterized in that, in above-mentioned steps (a), and the consisting of of described primary dcreening operation substratum: Xylo-Mucine, 5.0g; K
2hPO
4, 1.0g; NaNO
3, 3.0g; KCl, 0.5g; MgSO
4, 0.5g; FeSO
4, 0.01g; Agar, 17.0g; Distilled water, 1000mL, pH6.0～6.5.
The selection of aforesaid product neutral cellulase bacterial strain, is characterized in that, in above-mentioned steps (c), and the consisting of of described enzymatic production substratum: rice straw powder, 10g; (NH
4)
2sO
4, 4g; KH
2pO
4, 2g; MgSO
4, 0.5g; Distilled water, 1000mL, pH6.0～6.5.
A method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1, is characterized in that, said method comprising the steps of:
(1) producing enzyme cultivates
StreptomycesparvusS10A10 is inoculated in to No. 1 substratum of inclined-plane Gao Shi, and the liquid seed culture medium of transferring after 27 ℃ of cultivation 2d, cultivates 24h under 27 ℃ of conditions; With the 2% inoculum size enzymatic production substratum of transferring again, the suitableeest initial pH of fermention medium cultivates 5d under 6～6.5,27 ℃ of conditions;
(2) processing of extracellular products crude enzyme liquid
Rotating speed frozen centrifugation 10min by fermented liquid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Dialysed overnight in the citric acid-sodium citrate damping fluid that to get 20mL crude enzyme liquid be 6.2 in 0.05mol/L, pH; Use polyoxyethylene glycol concentrate dialysate, then through O.45 μ m filtering with microporous membrane removal of impurities, preserve filtrate;
(3) sieve chromatography
The abundant balance sephadex chromatography of the phosphate buffered saline buffer post SephadexG-100 post that is 6.2 with pH in advance, the citric acid-sodium citrate wash-out that is 6.2 with pH after loading, collects the elution peak that has enzyme to live, and is the cellulase after purifying.
A kind of aforesaid method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1, is characterized in that, in above-mentioned steps (1), and the consisting of of described No. 1 substratum of Gao Shi: Zulkovsky starch, 20g; KNO
3, 1g; K
2hPO
4, 0.5g; MgSO
47H
2o, 0.5g; NaCl, 0.5g; FeSO
47H
2o, 0.01g; Agar, 20g; Distilled water, 1000mL, pH6.0～6.5.
A kind of aforesaid method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1, is characterized in that, in above-mentioned steps (1), and the consisting of of described liquid seed culture medium: CMC-Na, 10g; KH
2pO
4, 4.0g; MgSO
4, 0.03g; Distilled water, 1000mL, pH6.0～6.5.
A kind of aforesaid method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1, is characterized in that, in above-mentioned steps (1), and the consisting of of described enzymatic production substratum: rice straw powder, 10g; (NH
4)
2sO
4, 4g; KH
2pO
4, 2g; MgSO
4, 0.5g; Distilled water, 1000mL, pH6.0～6.5.
The invention has the beneficial effects as follows:
(1) StreptomycesparvusS10A10 that the present invention screens belongs to composing type cellulase high-yield, compare with the bacterial isolates of enzyme with current industrial, the enzymatic production cycle is short, enzyme activity is high, and produce enzyme process without induction, therefore the cellulase of its generation is composing type cellulase, so this bacterium is applicable to the feature of large-scale fermentative production;
(2) the vigor specific activity of the total enzyme of cellulase extracting from StreptomycesparvusS10A10 of the present invention reaches 33.02U/mL, compares with report bacterial strain, has higher catalytic capability; This enzyme optimal reaction pH is 5.0, is rare acid composing type cellulase; This enzyme is preserved 2h enzyme activity in the damping fluid of pH4.0～8.0 can keep the more than 90% of the highest enzyme work, has embodied good pH stability; Optimal reactive temperature is 50～65 ℃; There is resistance to Na
+, K
+, Ca
2+, Ni
2+, Zn
2+, Mg
2+, Mn
2+, Ba
2+, Pb
2+, Fe
3+deng the superperformance of metal ion, EDTA is very micro-on enzymic activity impact, and this enzyme is nonmetallic ion cellulase, has shown good characteristic, at industrial circles such as weaving, food-processing and feeds, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is the transparent circle of StreptomycesparvusS10A10 bacterial strain of the present invention on primary dcreening operation substratum;
Fig. 2 is the scanning electron microscope form picture of StreptomycesparvusS10A10 bacterial strain of the present invention;
Fig. 3 be purifying of the present invention cellulase polyacrylamide gel electrophoresis figure (M is standard protein molecular weight, 3 for zymoprotein molecular weight be 82Kd endo cellulase.)；
Fig. 4 is the color atlas that SephadexG-100 column chromatography obtains cellulase Peak Activity;
Fig. 5 is the impact that pH lives on Mierocrystalline cellulose restriction endonuclease;
Fig. 6 is the impact that temperature is lived on Mierocrystalline cellulose restriction endonuclease;
Sequence table is the 16SrDNA Sequence Identification of StreptomycesparvusS10A10 bacterial strain.
Biological material specimens preservation:
StreptomycesparvusS10A10 has been preserved in Wuhan, China university, and " Chinese Typical Representative culture collection " center "; preservation address: Luojiashan, Wuchang, Wuhan City, Hubei Province; deposit number is that deposit number is CCTCCNO:M2014056, preservation date is on March 3rd, 2014.
Embodiment
For further disclosing technical scheme of the present invention, below in conjunction with accompanying drawing, describe embodiments of the present invention in detail:
In the present invention, used medium is:
No. 1 substratum of Gao Shi (Gause): Zulkovsky starch, 20g; KNO
3, 1g; K
2hPO
4, 0.5g; MgSO
47H
2o, 0.5g; NaCl, 0.5g; FeSO
47H
2o, 0.01g; Agar, 20g; Distilled water, 1000mL, pH6.0～6.5.
Primary dcreening operation substratum: Xylo-Mucine, 5.0g; K
2hPO
4, 1.0g; NaNO
3, 3.0g; KCl, 0.5g; MgSO
4, 0.5g; FeSO
4, 0.01g; Agar, 17.0g; Distilled water 1000mL, pH6.0～6.5.
Liquid seed culture medium: CMC-Na, 10g; KH
2pO
4, 4.0g; MgSO
4, 0.03g; Distilled water 1000mL, pH6.0～6.5.
Enzymatic production substratum: rice straw powder, 10g; (NH
4)
2sO
4, 4g; KH
2pO
4, 2g; MgSO
4, 0.5g; Distilled water 1000mL, pH6.0～6.5.
Embodiment 1
The screening process of StreptomycesparvusS10A10:
Get the aseptic grinding of black shield vinegarron (Typopeltisniger) enteron aisle and add in liquid seed culture medium after enrichment culture 1d, the bacterium liquid after enrichment culture is diluted to 10-4, get diluent, on primary dcreening operation substratum, be coated with dull and stereotyped cultivation.Cultivate after about 7d, with inoculating needle, choose the actinomycetes bacterium colony of different shape on same flat board, then be transferred to that on flat board, to carry out single bacterium colony separated, finally obtain 42 strain bacterial strains.Every strain bacterium is inoculated in respectively in liquid seed culture medium and enzymatic production substratum, in 28 ℃ of constant incubators, cultivate 36h, get fermented liquid and carry out the rapid screening of Congo red flat board, Congo red solution-dyed 30min through 1mg/mL, after the NaCl solution decolouring 30min of 1mol/L, measure dull and stereotyped upper bacterium colony hydrolysis circle and colony diameter, calculate the ratio of hydrolysis circle and bacterial strain diameter, filter out the bacterial strain that ratio is greater than 5, through experiment screening repeatedly, obtain a strain and in two kinds of cultivations, all can produce the bacterial strain (D/d>5) of obvious transparent circle, by its called after StreptomycesparvusS10A10.Fig. 1 is the transparent circle of StreptomycesparvusS10A10 bacterial strain on primary dcreening operation substratum, and Fig. 2 is the microscopic examination picture of StreptomycesparvusS10A10 bacterial strain.
Embodiment 2
From StreptomycesparvusS10A10, extract the method for neutral composing type endo cellulase:
1. producing enzyme cultivates
StreptomycesparvusS10A10 is inoculated in to No. 1 substratum of inclined-plane Gao Shi, and the seed culture medium of transferring after 27 ° of C cultivation 2d, cultivates 24h under 27 ° of C conditions; With the 2% inoculum size liquid fermentation medium of transferring again, the suitableeest initial pH of fermention medium is 6～6.5; Under 27 ° of C conditions, cultivate 5d.
2. the pre-treatment of extracellular products crude enzyme liquid
Rotating speed frozen centrifugation 10min by fermented liquid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Get 20mL crude enzyme liquid dialysed overnight in the citric acid-sodium citrate damping fluid of 0.05mol/L, pH value 6.2; Use polyoxyethylene glycol concentrate dialysate, then through O.45 μ m filtering with microporous membrane removal of impurities, preserve filtrate.
3. sieve chromatography
Use in advance the abundant balance sephadex chromatography of the phosphate buffered saline buffer post SephadexG-100 post of pH6.2, after loading, with the citric acid-sodium citrate wash-out of PH6.2, collect the elution peak (seeing Fig. 4) that has enzyme to live.
SephadexG-100 column chromatography condition is as follows:
The citric acid-sodium citrate damping fluid of level pad: 0.05mol/L, pH value 6.2;
The citric acid-sodium citrate damping fluid of elution buffer: 0.05mol/L, pH value 6.2;
Loading flow velocity: 1.0mL/min;
Elution flow rate: 1.5mL/min.
Be in charge of collection protein peak, detect enzyme activity and protein concentration, obtain the collection liquid of cellulase Peak Activity.
Enzyme activity determination method:
1. the mensuration of total enzyme (filter paper enzyme activity, FPA) vigor:
Get the crude enzyme liquid 0.5mL after diluting 10 times, the citrate buffer solution (pH value 4.8) and the 50mg Xinhua filter paper that add 1.0ml0.05mol/L, take and do not add substrate as contrast, 50 ℃ of reaction 60min, add 3mLDNS solution, boiling water bath 5min, then test tube is placed in to cooling bath, after test tube is cooling, add 18mL distilled water.With blank tube, contrast, adopt spectrophotometer to record the value at the OD at 540nm place.
2. the mensuration of Mierocrystalline cellulose restriction endonuclease (carboxymethylcelluloenzyme enzyme, CMCA) vigor:
Get the crude enzyme liquid 0.5mL after diluting 10 times, add 1.0mL, 1% carboxymethylcellulose sodium solution (pH value 4.8), take and do not add substrate as contrast, 50 ℃ of reaction 30min, add 3mLDNS solution, boiling water bath 5min, then test tube is placed in to cooling bath, after test tube is cooling, add 18mL distilled water.With blank tube, contrast, adopt spectrophotometer to record the value at the OD at 540nm place.
3. the mensuration of Mierocrystalline cellulose excision enzyme (avicelase) vigor:
Get the enzyme liquid after 3 times of 0.5mL dilutions, add 1.0mL, 1% microcrystalline cellulose cellulose solution (pH value 4.8), take and do not add substrate as contrast, 50 ℃ of reaction 60min, add 3mLDNS solution, boiling water bath 5min, again test tube is placed in to cooling bath, after test tube is cooling, adds 18mL distilled water.With blank tube, contrast, adopt spectrophotometer to record the value at the OD at 540nm place.
4. beta-glucosidase (BGA) vitality test:
Get the enzyme liquid after 3 times of 0.5mL dilutions, add 1.0mL, 1% saligenin solution (pH value 4.8), take and do not add substrate as contrast, 50 ℃ of reaction 30min, add 3mLDNS solution, boiling water bath 5min, again test tube is placed in to cooling bath, after test tube is cooling, adds 18mL distilled water.With blank tube, contrast, adopt spectrophotometer to record the value at the OD at 540nm place.
The method of calculation of enzyme activity:
The definition of enzyme activity: at pH4.8, under 50 ℃ of conditions, the enzyme amount that every 1mL enzyme liquid hydrolysis substrate in 1min generates 1 μ g glucose is called an enzyme activity unit (U).
Cellulase activity unit of force=G * n * 1000/ (0.5 * t)
G---the concentration of reduced sugar that measured OD value is found on glucose typical curve, mg/mL;
N---the extension rate of enzyme liquid;
1000---milligram is converted into microgram;
0.5---the volume of reaction enzymes liquid;
T---enzyme and substrate reactions time, min.
Get crude enzyme liquid and measure the vigor of enzyme, after measured, total enzyme, restriction endonuclease and the work of excision enzyme enzyme are respectively 33.02U/mL, 18.51U/mL and 23.92U/mL.
Respectively crude enzyme liquid, sample pre-treatments liquid and the sample after SephadexG-100 column chromatography are carried out the mensuration of total enzyme activity, as shown in table 1, result shows yield 78% after StreptomycesparvusS10A10 cellulase purifying, and yield is higher, and enzyme work is comparatively stable in purge process.
The restriction endonuclease vitality test of table 1 cellulase
Embodiment 3
The relevant nature of the neutral composing type endo cellulase extracting from StreptomycesparvusS10A10 bacterial strain:
1. the mensuration of zymoprotein molecular weight
With SDS-PAGE polyacrylamide gel electrophoresis, carry out according to a conventional method.Separation gel, concentrated gum concentration are respectively 12% and 5%, and electrode buffer is pH8.3Tris-Gly buffering, coomassie brilliant blue staining, and result is as shown in Figure 3.After electrophoresis, with GEL-DOC2000 (Bio-Rad), carry out gel image scanning.Use scanning system to carry software QuantityOne and carry out molecular weight determination, recording endo cellulase zymoprotein molecular weight is 82Kd.
2. the optimal pH of enzyme
By various bufferings, prepare the CMC-Na substrate of different pH values.Endo cellulase LC and different pH value CMC-Na substrate reactions by after extracting, react 30min under 65 ℃ of conditions, and detect CMCase vigor, as shown in Figure 5.Cellulase, at the higher vigor of having of neutral slant acidity, has very high vigor within the scope of pH5.O～7.O, reaches the more than 80% of the highest enzyme work, and its optimal reaction pH is 6.2, is neutral cellulase.
3. the pH stability of enzyme
Endo cellulase after extraction is dissolved in different pH damping fluids, under 65 ℃ of conditions, places after 2h, measures endo cellulase relative activity, as shown in Figure 5.Result shows that this endo cellulase has shown the stability of height within the scope of wider pH, in the damping fluid of pH5.0～8.0, can keep its highest enzyme to live more than 85%, show good pH stability, at weaving, papermaking and food-processing industry, had good application potential.
4. the optimal reactive temperature of enzyme
The pure enzyme of endo cellulase LC is positioned over respectively under differing temps and reacts 30min, measure its enzyme and live as shown in Figure 6.Result shows that endo cellulase has higher vigor when 50～65 ℃ of reactions, and its optimum temperuture is 50 ℃.
5. the impact of metal ion on cellulase productivity
At three concentration level lmmol/L, 5mmol/L, 10mmol/L, inquire into Na
+, K
+, Li
+-valence metal ion; Ca
2+, Ni
2+, Zn
2+, Mg
2+, Cu
2+, Fe
2+, Co
2+, Mn
2+, Ba
2+, Pb
2+divalent-metal ion; Fe
3+trivalent metal ion and the EDTA impact on CMCase vigor.At 4 ℃, place 30min, under 65 ℃ of conditions, react 30min, and detect CMCase vigor.Take and do not add the relative activity that the enzyme work of metal ion is blank determination enzyme.Under the metal ion existence condition of result lower concentration (lmmol/L), most of metal ion can obviously promote cellulase productivity, under the metal ion existence condition of intermediate concentration (5mmol/L), most of metal ion can improve cellulase activity.In addition experiment finds that the EDTA of different concns is little on enzyme activity impact, infers that endo cellulase is probably nonmetallic ion cellulase, and metal ion has certain promoter action to this enzyme.Show that this enzyme meets the basic demand of weaving, papermaking and food-processing industry application.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any deviates from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify, all should be equivalent substitute mode, is included in protection scope of the present invention.
Claims (10)
1. the streptomyces bacterial strain of neutral cellulase is produced in a strain, it is characterized in that: it on March 3rd, 2014 be preserved in Wuhan, China university " Chinese Typical Representative culture collection " center ", deposit number is CCTCC NO:M 2014056, Classification And Nomenclature is
streptomyces parvuss10A10.
2. neutral cellulase bacterial strain is produced in a strain according to claim 1, it is characterized in that: this bacterial strain is actinomycetes, can effectively obtain the neutral cellulase of high yield.
3. a selection for product neutral cellulase bacterial strain as claimed in claim 1, is characterized in that, said method comprising the steps of:
(a) get the aseptic grinding of insect gut and add in liquid seed culture medium after enrichment culture 1d, the bacterium liquid after enrichment culture is diluted to 10
-4, get diluent, on primary dcreening operation substratum, be coated with dull and stereotyped cultivation;
(b) cultivate after 7d, with inoculating needle, choose the actinomycetes bacterium colony of different shape on same flat board, then be transferred to that on flat board, to carry out single bacterium colony separated, finally obtain multi-strain bacteria;
(c) every strain bacterium is inoculated in respectively in liquid seed culture medium and enzymatic production substratum, in 28 ℃ of constant incubators, cultivate 36h, get fermented liquid and carry out the rapid screening of Congo red flat board, Congo red solution-dyed 30 min through 1 mg/mL, the NaCl solution of 1 mol/L decolours after 30 min, measure dull and stereotyped upper bacterium colony hydrolysis circle and colony diameter, calculate the ratio of hydrolysis circle and bacterial strain diameter, filter out the bacterial strain that ratio is greater than 5, through experiment screening repeatedly, obtain a strain and in two kinds of cultivations, all can produce the bacterial strain of obvious transparent circle, by its called after
streptomyces parvuss10A10.
4. the selection of product neutral cellulase bacterial strain according to claim 3, is characterized in that, in above-mentioned steps (a) and step (c), and the consisting of of described liquid seed culture medium: CMC-Na, 10 g; KH
2pO
4, 4.0 g; MgSO
4, 0.03 g; Distilled water, 1000 mL, pH 6.0～6.5.
5. the selection of product neutral cellulase bacterial strain according to claim 3, is characterized in that, in above-mentioned steps (a), and the consisting of of described primary dcreening operation substratum: Xylo-Mucine, 5.0 g; K
2hPO
4, 1.0 g; NaNO
3, 3.0 g; KCl, 0.5 g; MgSO
4, 0.5 g; FeSO
4, 0.01 g; Agar, 17.0 g; Distilled water, 1000 mL, pH 6.0～6.5.
6. the selection of product neutral cellulase bacterial strain according to claim 3, is characterized in that, in above-mentioned steps (c), and the consisting of of described enzymatic production substratum: rice straw powder, 10 g; (NH
4)
2sO
4, 4 g; KH
2pO
4, 2 g; MgSO
4, 0.5 g; Distilled water, 1000 mL, pH 6.0～6.5.
7. a method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1, is characterized in that, said method comprising the steps of:
(1) producing enzyme cultivates
Will
streptomyces parvuss10A10 is inoculated in No. 1 substratum of inclined-plane Gao Shi, and the liquid seed culture medium of transferring after 27 ℃ of cultivation 2d, cultivates 24h under 27 ℃ of conditions; With the 2% inoculum size enzymatic production substratum of transferring again, the suitableeest initial pH of fermention medium cultivates 5d under 6～6.5,27 ℃ of conditions;
(2) processing of extracellular products crude enzyme liquid
Rotating speed frozen centrifugation 10min by fermented liquid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Dialysed overnight in the citric acid-sodium citrate damping fluid that to get 20mL crude enzyme liquid be 6.2 in 0.05 mol/L, pH; Use polyoxyethylene glycol concentrate dialysate, then through O.45 μ m filtering with microporous membrane removal of impurities, preserve filtrate;
(3) sieve chromatography
The abundant balance sephadex chromatography of the phosphate buffered saline buffer post Sephadex G-100 post that is 6.2 with pH in advance, the citric acid-sodium citrate wash-out that is 6.2 with pH after loading, collects the elution peak that has enzyme to live, and is the cellulase after purifying.
8. a kind of method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1 according to claim 7, is characterized in that, in above-mentioned steps (1), and the consisting of of described No. 1 substratum of Gao Shi: Zulkovsky starch, 20 g; KNO
3, 1 g; K
2hPO
4, 0.5 g; MgSO
47H
2o, 0.5 g; NaCl, 0.5 g; FeSO
47H
2o, 0.01 g; Agar, 20 g; Distilled water, 1000 mL, pH 6.0～6.5.
9. a kind of method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1 according to claim 7, is characterized in that, in above-mentioned steps (1), and the consisting of of described liquid seed culture medium: CMC-Na, 10 g; KH
2pO
4, 4.0 g; MgSO
4, 0.03 g; Distilled water, 1000 mL, pH 6.0～6.5.
10. a kind of method of utilizing the product neutral cellulase bacterial strain production of cellulose enzyme described in claim 1 according to claim 7, is characterized in that, in above-mentioned steps (1), and the consisting of of described enzymatic production substratum: rice straw powder, 10 g; (NH
4)
2sO
4, 4 g; KH
2pO
4, 2 g; MgSO
4, 0.5 g; Distilled water, 1000 mL, pH 6.0～6.5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673713A (en) * | 2015-01-23 | 2015-06-03 | 深圳大学 | Basophilic streptomyce, neutral endoglucanase produced by basophilic streptomyce and application of basophilic streptomyce |
| CN105624066A (en) * | 2015-12-31 | 2016-06-01 | 中国科学院烟台海岸带研究所 | Cellulose producing actinomycete and application thereof |
| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
| CN109321503A (en) * | 2018-11-05 | 2019-02-12 | 南京农业大学 | A kind of separation method for the actinomyces promoting manioc waste substrate fermentation process |
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| CN102911953A (en) * | 2012-10-17 | 2013-02-06 | 华东理工大学 | Neutral and alkaline glucanase and method for preparing same |
| CN103160556A (en) * | 2011-12-15 | 2013-06-19 | 上海来益生物药物研究开发中心有限责任公司 | Application of Streptomyces parvus |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
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| CN102399706A (en) * | 2010-09-17 | 2012-04-04 | 上海来益生物药物研究开发中心有限责任公司 | Streptomyces parvus and application thereof |
| CN103160556A (en) * | 2011-12-15 | 2013-06-19 | 上海来益生物药物研究开发中心有限责任公司 | Application of Streptomyces parvus |
| CN102911953A (en) * | 2012-10-17 | 2013-02-06 | 华东理工大学 | Neutral and alkaline glucanase and method for preparing same |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673713A (en) * | 2015-01-23 | 2015-06-03 | 深圳大学 | Basophilic streptomyce, neutral endoglucanase produced by basophilic streptomyce and application of basophilic streptomyce |
| CN104673713B (en) * | 2015-01-23 | 2017-10-20 | 深圳大学 | A kind of basophilic streptomycete and its neutral endoglucanase of generation and application |
| CN105624066A (en) * | 2015-12-31 | 2016-06-01 | 中国科学院烟台海岸带研究所 | Cellulose producing actinomycete and application thereof |
| CN105624066B (en) * | 2015-12-31 | 2019-08-09 | 中国科学院烟台海岸带研究所 | Cellulase-producing actinomyces and application |
| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
| CN106434447B (en) * | 2016-09-26 | 2019-08-13 | 浙江大学舟山海洋研究中心 | The bacterial strain and screening technique of production salt tolerant neutral cellulase and application |
| CN109321503A (en) * | 2018-11-05 | 2019-02-12 | 南京农业大学 | A kind of separation method for the actinomyces promoting manioc waste substrate fermentation process |
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| CN103937732B (en) | 2016-05-25 |
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