CN103937732B - Produce the method for neutral cellulase bacterial strain and selection and its production of cellulose enzyme - Google Patents
Produce the method for neutral cellulase bacterial strain and selection and its production of cellulose enzyme Download PDFInfo
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Abstract
The invention discloses a strain and produce the streptomyces bacterial strain of neutral cellulase, it is characterized in that: it on March 3rd, 2014 be preserved in Wuhan, China university " Chinese Typical Representative culture collection " center ", does is deposit number CCTCC? NO:M? 2014056, Classification And Nomenclature is<i>Streptomyces? parvus?</i>S10A10. This bacterium source is in insect gut. The invention provides the rDNA sequence of the strain culture of qualification. The present invention also provides culture medium and the condition of culture of this bacterial strain, and zymologic property and the production method of this bacterial strain institute cellulase-producing. The neutral cellulase vigor that the present invention produces is high, and low production cost, fermentation period are short. The neutral cellulase obtaining is suitable as food and feed additive, improves food quality, promotes growth of animal, reduces feed coefficient, and can be used for 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 oneProduce the method for neutral cellulase bacterial strain and selection and its production of cellulose enzyme. The cellulase that the present invention produces is mainly appliedIn industries such as food processing, feed addictive and weavings.
Background technology
Cellulase is the general name that cellulose and derivative thereof is hydrolyzed into one group of enzyme of glucose. One group of complete cellulase systemConventionally formed by cellulolytic three fermentoids of mutual concerted catalysis: endo cellulase (CMCase), circumscribed cellulase and PortugalPolyglycoside enzyme. By the optimal pH of catalytic reaction, endo cellulase can be divided into: acidic incision cellulase (optimal pH 3～5),Neutral endo cellulase (optimal pH 6～8), alkaline endo cellulase (optimal pH 8～10). Current domestic commodity chemical fibreTie up plain enzyme and mostly be acid high temperature enzyme, the neutral cellulase of domestic use is nearly all external manufacturer production. Technical monopoly causesIts commercialization neutral cellulase is expensive, has restricted its marketing. Therefore, this area is in the urgent need to developing new productionThe bacterial strain of high vigor neutral cellulase.
If can produce microbial host fungi and the part bacterium of endo cellulase. Fungi institute cellulase-producing mostly is outside acid born of the same parentsEnzyme and product enzyme efficiency are high, and enzyme architecture is complete, but fungi institute cellulase-producing is all complex enzyme, and enzyme architecture complexity, pointSon amount 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 containingHave a fiber element enzyme), can simplify extraction step, but bacteria cellulose enzyme mostly is neutral or alkaline endocellular enzyme, adsorbs moreIn bacterium wall, and its enzyme is alive generally lower, and in addition, most of bacterial strain produces induction type endo cellulase, need to add inducer,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, provide a kind of produce neutral cellulase bacterial strain and selection andThe method of its production of cellulose enzyme, cellulase pH good stability, catalytic capability that this bacterial strain produces are strong, are applicable to higher enzyme anti-Answer 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 " Chinese Typical Representative culture collection " center ", preservation on March 3rd, 2014Be numbered 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 actinomyces, can effectively obtain the neutrality of high yieldCellulase.
A selection for product neutral cellulase bacterial strain as claimed in claim 1, is characterized in that, described method comprises followingStep:
(a) getting the aseptic grinding of insect gut adds in liquid seed culture medium after enrichment culture 1d, by the bacterium after enrichment cultureLiquid is diluted to 10-4, get dilution, on primary dcreening operation culture medium, be coated with dull and stereotyped cultivation;
(b) cultivate after 7d, choose the actinomyces bacterium colony of different shape on same flat board with transfer needle, then be transferred on flat board and carry outSingle bacterium colony separates, and finally obtains multi-strain bacteria;
(c) every strain bacterium is inoculated in respectively in liquid seed culture medium and enzymatic production culture medium, in 28 DEG C of constant incubators, trainsSupport 36h, get zymotic fluid and carry out the rapid screening of Congo red flat board, through the Congo red solution-dyed 30min of 1mg/mL, 1mol/LNaCl solution decolouring 30min after, measure dull and stereotyped upper bacterium colony hydrolysis circle and colony diameter, calculate hydrolysis circle and bacterial strain diameterRatio, filters out the bacterial strain that ratio is greater than 5, obtains a strain in two kinds of cultivations, all can produce obviously transparent through experiment screening repeatedlyThe bacterial strain of 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 instituteState consisting of of liquid seed culture medium: CMC-Na, 10g; KH2PO4，4.0g；MgSO4, 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 described primary dcreening operation culture mediumConsist of: sodium carboxymethylcellulose, 5.0g; K2HPO4，1.0g；NaNO3，3.0g；KCl，0.5g；MgSO4，0.5g；FeSO4, 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 described enzymatic production trainingSupport consisting of of base: rice straw powder, 10g; (NH4)2SO4，4g；KH2PO4，2g；MgSO4, 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, described inMethod comprises the following steps:
(1) producing enzyme cultivates
StreptomycesparvusS10A10 is inoculated in to No. 1 culture medium of inclined-plane Gao Shi, the liquid of transferring after 27 DEG C of cultivation 2dSeed culture medium, cultivates 24h under 27 DEG C of conditions; With the 2% inoculum concentration enzymatic production culture medium of transferring again, at the beginning of fermentation medium is the suitableeestBeginning pH cultivates 5d under 6～6.5,27 DEG C of conditions;
(2) processing of extracellular products crude enzyme liquid
Rotating speed refrigerated centrifuge 10min by zymotic fluid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Get 20mL crude enzyme liquid in0.05mol/L, pH are dialysed overnight in 6.2 citric acid-sodium citrate buffer solution; Use polyethylene glycol concentrate dialysate, thenThrough O.45 μ m filtering with microporous membrane removal of impurities, preserve filtrate;
(3) sieve chromatography
The abundant balance sephadex chromatography of the phosphate buffer post SephadexG-100 post that is 6.2 with pH in advance, loadingThe citric acid-sodium citrate wash-out that is 6.2 with pH afterwards, collects the eluting 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), the consisting of of described No. 1 culture medium of Gao Shi: soluble starch, 20g; KNO3，1g；K2HPO4，0.5g；MgSO4·7H2O，0.5g；NaCl，0.5g；FeSO4·7H2O, 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), the consisting of of described liquid seed culture medium: CMC-Na, 10g; KH2PO4，4.0g；MgSO4，0.03G; 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), the consisting of of described enzymatic production culture medium: rice straw powder, 10g; (NH4)2SO4，4g；KH2PO4，2g；MgSO4, 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, withCurrent industrial is compared with the bacterial isolates of enzyme, and the enzymatic production cycle is short, and enzyme activity is high, and produce enzyme process without induction, therefore itsThe cellulase producing is composing type cellulase, and therefore this bacterium is applicable to the feature of large-scale fermenting and producing;
(2) the vigor specific activity of the total enzyme of cellulase extracting from StreptomycesparvusS10A10 of the present invention reaches 33.02U/mL, compared with report bacterial strain, has higher catalytic capability; This enzyme optimal reaction pH is 5.0, is rare acidity compositionFiber type element enzyme; This enzyme is preserved 2h enzyme activity in the buffer solution of pH4.0～8.0 can keep the more than 90% of the highest enzyme work, bodyShow good pH stability; Optimal reactive temperature is 50～65 DEG C; There is resistance to Na+、K+、Ca2+、Ni2+、Zn2+、Mg2+、Mn2+、Ba2+、Pb2+、Fe3+Deng the superperformance of metal ion, EDTA is very micro-on enzymatic activity impact, and this enzyme is nonmetallic ion celluloseEnzyme, has shown good characteristic, has a good application prospect at industrial circles such as weaving, food processing and feeds.
Brief description of the drawings
Fig. 1 is the transparent circle of StreptomycesparvusS10A10 bacterial strain of the present invention on primary dcreening operation culture medium;
Fig. 2 is the ESEM form picture of StreptomycesparvusS10A10 bacterial strain of the present invention;
Fig. 3 is that (M is standard protein molecular weight, and 3 is zymoprotein for the cellulase polyacrylamide gel electrophoresis figure of purifying of the present inventionMolecular weight is 82Kd endo cellulase. );
Fig. 4 is the chromatogram that SephadexG-100 column chromatography obtains cellulase Peak Activity;
Fig. 5 is the impact that pH lives on cellulose restriction endonuclease;
Fig. 6 is the impact that temperature is lived on 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 " Chinese Typical Representative culture collection " center ", preservationAddress: Luojiashan, Wuchang, Wuhan City, Hubei Province, deposit number is that deposit number is CCTCCNO:M2014056, preservation dateIt is on March 3rd, 2014.
Detailed description of the invention
For further disclosing technical scheme of the present invention, describe embodiments of the present invention in detail below in conjunction with accompanying drawing:
In the present invention, used medium is:
No. 1 culture medium of Gao Shi (Gause): soluble starch, 20g; KNO3，1g；K2HPO4，0.5g；MgSO4·7H2O，0.5g；NaCl，0.5g；FeSO4·7H2O, 0.01g; Agar, 20g; Distilled water, 1000mL, pH6.0～6.5.
Primary dcreening operation culture medium: sodium carboxymethylcellulose, 5.0g; K2HPO4，1.0g；NaNO3，3.0g；KCl，0.5g；MgSO4，0.5g；FeSO4, 0.01g; Agar, 17.0g; Distilled water 1000mL, pH6.0～6.5.
Liquid seed culture medium: CMC-Na, 10g; KH2PO4，4.0g；MgSO4, 0.03g; Distilled water 1000mL, pH6.0～6.5。
Enzymatic production culture medium: rice straw powder, 10g; (NH4)2SO4，4g；KH2PO4，2g；MgSO4, 0.5g; SteamHeat up in a steamer 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,Bacterium liquid after enrichment culture is diluted to 10-4, gets dilution, on primary dcreening operation culture medium, be coated with dull and stereotyped cultivation. Cultivate after about 7d,Choose the actinomyces bacterium colony of different shape on same flat board with transfer needle, then be transferred to and on flat board, carry out single bacterium colony separation, finalTo 42 strain bacterial strains. Every strain bacterium is inoculated in respectively in liquid seed culture medium and enzymatic production culture medium, 28 DEG C of constant temperature cultureIn case, cultivate 36h, get zymotic fluid and carry out the rapid screening of Congo red flat board, through the Congo red solution-dyed 30min of 1mg/mL,After the NaCl solution decolouring 30min of 1mol/L, measure dull and stereotyped upper bacterium colony hydrolysis circle and colony diameter, calculate hydrolysis circle and bacteriumThe ratio of strain diameter, filters out the bacterial strain that ratio is greater than 5, obtains a strain all can produce in two kinds of cultivations through experiment screening repeatedlyThe obviously bacterial strain of transparent circle (D/d > 5), by its called after StreptomycesparvusS10A10. Fig. 1 is StreptomycesThe transparent circle of parvusS10A10 bacterial strain on primary dcreening operation culture medium, Fig. 2 is StreptomycesparvusS10A10 bacterial strainMicroexamination picture.
Embodiment 2
From StreptomycesparvusS10A10, extract the method for neutral composing type endo cellulase:
1. producing enzyme cultivates
StreptomycesparvusS10A10 is inoculated in to No. 1 culture medium of inclined-plane Gao Shi, and after 27 ° of C cultivate 2d, switching is plantedSub-culture medium, cultivates 24h under 27 ° of C conditions; With the 2% inoculum concentration liquid fermentation medium of transferring again, at the beginning of fermentation medium is the suitableeestBeginning pH is 6～6.5; Under 27 ° of C conditions, cultivate 5d.
2. the pre-treatment of extracellular products crude enzyme liquid
Rotating speed refrigerated centrifuge 10min by zymotic fluid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Get 20mL crude enzyme liquid inDialysed overnight in the citric acid-sodium citrate buffer solution of 0.05mol/L, pH value 6.2; Use polyethylene glycol concentrate dialysate, thenThrough 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 buffer post SephadexG-100 post of pH6.2, after loading, useThe citric acid-sodium citrate wash-out of PH6.2, collects the eluting peak (seeing Fig. 4) that has enzyme to live.
SephadexG-100 column chromatography condition is as follows:
The citric acid-sodium citrate buffer solution of level pad: 0.05mol/L, pH value 6.2;
The citric acid-sodium citrate buffer solution 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, add the citrate buffer solution (pH value 4.8) of 1.0ml0.05mol/LWith 50mg Xinhua filter paper, not add substrate as contrast, 50 DEG C of reaction 60min, add 3mLDNS solution, boiling water bath 5min,Again test tube is placed in to cold bath, after test tube is cooling, adds 18mL distilled water. Contrast with blank tube, adopt light splitting lightDegree instrumentation must be in the OD at 540nm place value.
2. the mensuration of 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),Not add substrate as contrast, 50 DEG C of reaction 30min, add 3mLDNS solution, boiling water bath 5min, then test tube is placed in coldIn water-bath, after test tube is cooling, add 18mL distilled water. Contrast with blank tube, adopt spectrophotometer to record at 540nmThe OD value at place.
3. the mensuration of 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), not addSubstrate is contrast, and 50 DEG C of reaction 60min, add 3mLDNS solution, boiling water bath 5min, then test tube is placed in to cold bath,After test tube is cooling, add 18mL distilled water. Contrast with blank tube, adopt spectrophotometer to record at 540nm placeOD value.
4. beta-glucosidase (BGA) vitality test:
Get the enzyme liquid after 3 times of 0.5mL dilutions, add 1.0mL, 1% salicin solution (pH value 4.8), not add substrateFor contrast, 50 DEG C of reaction 30min, add 3mLDNS solution, boiling water bath 5min, then test tube is placed in to cold bath, treatAfter test tube is cooling, add 18mL distilled water. Contrast with blank tube, adopt spectrophotometer to record the OD at 540nm placeValue.
The computational methods of enzyme activity:
The definition of enzyme activity: at pH4.8, under 50 DEG C of conditions, every 1mL enzyme liquid hydrolysis substrate in 1min generates 1 μ gThe enzyme amount of 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 calibration 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
Carry out according to a conventional method with SDS-PAGE polyacrylamide gel electrophoresis. Separation gel, concentrated gum concentration are respectively 12% and 5%,Electrode buffer is pH8.3Tris-Gly buffering, coomassie brilliant blue staining, and result is as shown in Figure 3. After electrophoresis, use GEL-DOC2000(Bio-Rad) carry out gel image scanning. Use scanning system to carry software QuantityOne and carry out molecular weight determination, record inscribeCellulose enzyme molecular weight of albumen is 82Kd.
2. the optimal pH of enzyme
Prepare the CMC-Na substrate of different pH values by various bufferings. By endo cellulase LC and different pH values after extractingCMC-Na substrate reactions, reacts 30min under 65 DEG C of conditions, and detects CMCase vigor, as shown in Figure 5. Cellulase inProperty slant acidity there is higher vigor, within the scope of pH5.O～7.O, there is very high vigor, reach that the highest enzyme lives 80% withUpper, 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 buffer solutions, under 65 DEG C of conditions, places after 2h, measures inscribe fiberElement enzyme relative activity, as shown in Figure 5. Result shows that this endo cellulase has shown the steady of height within the scope of wider pHQualitative, in the buffer solution of pH5.0～8.0, can keep its highest enzyme to live more than 85%, show good pH stability,Weaving, papermaking and food-processing industry have good application potential.
4. the optimal reactive temperature of enzyme
Pure endo cellulase LC enzyme is positioned over respectively under different temperatures and reacts 30min, measure its enzyme and live as shown in Figure 6. KnotFruit shows that endo cellulase has higher vigor in the time of 50～65 DEG C of reactions, and its optimum temperature is 50 DEG C.
5. the impact of metal ion on cellulase productivity
Inquire into Na at three concentration level lmmol/L, 5mmol/L, 10mmol/L+、K+、Li+-valence metal ion; Ca2+、Ni2+、Zn2+、Mg2+、Cu2+、Fe2+、Co2+、Mn2+、Ba2+、Pb2+Bivalent metal ion; Fe3+Trivalent metal ion and EDTA coupleThe impact of CMCase vigor. At 4 DEG C, place 30min, under 65 DEG C of conditions, react 30min, and detect CMCase vigor. With notThe enzyme that adds metal ion is lived as the relative activity of blank determination enzyme. Under the metal ion existence condition of result low 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 variable concentrations is little on enzyme activity impact, pushes awaySurveying endo cellulase is probably nonmetallic ion cellulase, and metal ion has certain facilitation to this enzyme. Show thisEnzyme 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, itsHe any deviates from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify, all should be etc.The substitute mode of effect, is included in protection scope of the present invention.
Claims (3)
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 CCTCCNO:M2014056, Classification And Nomenclature be streptomyces parvus (Streptomycesparvus）S10A10。
2. a method of utilizing the bacterial strain production of cellulose enzyme of the product neutral cellulase described in claim 1, is characterized in that, said method comprising the steps of:
(1) producing enzyme cultivates
By streptomyces parvus (Streptomycesparvus) S10A10 is inoculated in No. 1 culture medium of inclined-plane Gao Shi, cultivates the liquid seed culture medium of transferring after 2d for 27 DEG C, under 27 DEG C of conditions, cultivates 24h, the consisting of of described liquid seed culture medium: CMC-Na, 10g; KH2PO4，4.0g；MgSO4, 0.03g; Distilled water, 1000mL, pH6.0～6.5; With the 2% inoculum concentration enzymatic production culture medium of transferring again, the suitableeest initial pH of fermentation medium cultivates 5d under 6～6.5,27 DEG C of conditions, the consisting of of described enzymatic production culture medium: rice straw powder, 10g; (NH4）2SO4，4g；KH2PO4，2g；MgSO4, 0.5g; Distilled water, 1000mL;
(2) processing of extracellular products crude enzyme liquid
Rotating speed refrigerated centrifuge 10min by zymotic fluid with 4000r/min, gets supernatant, obtains crude enzyme liquid; Dialysed overnight in the citric acid-sodium citrate buffer solution that to get 20mL crude enzyme liquid be 6.2 in 0.05mol/L, pH; Use polyethylene glycol concentrate dialysate, then through 0.45 μ m filtering with microporous membrane removal of impurities, preserve filtrate;
(3) sieve chromatography
The abundant balance sephadex chromatography of the phosphate 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 eluting peak that has enzyme to live, and is the cellulase after purifying.
3. the method for a kind of enzyme bacterial strain production of cellulose enzyme that utilizes the product neutral fibre element described in claim 1 according to claim 2, is characterized in that, in above-mentioned steps (1), and the consisting of of described No. 1 culture medium of Gao Shi: soluble starch, 20g; KNO3，1g；K2HPO4，0.5g；MgSO4·7H2O，0.5g；NaCl，0.5g；FeSO4·7H2O, 0.01g; Agar, 20g; Distilled water, 1000mL, pH6.0～6.5.
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| 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 |
| 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 |
| 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 |
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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 |
| 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 |
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