CN105647904A - Method for screening cellulase producing strains and method for producing cellulase by means of fermentation - Google Patents
Method for screening cellulase producing strains and method for producing cellulase by means of fermentation Download PDFInfo
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Abstract
The invention discloses a method for screening cellulase producing strains and a method for producing cellulase by means of fermentation. The cellulase producing strains which are named as TP-02 are acquired from rotten wood in ecological forest in the Huangshang Mountain by means of primary screening, protoplast preparation, ultraviolet-irradiation mutation breeding and secondary screening. The methods have the advantages that the cellulase producing strains are high in cellulase producing capacity, bean pulp, ammonium sulfate, urea or ammonium chloride can be used as nitrogen sources, bran juice, lactose, starch sugar, rice straw or corn straw can be used as carbon sources, the nitrogen sources and the carbon sources are subjected to liquid-state fermentation to obtain the cellulase producing strains, the enzyme activity of filter paper can reach 16.98 IU/mL after the cellulase producing strains are cultivated for 72-84 hours, the enzyme activity of incision enzymes can reach 41.33 IU/mL, the enzyme activity of excision enzymes can reach 3.65 IU/mL, and the enzyme activity of beta-glucanase can reach 15.98 IU/mL.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the screening technique of a kind of cellulase producing strain and the method for fermenting and producing cellulase thereof.
Background technology
Lignocellulose material is to be distributed the material the widest, storage is the abundantest on the earth, is also the most cheap Renewable resource. Cellulase is then that a class can by the general name of cellulose degradation is glucose multicomponent enzyme system, they synergism, cellulose degradation is produced oligosaccharide and fiber two pool, finally it is hydrolyzed to the monosaccharide such as glucose, xylose, and then ethanol, food, single cell protein, pharmaceuticals and other chemical raw materials can be produced further. Therefore, cellulase is produced at alcohol fuel, papermaking, the industry such as feed manufacturing have a good application prospect.
The producing strains of cellulase includes antibacterial, fungus, yeast etc., but the strain being currently used primarily in cellulase production is mainly filamentous fungi. The cellulase systems that filamentous fungi produces is relatively complete, and the enzyme of its generation mostly is exoenzyme, it is simple to the separation and Extraction in later stage. The multienzyme complex that cellulase is made up of enzyme three kinds different: exoglucanase, endoglucanase and beta-glucosidase (BGL), three kinds of enzyme synergism complete cellulosic hydrolysis.
The application of current cellulase is still limited to high production cost problem to a certain extent. Cellulase height production cost problem main manifestations is following two aspect: one, the fermentation enzyme vigor of the producing bacterial strain of cellulase is relatively low, it is always up large-scale production urgent problem, is also that long-term constraints on fiber element enzyme produces in a large number and limits the principal element of its application; Its two, currently used filamentous fungi makes the cellulase fermentations time general longer due to the factor such as strain properties and condition of culture, high cost. At present the filamentous fungi about cellulase production and research mainly has trichoderma, aspergillus and Penicillium microorganism, and fermentation period is typically in more than 6d, has the features such as length consuming time, efficiency are low.
Summary of the invention
For above deficiency, the invention provides the screening technique of the bacterial strain of a kind of cellulase-producing, described bacterial strain is to be prepared by primary dcreening operation, protoplast from the ecological forest corruption wood of Mount Huang, ultraviolet lighting mutagenic and breeding, sieve the bacterial strain that a kind of name obtained is called the cellulase-producing of TP-02 again.
Present invention also offers and screened the bacterial strain obtained by above-mentioned screening technique as the application producing dimension element enzyme. This bacterial strain possesses the ability of High Cellulase Production, can bean cake, ammonium sulfate, carbamide or ammonium chloride be nitrogenous source, with wheat bran juice, lactose, starch sugar, rice straw or corn straw for carbon source through liquid fermentation, after cultivating 72��84 hours, its filter paper enzyme activity can reach 16.98IU/mL, restriction endonuclease enzyme is lived and is reached 41.33IU/mL, and excision enzyme enzyme is lived and reached 3.65IU/mL, and beta glucan glycosides enzyme enzyme activity reaches 15.98IU/mL.
Present invention also offers a kind of production method tieing up element enzyme, the method is relative to other prior aries, and its enzymatic production cycle is greatly shortened.
The technical scheme that the present invention takes is:
A kind of screening technique of cellulase producing strain, described screening technique comprises the following steps: prepared by primary dcreening operation, protoplast, ultraviolet lighting mutagenic and breeding, multiple sieve;
Described cellulase producing strain on July 16th, 2015 by China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica; Deposit number is CGMCCNo.11119; Classification And Nomenclature is that Rhizopus stolonifer is nodded mutation Rhizopusstolonifervar.reflexus.
Described screening technique specifically includes following steps:
A. primary dcreening operation culture medium primary dcreening operation, the composition of described primary dcreening operation culture medium is: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL;
B. preparing protoplast, the spore of the bacterial strain obtained by primary dcreening operation is cultivated in PDA culture medium, the mycelia oscillation incubation in shell-broken liquid obtained after purified, centrifugal, obtains protoplast after filtration, centrifugal purification; The composition of described shell-broken liquid is Snailase: cellulase=3:1;
C. mutagenic and breeding, the protoplast 15W ultra violet lamp obtained by step B carries out mutagenic treatment, and treatment fluid is coated height and oozed on primary dcreening operation culture medium flat plate, and after cultivation, bacterium colony is transferred to the cultivation of PDA slant medium;
Described height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol; Height is used to ooze the physiological osmotic pressure that primary dcreening operation culture medium flat plate can remain suitable, it is to avoid protoplast crushes.
D. sieve again; The spore suspension of the culture on PDA slant medium obtained by step C is inoculated in liquid seed culture medium and cultivates, and seed culture fluid is inoculated in liquid fermentation culture medium by 1:10 volume ratio and cultivates, cross the mensuration to tunning enzymolysis reducing sugar yield, filter out the bacterial strain that enzymatic activity is high, be cellulase producing strain TP-02.
The composition of described liquid seed culture medium is: 10% wheat bran diffusion juice, and its preparation method, for weighing 100g wheat bran, adds 800mL distilled water and boils 30min, and 4 layers of filtered through gauze are settled to 1000mL.
The composition of described liquid fermentation culture medium is: every L culture medium contains: the corn straw of 80��120 orders, Caulis et Folium Oryzae or straw 5-10g, 5% wheat bran diffusion juice 80-100g, (NH4)2SO420-30g, KH2PO45-10g, MgSO4��7H2O5-10g, CaCl210-20g, Tween800.15-0.25g, trace element: FeSO4��7H2O0.003-0.005g, MnSO4��H2O0.001-0.00156g, ZnSO4��7H2O0.001-0.0014g, CoCl2��6H2O0.001-0.002g, pH5.0.
Present invention also offers and screen the bacterial strain obtained according to above-mentioned screening technique as the application producing dimension element enzyme.
Present invention also offers a kind of production method tieing up element enzyme, said method comprising the steps of:
(1) the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained by liquid seed culture medium is used to carry out seed culture,
To seed culture fluid;
(2) seed culture fluid is inoculated in liquid fermentation culture medium and cultivates, liquid fermentation cellulase-producing.
Described step (1) specifically includes following steps: the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained cultivates through PDA slant medium, liquid seed culture medium after first, and gained seed liquor is saved in-80 DEG C of ultralow temperature glycerol freezing pipes; The seed liquor preserved in-80 DEG C of ultralow temperature glycerol freezing pipes be can be prepared by seed culture fluid through PDA slant medium, liquid seed culture medium after cultivating after first.
In described step (2), the ratio of seed culture fluid and the volume of liquid fermentation culture medium is 1:9��10.
Condition of culture in described step (2) is 30 DEG C, 200��350r/min cultivates 84��96 hours.
Further, the condition of culture in described step (2) also includes: speed of agitator is 450r/min, and ventilation is 4.5vvm, and tank pressure is 0.03MPa, pH is 5.0��5.5 scopes, controls pH in 5.0��5.5 scopes by Feeding ammonia water in this process.
Relative to prior art, the invention have the advantages that
1. growth speed is fast, produces enzyme speed height, and fermentation time is short, can terminate fermentation in 4d;
2. strain is wild mushroom, flushes, not easily microbiological contamination, it is easy to pure culture;
3. ferment strain available agricultural by-products such as wheat bran juice, rice straw (or Wheat Straw, corn straw) cellulase-producing, and nutrient matrix utilizes scope wide and composition is cheap, be easy to get;
4. repeating shake flask fermentation stable performance, its filter paper enzyme activity reaches 13.16IU/mL, and restriction endonuclease enzyme is lived and reached 45.81IU/mL, and excision enzyme enzyme is lived and reached 0.537IU/mL, and glucosan glycosides enzyme enzyme activity reaches 12.45IU/mL;
5. after cultivating 72 hours in fermentation tank, its its filter paper enzyme activity can reach 16.98IU/mL, and restriction endonuclease enzyme is lived and reached 41.33IU/mL, and excision enzyme enzyme is lived and reached 3.65IU/mL, and glucosan glycosides enzyme enzyme activity reaches 15.98IU/mL;
6. fermentation pH wide accommodation, technology controlling and process is simple.
Accompanying drawing explanation
Fig. 1 is primary dcreening operation bacterium colony hydrolysis circle in culture medium;
Fig. 2 is TP-02 strain enzyme-producing characteristic research in embodiment 5;
Fig. 3 is TP-02 strain enzyme-producing characteristic research in embodiment 6.
Detailed description of the invention
Embodiment 1
A kind of screening technique of cellulase producing strain, described screening technique comprises the following steps:
Primary dcreening operation:
Take the 1g material (Mount Huang ecological forest corruption wood) containing purpose strain to pulverize; Adding 10mL normal saline, vibrate 1h. Above-mentioned suspension is all added in 200mLPDA culture medium, 30 DEG C, 180rpm shaken cultivation 12h;
Above-mentioned culture utilize normal saline dilute 10 times successively to 10-3��10-4��10-5, and it being respectively coated in PDA plate culture medium, 30 DEG C of quiescent culture, to there being bacterium colony to occur, choose typical case's single bacterium colony switching PDA inclined-plane, and purity test is done in film-making simultaneously, it is thus achieved that pure culture body; By pure culture body dibbling to primary dcreening operation culture medium, the composition of described primary dcreening operation culture medium is: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g;Agar 10-20g; MandelS inorganic nutrient salt 1000mL, with the Congo red dye liquor dyeing 2h of 1mg/mL after 28 DEG C of cultivation 48h, again with the washed many dye liquors of NaOH solution of 1mol/L after the dye liquor that inclines, it is suppressed that enzymatic activity, fixes and is hydrolyzed circle size. If thalline energy eccrine fiber element enzyme, then there will be in periphery of bacterial colonies and be hydrolyzed circle clearly, according to the size selection high dynamic strain of hydrolysis circle with colony diameter ratio (LD ratio). Choose the big bacterium colony transposing of LD ratio to cultivate to PDA test tube slant and save backup (see Fig. 1).
Prepared by protoplast:
By the spore normal saline eluting of the rhizopus stolonifer bacterial strain of PDA test tube slant preservation, after mixing, it is diluted to 1 �� 105Individual/mL; Being transferred by above-mentioned spore suspension in the 500mL conical flask containing 100mLPDA culture medium, adjusting spore concentration is 107Individual/mL, cultivates 12h on the shaking table that rotating speed is 150r/min; Gained culture is centrifuged in 3500r/min, takes precipitation, and with brine twice, centrifugal, then wash a centrifuging and taking with 0.6mol/LKCl hyperosmotic solution and precipitate;
Electronic balance is utilized accurately to weigh 2g mycelium after treatment, add 20mL3mg/mL shell-broken liquid, the composition of shell-broken liquid is Snailase: cellulase=3:1, fully mixing, in 30 DEG C, 160rpm concussion is hatched, and the generation situation counting of protoplast is once observed every 0.5h microscopy, determine enzymolysis time, with four layers of sterile microscope paper, protoplasm body fluid is filtered, remove mycelia fragment and take filtrate, 3500r/min is centrifuged 10min, collect protoplast, wash 2 times with organic sugar alcohol homeo-osmosis agent, obtain the protoplast of purification.
Mutagenic and breeding:
Take the protoplast of the above-mentioned purification of 5mL in aseptic 9cm culture dish, mutation is carried out with 15W uviol lamp distance 30cm place's irradiation, uviol lamp preheats 30min in advance so that ultraviolet lighting is stable, take the treatment fluid of different irradiation time, coating height and ooze on primary dcreening operation culture medium flat plate, height oozes the composition of primary dcreening operation culture medium flat plate and is: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol; Dark 30 DEG C of constant temperature culture of lucifuge, grow after bacterium colony until flat board, PDA slant culture of transferring;
Utilizing CMC-Congo red flat band method, Preliminary screening goes out the bacterial strain that transparent circle is bigger with colony diameter, and the method sieved again by shake flask fermentation selects the mutant of high active cellulase.
Multiple sieve:
Being washed down by PDA slant culture normal saline, abundant mixing is suitably diluted to 1 �� 105Individual/mL, prepares into spore suspension; Spore suspension is inoculated in the 250mL triangular flask including 50mL liquid seed culture medium, the composition of liquid seed culture medium is that 10% wheat bran diffusion juice (weighs 100g wheat bran, add 800mL distilled water and boil 30min, 4 layers of filtered through gauze, it is settled to 1000mL), adjusting spore concentration is 107Individual/mL, cultivates 12h on the shaking table that rotating speed is 150r/min, obtains seed culture fluid;
In 500mL triangular flask, add the liquid fermentation culture medium of about 200mL, access 20mL seed culture fluid, 30 DEG C, 180rpm shaken cultivation 12h. The composition of described liquid fermentation culture medium is: every L culture medium contains: the corn straw of 80��120 orders, Caulis et Folium Oryzae or straw 5-10g, 5% wheat bran diffusion juice 80-100g, (NH4)2SO420-30g, KH2PO45-10g, MgSO4��7H2O5-10g, CaCl210-20g, Tween800.15-0.25g, trace element: FeSO4��7H2O0.003-0.005g, MnSO4��H2O0.001-0.00156g, ZnSO4��7H2O0.001-0.0014g, CoCl2��6H2O0.001-0.002g, pH5.0.
By the mensuration to tunning enzymolysis reducing sugar yield, filter out the high bacterial strain of enzymatic activity and enter the research of next round fermentating enzyme-producing condition.
Table 1 hydrolysis circle size and enzyme activity relation just
The bacterial strain TP-02 screened is carried out continuous passage 10 generation, by the mode of liquid fermentation, measure its enzyme to live, studying its hereditary stability as shown in table 1, as shown in Table 1, mutant TP-02 is through passing 10 cultures, FPA and CMC enzyme activity between any 2 generations, difference is all in 10%, it was shown that this strain enzyme-producing stable performance, can as follow-up experiment starting strain.
Table 2 mutant TP-02 hereditary stability
| Algebraically | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| FPA(IU/mL) | 5.2 | 5.3 | 5.1 | 5.2 | 5.0 | 4.8 | 4.9 | 5.3 | 5.2 | 5.2 |
| CMC(IU/mL) | 20.5 | 21.2 | 20.8 | 21.0 | 20.2 | 19.8 | 20.2 | 22.1 | 21.8 | 21.5 |
Strain identification:
Obtain the ITS-5.8srDNA sequence of this strain through PCR and utilize nucleotide comparison in order-checking with NCBI. From NCBI, obtain the recognised standard sequence data of relevant bacteria species, carry out multiple analysis comparison with ClustalX1.8. Utilize MEGA3.1, PAUP software, with the Related species trichoderma reesei of Rhizopus for outer group, build MP and ML phylogenetic tree. This research strain and rhizopus stolonifer nod rhizopus sequence homology rate up to 99.8%. It is generally acknowledged homology more than 98% it is believed that belong to same kind. Therefore it is accredited as Rhizopus rhizopus stolonifer subspecies according to molecular level to nod rhizopus. Combining form qualification result, Preliminary Identification TP-02 bacterial strain is that Zygomycotina, Phycomycetes, Mucoales, Mucoraceae, rhizopus, rhizopus stolonifer subspecies are nodded rhizopus, and its gene order table is such as shown in SEQUENCELISTING1.
Embodiment 2
A kind of screening technique of cellulase producing strain, described screening technique comprises the following steps:
The other the same as in Example 1, simply the composition of primary dcreening operation culture medium therein is: every L culture medium contains: CMC-Na5g; Glucose 3g; Agar 10g; MandelS inorganic nutrient salt 1000mL;
Height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na5g; Glucose 3g; Agar 10g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;
The composition of liquid fermentation culture medium is: every L culture medium contains: the corn straw 5g of 80��120 orders, 5% wheat bran diffusion juice 80g, (NH4)2SO420g, KH2PO45g, MgSO4��7H2O5g, CaCl210g, Tween800.15g, trace element: FeSO4��7H2O0.003g, MnSO4��H2O0.001g, ZnSO4��7H2O0.001g, CoCl2��6H2O0.001g, pH5.0.
Embodiment 3
A kind of screening technique of cellulase producing strain, described screening technique comprises the following steps:
The other the same as in Example 1, simply the composition of primary dcreening operation culture medium therein is: every L culture medium contains: CMC-Na10g; Glucose 5g; Agar 20g; MandelS inorganic nutrient salt 1000mL;
Height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na10g; Glucose 5g; Agar 20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;
The composition of liquid fermentation culture medium is: every L culture medium contains: the Caulis et Folium Oryzae 10g of 80��120 orders, 5% wheat bran diffusion juice 100g, (NH4)2SO430g, KH2PO410g, MgSO4��7H2O10g, CaCl220g, Tween800.25g, trace element: FeSO4��7H2O0.005g, MnSO4��H2O0.00156g, ZnSO4��7H2O0.0014g, CoCl2��6H2O0.002g, pH5.0.
Embodiment 4
A kind of screening technique of cellulase producing strain, described screening technique comprises the following steps:
The other the same as in Example 1, simply the composition of primary dcreening operation culture medium therein is: every L culture medium contains: CMC-Na8g;Glucose 4g; Agar 15g; MandelS inorganic nutrient salt 1000mL;
Height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na8g; Glucose 4g; Agar 15g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;
The composition of liquid fermentation culture medium is: every L culture medium contains: the straw 8g of 80��120 orders, 5% wheat bran diffusion juice 90g, (NH4)2SO425g, KH2PO48g, MgSO4��7H2O8g, CaCl215g, Tween800.2g, trace element: FeSO4��7H2O0.004g, MnSO4��H2O0.0014g, ZnSO4��7H2O0.0012g, CoCl2��6H2O0.0015g, pH5.0.
Embodiment 5
A kind of production method tieing up element enzyme, said method comprising the steps of:
The preparation method of seed is with embodiment 2;
Liquid fermentation produces cellulase:
Drawing 5mL seed culture fluid and access 45mL liquid fermentation culture medium, inoculation post-fermentation and culture base dress base liquid amount is the bottled 50mL culture medium of 250mL triangle. Inoculation is placed in concussion constant temperature culture and cultivates, and cultivation temperature is 30 DEG C, and shaking speed is 200rpm/min, and total incubation time is 4d, and period every 12h draws 1mL liquid from fermentation liquid and its enzyme activity is measured. After fermentation culture 84h, fermentation liquid cellulase filter paper enzyme activity (FPA), restriction endonuclease enzyme live, excision enzyme enzyme live and beta glucan glycosides enzyme enzyme reach maximum, respectively 13.16IU/mL, 45.81IU/mL, 0.537IU/mL, 12.45IU/mL, as shown in Figure 2.
The composition of described liquid fermentation culture medium is: every L culture medium contains: the straw 5g of 80��120 orders, 5% wheat bran diffusion juice 100g, (NH4)2SO426g, KH2PO48g, MgSO4��7H2O9g, CaCl218g, Tween800.2g, trace element: FeSO4��7H2O0.004g, MnSO4��H2O0.0015g, ZnSO4��7H2O0.0013g, CoCl2��6H2O0.0015g, pH5.0.
Embodiment 6
A kind of production method tieing up element enzyme, said method comprising the steps of:
Spawn preparation:
Bacterial strain TP-02 is cultivated in PDA slant medium after 3d with spore under aseptic washing, use blood counting chamber to calculate spore count and be also diluted to 1 �� 105Individual/mL. Access in liquid amount 200mL (500mL triangular flask) liquid seed culture medium by 10% inoculum concentration, in 30 DEG C, 200rpm when cultivate 24h after standby as seed liquor, and gained seed liquor is saved in-80 DEG C of ultralow temperature glycerol freezing pipes;
Take-80 DEG C of ultralow temperature glycerol freezing pipes and put room-temperature dissolution PDA slant medium of ruling, with mycelia or spore under aseptic washing after cultivating 3d in 30 DEG C, each flat board uses 5mL sterilized water to wash, the mycelia suspension obtained after drawing 2mL washing accesses in 48mL liquid seed culture medium, after inoculation, seed culture medium liquid amount is the bottled 50mL culture medium of 250mL triangle, and in 30 DEG C, shaking speed is 200rpm, after constant temperature culture 24h, it is shake flask fermentation and produces the seed culture fluid of enzyme.
Liquid fermentation produces cellulase:
3L fermentation tank fills 2L liquid fermentation culture medium, accesses 200mL seed culture fluid and ferment. Fermentation condition: cultivation temperature is 30 DEG C, speed of agitator is 450r/min, and ventilation is 4.5vvm, and tank pressure is 0.03MPa, controls pH in 5.0��5.5 scopes by Feeding ammonia water, and concrete PH can be 5.0,5.1,5.2,5.3,5.4,5.5 these numerical value. Under above-mentioned condition of culture, cultivate 88h, period every 4h and from fermentation liquid, draw 1mL liquid and its enzyme activity is measured. After finding cultivation 84h, filter paper enzyme activity reaches 16.98IU/mL, and restriction endonuclease enzyme is lived and reached 41.33IU/mL, and excision enzyme enzyme is lived and reached 3.65IU/Ml, and glucosan glycosides enzyme enzyme activity reaches 15.98IU/mL, as shown in Figure 3.
The composition of described liquid fermentation culture medium is: every L culture medium contains: the corn straw 8g of 80��120 orders, 5% wheat bran diffusion juice 90g, (NH4)2SO420g, KH2PO45g, MgSO4��7H2O7g, CaCl215g, Tween800.15g, trace element: FeSO4��7H2O0.003g, MnSO4��H2O0.001g, ZnSO4��7H2O0.001g, CoCl2��6H2O0.001g, pH5.0.
Embodiment 7
The detection method of cellulase
The mensuration of filter paper enzyme activity: accurately draw the enzyme liquid 0.5mL of suitably dilution 10-50 times, put into the 0.05mol/L citric acid sodium citrate buffer of the filter paper of the 1cm �� 6cm 0.05mol/L citric acid sodium citrate buffer in pH4.8 and 0.5mLpH4.8,3mLDNS reagent boiling water bath 10min is added after being incubated 30min in 50 DEG C, it is diluted with water to 25mL after cooling, measures 520nm place light absorption value.
Beta-glucosidase: accurately draw the enzyme liquid 0.5mL of suitably dilution 10-50 times, add the 0.05mol/L citric acid sodium citrate buffer that 1mL mass fraction is 1% salicin (being dissolved in the 0.05mol/L citric acid sodium citrate buffer of pH4.8) and 0.5mLpH4.8,3mLDNS reagent boiling water bath 10min is added after being incubated 30min in 50 DEG C, it is diluted with water to 25mL after cooling, measures 520nm place light absorption value.
Restriction endonuclease enzyme activity determination: accurately draw the enzyme liquid 0.5mL of suitably dilution 10-50 times, add the 0.05mol/L citric acid sodium citrate buffer that 1mL mass fraction is 1%CMC (being dissolved in the 0.05mol/L citric acid sodium citrate buffer of pH4.8) and 0.5mLpH4.8,3mLDNS reagent boiling water bath 10min is added after being incubated 30min in 50 DEG C, it is diluted with water to 25mL after cooling, measures 520nm place light absorption value.
Excision enzyme enzyme activity determination: really draw the enzyme liquid 0.5mL of suitably dilution 10-50 times, add the 0.05mol/L citric acid sodium citrate buffer that 1mL mass fraction is 2% avicelase (being dissolved in the 0.05mol/L citric acid sodium citrate buffer of pH4.8) and 0.5mLpH4.8,3mLDNS reagent boiling water bath 10min is added after being incubated 30min in 50 DEG C, it is diluted with water to 25mL after cooling, measures 520nm place light absorption value.
Cellulase activity unit definition: under the above-described reaction conditions, the enzyme amount needed for hydrolysis 1 ��m of ol glucose of generation per minute is 1 enzyme iu (IU) alive.
The detailed description that the method for above-mentioned screening technique with reference to embodiment to cellulase producing strain and fermenting and producing cellulase thereof carries out; it is illustrative rather than determinate; can according to restriction scope list several embodiments; therefore without departing from changing and modifications under present general inventive concept, should belong within protection scope of the present invention.
Claims (10)
1. the screening technique of a cellulase producing strain, it is characterised in that described screening technique comprises the following steps: prepared by primary dcreening operation, protoplast, ultraviolet lighting mutagenic and breeding, multiple sieve;
Described cellulase producing strain is now by China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, deposit number is No.11119, and Classification And Nomenclature is that Rhizopus stolonifer is nodded mutation Rhizopusstolonifervar.refiexus.
2. screening technique according to claim 1, it is characterised in that described screening technique specifically includes following steps:
A. primary dcreening operation culture medium primary dcreening operation, the composition of described primary dcreening operation culture medium is: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g;MandelS inorganic nutrient salt 1000mL;
B. preparing protoplast, the spore of the bacterial strain obtained by primary dcreening operation is cultivated in PDA culture medium, the mycelia oscillation incubation in shell-broken liquid obtained after purified, centrifugal, obtains protoplast after filtration, centrifugal purification; The composition of described shell-broken liquid is Snailase: cellulase=3:1;
C. mutagenic and breeding, the protoplast 15W ultra violet lamp obtained by step B carries out mutagenic treatment, and treatment fluid is coated height and oozed on primary dcreening operation culture medium flat plate, and after cultivation, bacterium colony is transferred to the cultivation of PDA slant medium;
Described height oozes the composition of primary dcreening operation culture medium flat plate: every L culture medium contains: CMC-Na5-10g; Glucose 3-5g; Agar 10-20g; MandelS inorganic nutrient salt 1000mL; KCl0.6mol;
D. sieve again; The spore suspension of the culture on PDA slant medium obtained by step C is inoculated in liquid seed culture medium and cultivates, and seed culture fluid is inoculated in liquid fermentation culture medium by 1:10 volume ratio and cultivates, cross the mensuration to tunning enzymolysis reducing sugar yield, filter out the bacterial strain that enzymatic activity is high, be cellulase producing strain TP-02.
3. screening technique according to claim 2, it is characterised in that the composition of described liquid seed culture medium is: 10% wheat bran diffusion juice.
4. screening technique according to claim 2, it is characterised in that the composition of described liquid fermentation culture medium is: every L culture medium contains: the corn straw of 80��120 orders, Caulis et Folium Oryzae or straw 5-10g, 5% wheat bran diffusion juice 80-100g, (NH4)2SO420-30g, KH2PO45-10g, MgSO4��7H2O5-10g, CaCl210-20g, Tween800.15-0.25g, trace element: FeSO4��7H2O0.003-0.005g, MnSO4��H2O0.001-0.00156g, ZnSO4��7H2O0.001-0.0014g, CoCl2��6H2O0.001-0.002g, pH5.0.
5. the bacterial strain that screening technique according to claim 1 screening obtains is as the application producing dimension element enzyme.
6. the production method tieing up element enzyme, it is characterised in that said method comprising the steps of:
(1) use the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained by liquid seed culture medium to carry out seed culture, obtain seed culture fluid;
(2) seed culture fluid is inoculated in liquid fermentation culture medium and cultivates, liquid fermentation cellulase-producing.
7. production method according to claim 6, it is characterized in that, described step (1) specifically includes following steps: the bacterial strain TP-02 that screening technique according to claim 1 screening is obtained cultivates through PDA slant medium, liquid seed culture medium after first, and gained seed liquor is saved in-80 DEG C of ultralow temperature glycerol freezing pipes;
The seed liquor preserved in-80 DEG C of ultralow temperature glycerol freezing pipes be can be prepared by seed culture fluid through PDA slant medium, liquid seed culture medium after cultivating after first.
8. production method according to claim 6, it is characterised in that in described step (2), the ratio of seed culture fluid and the volume of liquid fermentation culture medium is 1:9��10.
9. production method according to claim 6, it is characterised in that the condition of culture in described step (2) is 30 DEG C, 200��350r/min cultivates 84��96 hours.
10. production method according to claim 9, it is characterised in that the condition of culture in described step (2) also includes: in fermentation tank, ventilation is 4.5vvm, tank pressure is 0.03MPa, pH is 5.0��5.5 scopes.
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| CN110129407A (en) * | 2019-05-30 | 2019-08-16 | 红河学院 | The separation of corn stover efficient degradation fungi and identification method |
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| CN107674838A (en) * | 2017-09-06 | 2018-02-09 | 中国农业大学 | Fungi rapid saving method |
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| CN108485984A (en) * | 2018-02-08 | 2018-09-04 | 中国科学院天津工业生物技术研究所 | The high-throughput screening method of cellulase high-yield |
| CN110129407A (en) * | 2019-05-30 | 2019-08-16 | 红河学院 | The separation of corn stover efficient degradation fungi and identification method |
| CN110144298A (en) * | 2019-06-06 | 2019-08-20 | 劲牌有限公司 | A kind of Novel Rhizopus oryzae bacterial strain G1 and its cultural method and application |
| CN111909968A (en) * | 2020-05-18 | 2020-11-10 | 南阳师范学院 | Symbiotic mycelium pellet straw biogas preparation method based on genome rearrangement technology |
| CN112920973A (en) * | 2021-03-22 | 2021-06-08 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
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