CN101463327A - Eupenicillium javanicum strain, and screening culture method and use thereof - Google Patents
Eupenicillium javanicum strain, and screening culture method and use thereof Download PDFInfo
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Abstract
The invention discloses a Eupenicillium javanicum bacterial strain and a screening method thereof. The preserving number of the bacterial strain is CCTCC M208204. The bacterial strain of the Eupenicillium javanicum can be used for producing cellulose, the step of which comprises that: the bacterial strain is inoculated into a seed culture medium and is cultured for 48-84 hours in the temperature of 25-32 DEG C so as to obtain the seed liquid; the seed liquid is inoculated into a fermentation medium according to 3-10% of inoculums concentration; the initial pH value of the fermentation medium is 4.5-6.0; the fermentation medium is cultured for 96-12 hours in the temperature of 25-32 DEG C so as to obtain fermentation liquor; the crude enzyme liquid containing cellulase is obtained by the centrifugation of the fermentation liquor. The cellulose produced by the Eupenicillium javanicum bacterial strain has the advantages of high activity, low cost, etc.
Description
Technical field
The present invention relates to bacterial strain and the screening and culturing methods and applications thereof of a kind of fungi, relate in particular to a kind of bacterial strain and screening and culturing methods and applications thereof of producing high active cellulase.
Background technology
At present, cellulase has been widely used in brewageing, fruit juice and each technical field such as vegetables juice processing, grain processing, food, feed, papermaking, Chinese herbal medicine effective ingredients extraction, plant genetic engineering, cell engineering and Oil extraction, the main application of cellulase is to realize cellulosic decomposition and conversion, the mechanism of cellulase effect mainly is to make the β-1 that connects glucose molecule by hydrolysis, the fracture of 4-glycosidic link, Mierocrystalline cellulose decomposes and changes into the small molecules carbohydrate the most at last.Current, those skilled in the art generally believe that degraded cellulose needs three kinds of function differences but the organized enzyme component of tool complementary action: inscribe β-1,4-dextranase (Cx enzyme), circumscribed β-1,4-dextranase (C at least fully
1Enzyme) and beta-glucosidase.Yet, those skilled in the art still fail to develop a kind of can high cellulase-producing, and make the activity of above-mentioned three kinds of constitutive enzymes reach system enzyme method simultaneously than high target.
Along with the development of biotechnology, utilize the technology of production by biological enzyme to progress into people's the visual field.Current, can be used in the production of cellulose enzyme and the stronger bacterial classification of gained cellulase capacity of decomposition mainly comprises: the bacterial strain of Trichoderma (Trichoderma), the bacterial strain of Aspergillus (Aspergillus), the bacterial strain of the bacterial strain of Penicillium (Penicillium) and the branch mould genus of top spore (Acremonium), but the cellulase activity of the bacterial strain production of these bacterial classifications preparation is high not enough, even the activity of a certain constitutive enzyme can reach effect preferably in the cellulase that above-mentioned bacterial strains makes, but also can form restriction to the overall activity of cellulase because the activity of other constitutive enzymes is low.Therefore, to those skilled in the art, cultivating and filtering out a kind of high enzyme work (is Cx enzyme, C
1The activity of enzyme and three kinds of constitutive enzymes of beta-glucosidase reaches higher index simultaneously) cellulose-decomposing bacterium, to cellulase from now in the widespread use of each technical field with significant.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide a kind of Eupenicillium javanicum strain with and the application on preparation production of cellulose enzyme of screening and culturing method and this bacterial strain, by optimizing the fermentation process of this bacterial strain production of cellulose enzyme, to prepare the cellulase that activity is high, cost is low.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of Eupenicillium javanicum strain, the preservation name that it is characterized in that described Eupenicillium javanicum strain is called Java penicillium (Eupenicillium javanicum) ZN-205, depositary institution is Chinese typical culture collection center, and preserving number is CCTCC M208204.
The bacterium colony of above-mentioned Eupenicillium javanicum strain is dark green, and the bacterium colony back side is tenne, no transudate; The mycelia quality is carpet-like, numerous and diverse, transparent at the visible mycelia branch of microscopically, have every, cell walls is smooth; The conidiophore clump Shu Shusong of described bacterial strain, bunch shape is arranged, and is uprightly born by mycelia, and is colourless; Main branch is tree-shaped, and level branch is many times arranged on it, and to giving birth to or 2～3 grades of branches of alternate, end is a stigma, the stigma doleiform, and the bottle stalk is short, and base portion attenuates, and expand the centre, stretches out with wide-angle; Spherical in shape or the oblong of conidium is gathered into the conidial head of spherical green, and the conidium wall is smooth, and spore is little.The sporophore form of above-mentioned Java penicillium CCTCC M208204 as shown in Figure 1; Colonial morphology after cultivating on potato dextrose agar (PDA) substratum as shown in Figure 2; Through molecular biology identification, its 18S rDNA sequence total length is 1687bp.
The present invention also provides a kind of screening and culturing method of above-mentioned Eupenicillium javanicum strain, specifically may further comprise the steps:
(1) primary dcreening operation: the bacterial strain of collecting is put into enrichment medium, shaking culture 3～5d under 28 ℃, the condition of 150r/min, dilution is coated on Xylo-Mucine (CMC-Na) substratum then, and picking list bacterium colony diluted coating once more after 4d was cultivated in 30 ℃ of inversions, up to obtaining pure bacterial strain; The Congo red solution-dyed with 1mg/mL is cultivated behind the 4d in the pure bacterial strain dibbling that separation is obtained to the CMC-Na substratum, clean with distilled water again, with the NaCl solution decolouring of 1mol/L, compare the ratio of each colony diameter, transparent circle diameter and colony diameter and transparent circle diameter then at last;
(2) final election: each bacterial strain behind the primary dcreening operation is inoculated into liquid by 5% inoculum size (inoculum size is meant that the inoculating strain volume accounts for the percent by volume for the treatment of fermention medium) produces in the enzyme substratum shaking culture 5d under 30 ℃, 150r/min, then that nutrient solution is centrifugal under the condition of 5000r/min, get its supernatant liquor, carboxymethylcelluloenzyme enzyme work, filter paper enzyme activity and the beta-glucosidase of mensuration and each bacterium colony of comparison are lived;
(3) filter out the bacterial strain that satisfies following two conditions simultaneously:
(a) ratio of the colony diameter of measuring behind the described primary dcreening operation, transparent circle diameter and colony diameter and transparent circle diameter all shows maximum;
(b) carboxymethylcelluloenzyme enzyme work, filter paper enzyme activity and beta-glucosidase all performance maximums alive of measuring after the described final election.
The present invention also provides the application of a kind of above-mentioned Eupenicillium javanicum strain on the production of cellulose enzyme.
Described Eupenicillium javanicum strain is by following steps cellulase-producing in next life:
Described bacterial strain is inserted in the seed culture medium, under 25～32 ℃ of conditions, cultivate and got seed liquor in 48～84 hours; By in 3～10% the inoculum size access fermention medium, the initial pH value of fermention medium is 4.5～6.0, cultivates to get fermented liquid in 96～120 hours under 25～32 ℃ of conditions with seed liquor; Fermented liquid is through the centrifugal crude enzyme liquid that obtains cellulase.
The above-mentioned crude enzyme liquid that makes has following zymologic property: the suitableeest action pH value of the carboxymethylcelluloenzyme enzyme of cellulase (the CMC enzyme is lived) alive is 5, between pH value 4～5, the stability of enzyme better, less than 4 with greater than 5 zone, the stability of enzyme is sharply decline all in pH value; Optimum temperature is 60 ℃.The suitableeest action pH value of the filter paper enzyme activity of cellulase (FPA enzyme live) is 5, between pH value 5～7, enzyme stable relatively good, and when the pH value greater than 7 with less than 5 the time, sharply decline of enzyme work; Optimum temperature is 50 ℃.
Above-mentioned seed culture medium is made up of the component of following massfraction: CMC-Na1～2%, MgSO
47H
2O 0.02～0.1%, KH
2PO
40.05 NH～0.5%,
4NO
30.05～0.2% and the water of surplus.Above-mentioned enrichment medium and liquid produce the enzyme substratum and are common substratum, and its component also can be identical with the component of seed culture medium.
Above-mentioned fermention medium is made up of the component of following massfraction: straw powder 0.5～1.5%, bran powder 0.1～0.5%, KNO
30.5 KH～2%,
2PO
40.05 CaCl～0.5%,
22H
2O 0.01～0.1%, MgSO
47H
2The water of O 0.02～0.1%, tween 0.1～0.8% and surplus.
Rotating speed all is controlled at 160～200r/min when cultivating when cultivating in the above-mentioned seed culture medium, in the fermention medium; Rotating speed was controlled at 1000～8000r/min when fermented liquid was centrifugal.
Compared with prior art, the invention has the advantages that: filter out a kind of high enzyme cellulose-decomposing bacterium---Java penicillium CCTCC M208204 alive by research, the present invention by optimizing this bacterial strain production of cellulose enzyme fermentation process and create good condition of enzyme production, can prepare active high, cellulase that cost is low.The method that the present invention screens Java penicillium CCTCCM208204 is simple relatively, easily operation; Utilize Java penicillium CCTCC M208204 of the present invention to produce the cellulase of preparation, cellulosic material can be converted into small molecular sugar.Therefore, application of the present invention may become the new source of opening up feed, zymotechnique raw material and human food prods, and all significant to processing of waste, elimination public hazards, protection environment etc.
Eupenicillium javanicum strain of the present invention is delivered Chinese typical culture collection center (CCTCC) preservation on November 6th, 2008, and deposit number is CCTCC M208204.
Description of drawings
Fig. 1 is the sporophore aspect graph of Eupenicillium javanicum strain of the present invention;
Fig. 2 is the colonial morphology figure of Eupenicillium javanicum strain of the present invention after cultivating on the PDA substratum;
Fig. 3 is the photo of Eupenicillium javanicum strain of the present invention by Congo red plate isolation screening.
Embodiment
Embodiment 1:
From rot trees and the soil screening and isolate a plant height vigor cellulase production bacterium ZN-205 of Yue Lu mountain, Changsha forest, concrete grammar is as follows:
(1) primary election: the bacterial strain sample of collecting is put into the triangular flask that contains the 50mL enrichment medium, shaking culture 4d under 28 ℃, the condition of 150r/min, dilution is coated on the CMC-Na substratum then, and picking list bacterium colony diluted coating once more after 4d was cultivated in 30 ℃ of inversions, till obtaining pure bacterium colony.The Congo red solution-dyed 30min with 1mg/mL is cultivated behind the 4d in the pure bacterial strain dibbling that separation is obtained to the CMC-Na substratum, clean with distilled water again, at last with the NaCl solution of 1mol/L decolouring 30min, with the ratio size of the diameter of transparent circle and colony diameter as the standard of screening.By Congo red plate isolation screening (see figure 3), obtain bigger 8 bacterium colonies (seeing Table 1) of transparent circle diameter and colony diameter ratio, the result shows, relative other bacterial strains, bacterial strain ZN-205 growing state is best, and its colony diameter, transparent circle diameter and transparent circle diameter and colony diameter ratio also are maximum.Further with the inoculation that screens in the filter paper liquid nutrient medium, under 30 ℃, the condition of 150r/min, cultivate 5d, with the filter paper liquid nutrient medium of not inoculating any bacterial strain as blank, the triplicate experiment, with the rate of weight loss (X) of filter paper standard, X=(M-M as current screening
1(wherein M is initial filter paper quality to)/M, M
1Quality after the filter paper drying of back is to degrade), the results are shown in Table 2, other seven strains bacterium relatively, the rate of weight loss maximum behind the bacterial strain ZN-205 degraded filter paper illustrates that its enzyme lives the highest.
The Congo red plate isolation The selection result of table 1
| The screening bacterial strain | Colony diameter D/cm | Transparent circle diameter d/cm | d/D |
| ZN-1 | 0.61 | 1.6 | 2.63 |
| ZN-39 | 0.8 | 1.62 | 2.1 |
| ZN-72 | 0.6 | 1.07 | 1.79 |
| ZN-205 | 1.1 | 2.97 | 2.7 |
| ZN-85 | 0.4 | 0.97 | 2.42 |
| ZN-201 | 1.0 | 2.5 | 2.5 |
| ZN-73 | 0.5 | 0.89 | 1.78 |
| ZN-207 | 0.7 | 1.54 | 2.2 |
Table 2 filter paper rate of weight loss result
| Bacterial strain | ZN-1 | ZN-39 | ZN-72 | ZN-205 | ZN-85 | ZN-201 | ZN-73 | ZN-207 |
| Rate of weight loss | 34.04％ | 27.6％ | 25.6％ | 35.4％ | 27.1％ | 24.3％ | 30.7％ | 26.6％ |
(2) final election: the bacterial strain that primary dcreening operation is obtained is inoculated into liquid by 5% inoculum size and produces in the enzyme substratum shaking culture 5d under 30 ℃, 150r/min, then with nutrient solution centrifugal 10min under the condition of 5000r/min, get its supernatant liquor and measure each enzyme size alive, with Trichodermareesei (Trichoderma reesei) RUT-C30 is the contrast strain, and each bacterial strain crude enzyme liquid enzyme work sees Table 3.The result shows that each enzyme of ZN-205 is lived all better.
Each bacterial strain crude enzyme liquid enzyme of table 3 IU/ml alive
| Bacterial strain | ZN-1 | ZN-39 | ZN-72 | ZN-205 | ZN-85 | ZN-201 | ZN-73 | ZN-207 | Trichodermareesei RUT-C30 |
| The CMC enzyme is lived | 3.71 | 1.85 | 1.74 | 5.37 | 2.27 | 2.6 | 1.87 | 2.29 | 10.4 |
| The FPA enzyme is lived | 1.59 | 1.04 | 0.65 | 2.11 | 1.71 | 0.84 | 0.71 | 1.71 | 1.91 |
| Beta-glucosidase | 1.05 | 0.99 | 0.59 | 1.06 | 0.08 | 0.98 | 1.02 | 0.29 | 0.33 |
Above-mentioned enrichment medium is identical with the component that liquid produces the enzyme substratum, and the massfraction of each component is: CMC-Na1.5%, MgSO
47H
2O 0.05%, KH
2PO
40.1%, NH
4NO
30.1% and the water of surplus.
Bacterial strain ZN-205 is the Java penicillium through form and Molecular Identification (qualification result is as follows), this strain name is Java penicillium (Eupenicillium javanicum) ZN-205, depositary institution is Chinese typical culture collection center, and preserving number is CCTCC M208204.
Identification of morphology: the inoculation that separation is obtained goes up the feature of cultivating observation bacterium colony after 3 days to potato dextrose agar (PDA) flat board, on flat board, choose thalline a little, be applied on the slide glass and place the microscopically observation.It is dark green that bacterium colony is, and the bacterium colony back side is tenne, no transudate; The mycelia quality is carpet-like, numerous and diverse, transparent at the visible mycelia branch of microscopically, have every, cell walls is smooth; Bacterial strain conidiophore clump Shu Shusong, bunch shape is arranged, and is uprightly born by mycelia, and is colourless; Main branch is tree-shaped, and level branch is many times arranged on it, and to giving birth to or 2～3 grades of branches of alternate, end is a stigma, the stigma doleiform, and the bottle stalk is short, and base portion attenuates, and expand the centre, stretches out with wide-angle; Spherical in shape or the oblong of conidium is gathered into the conidial head of spherical green, and the conidium wall is smooth, and spore is little.
Molecular Identification: the inoculation that separation obtains was cultivated 3 days to potato dextrose agar (PDA) flat board, get mycelium extracting DNA, with 18S rDNA gene universal primer (upstream primer 5 ＇-ATTGGAGGGCAAGTCTGGTG-3 ＇ that increase, downstream primer 5 ＇-CCGATCCCTAGTCGGCATAG-3 ＇), the amplification segment is by the order-checking of Shanghai bio-engineering corporation, and it is as follows to obtain 18S rDNA gene complete sequence:
acttcctcta atgaccgagt ttgaccaact ttccggctct ggggggtcgt tgccaaccct
cctgagccag tccgaaggcc tcactgagcc attcaatcgg tagtagcgac gggcggtgtg
tacaaagggc agggacgtaa tcggcacgag ctgatgactc gtgcctacta ggcattcctc
gttgaagagc aataattgca atgctctatc cccagcacga cagggtttaa caagattacc
cagacctctc ggccaaggtg atgtactcgc tggccctgtc agtgtagcgc gcgtgcggcc
cagaacatct aagggcatca cagacctgtt attgccgcgc acttccatcg gcttgagccg
atagtccccc taagaagcca gcggcccgca aatgcggacc gggctattta agggccgagg
tctcgttcgt tatcgcaatt aagcagacaa atcactccac caactaagaa cggccatgca
ccaccatcca aaagatcaag aaagagctct caatctgtca atccttattt tgtctggacc
tggtgagttt ccccgtgttg agtcaaatta agccgcaggc tccacgcctt gtggtgccct
tccgtcaatt tctttaagtt tcagccttgc gaccatactc cccccagaac ccaaaaactt
tgatttctcg taaggtgccg aacgggtcat catagaatcc cgtccgatcc ctagtcggca
tagtttatgg ttaagactac gacggtatct gatcgtcttc gatcccctaa ctttcgttcc
ctgattaatg aaaacatcct tggcgaatgc tttcgcagta gttagtcttc agcaaatcca
agaatttcac ctctgacagc tgaatactga cgcccccgac tatccctatt aatcattacg
gcggtcctag aaaccaacaa aatagaaccg cacgtcctat tctattattc catgctaatg
tattcgagca aaggcctgct ttgaacactc taattttttc acagtaaaag tcctggttcc
ccccacagcc agtgaaggcc atgaggttcc ccagaaggaa aggtccagcc ggaccagtac
tcgcggtgag gcggaccggc cagccagacc caaggttcaa ctacgagctt tttaactgca
acaactttaa tatacgctat tggagctgga attaccgcgg ctgctggcac cagacttgcc
ctccaattgt tcctcgttaa gggatttaaa ttgttctcat tccaattacg agacccaaaa
gagccccgta tcagtattta ttgtcactac ctccccgtat cgggattggg taatttgcgc
gcctgctgcc ttccttggat gtggtagccg tttctcaggc tccctctccg gaatcgaacc
ctaattcccc gttacccgtt gccaccatgg taggccacta tcctaccatc gaaagttgat
agggcagaaa tttgaatgaa ccatcgccgg cgcaaggcca tgcgattcgt taagttatta
tgattcacca aggagccccg aagggcgttg gttttttatc taataaatac accccttcct
gaagtcgggg tttttagcat gtattagctc tagaattacc acaggtatcc atgtagtaag
gtactatcaa ataaacgata actgatttaa tgagccattc gcagtttcac agtataaagt
gcttata
Above-mentioned sequence total length 1687bp carries out the Blast analysis through landing GeneBank, and the 18S rDNA gene order of bacterial strain ZN-205 and Java penicillium 18S rDNA gene order homology are up to 99%.
Embodiment 2:
A kind of Eupenicillium javanicum strain of the present invention, it is by following steps cellulase-producing in next life:
The bacterial strain that screens, turn out is inserted in the seed culture medium, under 30 ℃, the condition of 175r/min, cultivated 72 hours seed liquor; Seed liquor is inserted in the fermention medium (adorning the fermention medium of 20～50% volumes in fermentation flask) by 5% inoculum size, and the initial pH value of fermention medium is 5.5, under 30 ℃, the condition of 175r/min, cultivated 120 hours fermented liquid; Fermented liquid after the fermentation is through the centrifugal crude enzyme liquid that obtains cellulase of 4000r/min.After measured, the CMC enzyme of this bacterial strain crude enzyme liquid is lived and is 24.52IU/ml, the FPA enzyme is lived to 2.68IU/ml, beta-glucosidase are 2.03IU/ml, compares (seeing Table 4) with existing other strain enzyme-producings, and its CMC enzyme of the cellulase that bacterial strain of the present invention produced is lived the highest; Though the FPA enzyme is lived and the work of beta-glucosidase enzyme is not the highest, three kinds of enzyme globality and harmonies alive behave oneself best.
Table 4 Java penicillium ZN-205 and other strain enzyme-producings are relatively
Annotate: mensuration was not made in "--------" expression
The component of above-mentioned seed culture medium and the massfraction of each component are: Xylo-Mucine (CMC-Na) 1.5%, MgSO
47H
2O 0.05%, KH
2PO
40.1%, NH
4NO
30.1% and the water of surplus.
The component of above-mentioned fermention medium and the massfraction of each component are: straw powder 0.7%, bran powder 0.3% (straw powder and wheat bran ratio are 7:3), KNO
31% (nitrogenous source interpolation concentration), KH
2PO
40.4%, CaCl
22H
2O 0.03%, MgSO
47H
2The water of O0.03%, tween 0.4% and surplus.
The substratum that the inventive method is used with straw and wheat bran (0.5 yuan/kg) do carbon source, with KNO
3(5 yuan/kg) make nitrogenous source, compare usually with Microcrystalline Cellulose (5 yuan/kg) be carbon source, peptone (50 yuan/kg) be nitrogenous source, have advantages such as price is low, the source is wide, the cost of technical solution of the present invention is reduced, operation is more simple and easy to do.
Be to adopt the plain enzyme activity determination method of the middle specific fibre of China's light industry standard (QB2583-2003) to carry out enzyme activity determination in the foregoing description.Enzyme unit alive adopts international define method, and producing the required enzyme amount of 1 micromolar glucose with 1mL enzyme liquid per minute is an enzyme activity unit IU.
＜110〉Sino-South African Forestry University of Science and Technology
＜120〉Eupenicillium javanicum strain and screening and culturing methods and applications thereof
<160>1687
<210>1
<211>1687bp
<212>DNA
＜213〉Java penicillium (Eupenicillium javanicum)
<220>
<221>RRNA
<222>(1)...(1687)
<400>
acttcctcta atgaccgagt ttgaccaact ttccggctct ggggggtcgt tgccaaccct 60
cctgagccag tccgaaggcc tcactgagcc attcaatcgg tagtagcgac gggcggtgtg 120
tacaaagggc agggacgtaa tcggcacgag ctgatgactc gtgcctacta ggcattcctc 180
gttgaagagc aataattgca atgctctatc cccagcacga cagggtttaa caagattacc 240
cagacctctc ggccaaggtg atgtactcgc tggccctgtc agtgtagcgc gcgtgcggcc 300
cagaacatct aagggcatca cagacctgtt attgccgcgc acttccatcg gcttgagccg 360
atagtccccc taagaagcca gcggcccgca aatgcggacc gggctattta agggccgagg 420
tctcgttcgt tatcgcaatt aagcagacaa atcactccac caactaagaa cggccatgca 480
ccaccatcca aaagatcaag aaagagctct caatctgtca atccttattt tgtctggacc 540
tggtgagttt ccccgtgttg agtcaaatta agccgcaggc tccacgcctt gtggtgccct 600
tccgtcaatt tctttaagtt tcagccttgc gaccatactc cccccagaac ccaaaaactt 660
tgatttctcg taaggtgccg aacgggtcat catagaatcc cgtccgatcc ctagtcggca 720
tagtttatgg ttaagactac gacggtatct gatcgtcttc gatcccctaa ctttcgttcc 780
ctgattaatg aaaacatcct tggcgaatgc tttcgcagta gttagtcttc agcaaatcca 840
agaatttcac ctctgacagc tgaatactga cgcccccgac tatccctatt aatcattacg 900
gcggtcctag aaaccaacaa aatagaaccg cacgtcctat tctattattc catgctaatg 960
tattcgagca aaggcctgct ttgaacactc taattttttc acagtaaaag tcctggttcc 1020
ccccacagcc agtgaaggcc atgaggttcc ccagaaggaa aggtccagcc ggaccagtac 1080
tcgcggtgag gcggaccggc cagccagacc caaggttcaa ctacgagctt tttaactgca 1140
acaactttaa tatacgctat tggagctgga attaccgcgg ctgctggcac cagacttgcc 1200
ctccaattgt tcctcgttaa gggatttaaa ttgttctcat tccaattacg agacccaaaa 1260
gagccccgta tcagtattta ttgtcactac ctccccgtat cgggattggg taatttgcgc 1320
gcctgctgcc ttccttggat gtggtagccg tttctcaggc tccctctccg gaatcgaacc 1380
ctaattcccc gttacccgtt gccaccatgg taggccacta tcctaccatc gaaagttgat 1440
agggcagaaa tttgaatgaa ccatcgccgg cgcaaggcca tgcgattcgt taagttatta 1500
tgattcacca aggagccccg aagggcgttg gttttttatc taataaatac accccttcct 1560
gaagtcgggg tttttagcat gtattagctc tagaattacc acaggtatcc atgtagtaag 1620
gtactatcaa ataaacgata actgatttaa tgagccattc gcagtttcac agtataaagt 1680
gcttata 1687
Claims (7)
1, a kind of Eupenicillium javanicum strain, the preservation name that it is characterized in that described Eupenicillium javanicum strain is called Java penicillium (Eupenicillium javanicum) ZN-205, depositary institution is Chinese typical culture collection center, and preserving number is CCTCCM208204.
2, Eupenicillium javanicum strain according to claim 1 is characterized in that: the bacterium colony of described Eupenicillium javanicum strain is dark green, and the bacterium colony back side is tenne, no transudate; The mycelia quality is carpet-like, microscopically sees that the mycelia branch is numerous and diverse, transparent, have every, cell walls is smooth; The conidiophore clump Shu Shusong of described bacterial strain, bunch shape is arranged, and is uprightly born by mycelia, and is colourless; Main branch is tree-shaped, and secondary branch is arranged on it, and end is a stigma, the stigma doleiform, and the bottle stalk is short, and base portion attenuates, and expand the centre; Conidium is spherical in shape or oval, is gathered into the conidial head of spherical green, and the conidium wall is smooth.
3, a kind of screening and culturing method of Eupenicillium javanicum strain as claimed in claim 1 or 2 specifically may further comprise the steps:
(1) primary dcreening operation: the bacterial strain of collecting is put into enrichment medium, shaking culture 3～5d under 28 ℃, the condition of 150r/min, dilution is coated on the Xylo-Mucine substratum then, and picking list bacterium colony diluted coating once more after 4d was cultivated in 30 ℃ of inversions, up to obtaining pure bacterial strain; The Congo red solution-dyed with 1mg/mL is cultivated behind the 4d in the pure bacterial strain dibbling that separation is obtained to the Xylo-Mucine substratum, clean with distilled water again, with the NaCl solution decolouring of 1mol/L, compare the ratio of each colony diameter, transparent circle diameter and colony diameter and transparent circle diameter then at last;
(2) final election: each bacterial strain behind the primary dcreening operation is inoculated into liquid by 5% inoculum size produces in the enzyme substratum shaking culture 5d under 30 ℃, 150r/min, then that nutrient solution is centrifugal under the condition of 5000r/min, get its supernatant liquor, carboxymethylcelluloenzyme enzyme work, filter paper enzyme activity and the beta-glucosidase of mensuration and each bacterium colony of comparison are lived;
(3) filter out the bacterial strain that satisfies following two conditions simultaneously:
(a) ratio of the colony diameter of measuring behind the described primary dcreening operation, transparent circle diameter and colony diameter and transparent circle diameter all shows maximum;
(b) carboxymethylcelluloenzyme enzyme work, filter paper enzyme activity and beta-glucosidase all performance maximums alive of measuring after the described final election.
4, the application of a kind of Eupenicillium javanicum strain as claimed in claim 1 or 2 on the production of cellulose enzyme.
5, application according to claim 4 is characterized in that described Eupenicillium javanicum strain is by following steps cellulase-producing in next life:
Described bacterial strain is inserted in the seed culture medium, under 25～32 ℃ of conditions, cultivate and got seed liquor in 48～84 hours; By in 3～10% the inoculum size access fermention medium, the initial pH value of fermention medium is 4.5～6.0, cultivates to get fermented liquid in 96～120 hours under 25～32 ℃ of conditions with seed liquor; Fermented liquid is through the centrifugal crude enzyme liquid that obtains cellulase.
6, application according to claim 5 is characterized in that:
Described seed culture medium is made up of the component of following massfraction: Xylo-Mucine 1～2%, MgSO
47H
2O0.02～0.1%, KH
2PO
40.05 NH～0.5%,
4NO
30.05～0.2% and the water of surplus;
Described fermention medium is made up of the component of following massfraction: straw powder 0.5～1.5%, bran powder 0.1～0.5%, KNO
30.5 KH～2%,
2PO
40.05 CaCl～0.5%,
22H
2O0.01～0.1%, MgSO
47H
2The water of O0.02～0.1%, tween 0.1～0.8% and surplus.
7, application according to claim 5 is characterized in that: when described seed culture medium is cultivated and fermention medium when cultivating, rotating speed is controlled at 160～200r/min; When fermented liquid was centrifugal, rotating speed was controlled at 1000～8000r/min.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101921723A (en) * | 2010-08-25 | 2010-12-22 | 广东省微生物研究所 | Chryseobacterium rhizosphaerae and application thereof |
| CN102115772A (en) * | 2010-12-16 | 2011-07-06 | 浙江大学 | Method for synthesizing 8-isopentene group naringenin by catalyzing isoxanthohumol with microbial cells |
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| CN102533563A (en) * | 2011-11-08 | 2012-07-04 | 中国科学院微生物研究所 | Celluase producing bacterium and application thereof |
| CN102586135A (en) * | 2011-12-30 | 2012-07-18 | 南京理工大学 | Bacteria and breeding method and method for producing cellulase thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101921723A (en) * | 2010-08-25 | 2010-12-22 | 广东省微生物研究所 | Chryseobacterium rhizosphaerae and application thereof |
| CN102115772A (en) * | 2010-12-16 | 2011-07-06 | 浙江大学 | Method for synthesizing 8-isopentene group naringenin by catalyzing isoxanthohumol with microbial cells |
| CN102329737A (en) * | 2011-08-30 | 2012-01-25 | 东华大学 | Eupenicillium javanicum DB4 strain as well as preparation and application thereof |
| CN102329737B (en) * | 2011-08-30 | 2013-01-16 | 东华大学 | Eupenicillium javanicum DB4 strain as well as preparation and application thereof |
| CN102533563A (en) * | 2011-11-08 | 2012-07-04 | 中国科学院微生物研究所 | Celluase producing bacterium and application thereof |
| CN102533563B (en) * | 2011-11-08 | 2013-04-10 | 中国科学院微生物研究所 | Celluase producing bacterium and application thereof |
| CN102586135A (en) * | 2011-12-30 | 2012-07-18 | 南京理工大学 | Bacteria and breeding method and method for producing cellulase thereof |
| CN102787104A (en) * | 2012-07-20 | 2012-11-21 | 中南林业科技大学 | High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber |
| CN115160035A (en) * | 2022-06-26 | 2022-10-11 | 江西科技师范大学 | Preparation method of microbial fertilizer |
| CN115160035B (en) * | 2022-06-26 | 2024-05-14 | 江西科技师范大学 | Preparation method of microbial fertilizer |
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