CN102061270B - Streptomyces thermocarboxydus strain and cultivating method thereof - Google Patents
Streptomyces thermocarboxydus strain and cultivating method thereof Download PDFInfo
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- CN102061270B CN102061270B CN2010101582092A CN201010158209A CN102061270B CN 102061270 B CN102061270 B CN 102061270B CN 2010101582092 A CN2010101582092 A CN 2010101582092A CN 201010158209 A CN201010158209 A CN 201010158209A CN 102061270 B CN102061270 B CN 102061270B
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- monoxide streptomycete
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- 238000000034 method Methods 0.000 title claims abstract description 10
- 241000607101 Streptomyces thermocarboxydus Species 0.000 title claims abstract description 5
- 239000007787 solid Substances 0.000 claims abstract description 8
- 241001655322 Streptomycetales Species 0.000 claims description 24
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 claims description 22
- 229910002091 carbon monoxide Inorganic materials 0.000 claims description 22
- 230000001580 bacterial effect Effects 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 230000006872 improvement Effects 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 abstract description 12
- 239000001913 cellulose Substances 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 229940088598 enzyme Drugs 0.000 abstract description 10
- 239000007788 liquid Substances 0.000 abstract description 8
- 108010059892 Cellulase Proteins 0.000 abstract description 7
- 229940106157 cellulase Drugs 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 3
- 235000010980 cellulose Nutrition 0.000 description 11
- 108010085318 carboxymethylcellulase Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
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- 239000000835 fiber Substances 0.000 description 2
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- 239000002609 medium Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 238000005297 material degradation process Methods 0.000 description 1
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- 229920001542 oligosaccharide Polymers 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
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Abstract
The invention discloses a streptomyces thermocarboxydus strain and a cultivating method thereof. The strain provided by the invention is named as the streptomyces thermocarboxydus Str430 and collected in China Center for Type Culture Collection on January 26, 2010, and the collection number is CCTCC NO: M2010026. The strain provided by the invention is cultivated through two modes comprising liquid cultivation and solid cultivation, and the obtained strain can generate cellulase, can hydrolyze cellulose, and has the advantages of relatively strong enzyme generation capability, relatively short production cycle and wide application prospects in industrial production.
Description
Technical field
The invention belongs to microbial technology field, mainly is a kind of cellulase that produces, and can make a kind of actinomycetes strain of cellulose hydrolysis, the invention still further relates to the cultural method of this bacterial strain.
Background technology
Mierocrystalline cellulose is the intravital structural polysaccharide of plant, and the branches and leaves of plant, stalk etc. all contain a large amount of Mierocrystalline celluloses.1,500 hundred million tons of vegetalitas organism of annual in the world generation; Wherein making an appointment with half the is cellulosic material; Natural cellulose is degraded to available sugar; Further be converted into fuel alcohol and food such as combustion gas equal energy source material or tropina again, not only can alleviate grain and energy dilemma that the world today faces, also can reduce the environmental pollution that cellulose wastes causes.Owing to the microorganism growth cycle is short, growth is fast, and therefore, the cellulosic purpose bacterial strain of screening ability efficient degradation is most important.Be sugar how with the cellulosic material Degradation and Transformation, ethanol, the small organic molecule that methane etc. utilize easily is one of current international key subjects.At present, being used to produce commercial bacterial strain with enzyme mainly is some fungies of aspergillus and Trichoderma, and is main with solid fermentation, and the production cycle is long, and extraction is difficult.Up to the present, do not see the actinomycetic bacterial strain report that is useful on the industrial production cellulase as yet.
Summary of the invention
The object of the invention provides a kind of production of cellulose enzyme that can be used for for the deficiency that overcomes above-mentioned prior art just, and can promote cellulose degradation to be converted into the thermophilic carbon monoxide streptomycete of actinomycetes strain of monose effectively.
Another object of the present invention provides the cultural method of this thermophilic carbon monoxide streptomycete.
A kind of streptomycete bacterial strain; Called after is thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) Str430; This bacterial classification has been deposited in Chinese typical culture collection center, preservation day: on 01 26th, 2010, and preserving number:: CCTCC M 2010026.The application attaches the sample detection report (No:209043) that this unit provides.
Str430 bacterium colony circle of the present invention, surperficial fine hair shape, grey, base portion brown of light color, aerial hyphae is luxuriant and curl, has the two-wheeled branch, produces ellipsoidal conidium.
Str430 strain culturing mode of the present invention is:
(1) liquid culture: in fermention medium, under 30 ℃ of temperature, cultivated inoculation on the shaking table of rotating speed 100r 32～36 hours.
(2) solid culture: in the Gause I substratum, in 30 ℃ of incubators, cultivate inoculation 3～4 days.
Can the eccrine fiber plain enzyme of Str430 of the present invention; Its mechanism of action is to cause cellulosic dispersion and take off fibrillation, cellulosic crystalline texture is upset caused distortion, and cellulase can be goed deep between the cellulosic molecule interface; Thereby the pressure of Mierocrystalline cellulose hole wall, chamber wall and microfissure wall is increased; The intervention of water molecules makes the hydrogen bond between the cellulosic molecule be destroyed again, produces the micro-crystallization of the fiber of part solubility, is beneficial to further degraded and forms cell-oligosaccharide and glucose.
Description of drawings
Fig. 1 is the microgram of thermophilic carbon monoxide streptomycete Str430 aerial hyphae (1000 *) at opticmicroscope.
Fig. 2 is the microgram of thermophilic carbon monoxide streptomycete Str430 fibrillae of spores (2000 *) at opticmicroscope.
Fig. 3 is the Electronic Speculum figure of thermophilic carbon monoxide streptomycete Str430 mycelia.
Fig. 4 is the Electronic Speculum figure of thermophilic carbon monoxide streptomycete Str430 spore.
Describe the present invention below in detail.
Embodiment
From the mashed straw topsoil of Hunan Province's Huaihua City, be separated to thermophilic carbon monoxide streptomycete Str430, it separates and screening process is:
1. collection soil sample;
2. the cellulose solids flat board carries out scalping;
3. the triangular flask liquid fermenting carries out multiple sieve;
4. obtain superior strain Str430;
5. the Physiology and biochemistry of bacterial strain Str430 and molecular level are identified, confirm to obtain bacterial strain Str430: thermophilic carbon monoxide streptomycete.
The cultivation of its bacterial strain is adopted:
(1), liquid culture: be inoculated in fermention medium (starch 10g, Semen Maydis powder 10g, glucose 5g, yeast extract paste 10g, (NH
4)
2SO
45g, KH
2PO
45g, K
2HPO
45g, CaCO
31g, MgSO
40.5g, CMC-Na 5g, pH7.5, add water 1000ml sterilization) in, under 30 ℃ of temperature, cultivated on the shaking table of rotating speed 100r 32～36 hours.The preparation method of cellulase solution is the bacterium liquid 4000r/min with liquid culture; Under 4 ℃ of conditions, frozen centrifugation 15 minutes is also got its supernatant, promptly gets crude enzyme liquid; Measure reducing sugar content in the crude enzyme liquid through the DNS method, draw the vigor of CMCase and FPase enzyme indirectly.
(2), solid culture: 1. be inoculated in Gause I substratum (Zulkovsky starch 20g, KNO
31g, NaCl 0.5g, K
2HPO
43H
2O 0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g, agar 20g, pH7.2～7.4 add water 1000ml sterilization) in, in 30 ℃ of incubators, cultivated 3～4 days.2. the bacterium colony dibbling is to solid plate (improvement Cha Shi substratum: CMC-Na 15g, NaNO
32g, NaCl 2g, MgSO
40.5g, FeSO
40.1g, Congo red 0.2g, agar 20g, zero(ppm) water 1000mL, pH nature) on, cultivated 3～4 days in 30 ℃ of incubators.
Str430 bacterium colony circle, surperficial fine hair shape, grey, the base portion brown of light color that cultivation obtains, aerial hyphae is luxuriant and curl, has the two-wheeled branch, produces conidium and for oval.
Understand the relevant nature of thermophilic carbon monoxide streptomycete Str430 below through experiment.
1. cellulose hydrolysis experiment: degraded cellulose is thermophilic, and carbon monoxide streptomycete Str430 separates from the laboratory by the contriver obtains; And with the bacterium colony dibbling to solid improvement Cha Shi culture medium flat plate; Cultivated 3～4 days in 30 ℃ of incubators; In periphery of bacterial colonies tangible hydrolysis circle is arranged, diameter is 10mm.
2. cellulase activity experiment: with thermophilic carbon monoxide streptomycete Str430; Be inoculated in the shake flask fermentation substratum, under 30 ℃ of temperature, cultivated on the shaking table of rotating speed 100r 32～36 hours; Bacterium liquid 4000r/min with liquid culture; Under 4 ℃ of conditions, frozen centrifugation 15 minutes is also got its supernatant, promptly gets crude enzyme liquid.Measure reducing sugar content in the crude enzyme liquid through the DNS method, draw the vigor of CMCase and FPase enzyme indirectly, the CMCase vigor is that its ph optimum of 19.87IU/mL is 5.0; Be 888.89IU/mL to the maximum and PFase is alive, pairing pH is 7.0.
Experiment showed, that this thermophilic carbon monoxide streptomycete bacterial strain Str430 can produce cellulase, hydrocellulose, its CMCase vigor are that its ph optimum of 19.87IU/mL is 5.0; And the PFase vigor is the pairing pH of 888.89IU/mL to the maximum is 7.0.Thermophilic carbon monoxide streptomycete bacterial strain Str430 enzymatic productivity is stronger, and the production cycle is shorter, has wide practical use in industrial production to aforementioned.
Appendix: Chinese typical culture collection center sample detection report (No:209043)
Claims (4)
1. streptomycete bacterial strain, called after is thermophilic carbon monoxide streptomycete bacterial strain (Streptomyces thermocarboxydus) Str430, it was deposited in Chinese typical culture collection center on 01 26th, 2010, and deposit number is CCTCC M 2010026.
2. a kind of thermophilic carbon monoxide streptomycete bacterial strain according to claim 1 is characterized in that: described Str430 bacterium colony is circular, surperficial fine hair shape; Grey, base portion brown of light color, the luxuriant and curl of aerial hyphae; Have the two-wheeled branch, produce ellipsoidal conidium.
3. thermophilic carbon monoxide streptomycete bacterial strain according to claim 1; Its training method comprises liquid culture: the thermophilic carbon monoxide streptomycete Str430 inoculation that is separated in fermention medium; Under 30 ℃ of temperature, cultivated on the shaking table of rotating speed 100r 32～36 hours; Said fermention medium is: starch 10g, Semen Maydis powder 10g, glucose 5g, yeast extract paste 10g, (NH
4)
2SO
45g, KH
2PO
45g, K
2HPO
45g, CaCO
31g, MgSO
40.5g, CMC-Na 5g, pH7.5, add water 1000ml sterilization.
4. thermophilic carbon monoxide streptomycete bacterial strain according to claim 1, its training method comprises solid culture: 1. the thermophilic carbon monoxide streptomycete Str430 inoculation that is separated in the Gause I substratum, in 30 ℃ of incubators, cultivated 3～4 days; Said Gause I substratum is: Zulkovsky starch 20g, KNO
3Lg, NaCl 0.5g, K
2HPO
43H
2O 0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g, agar 20g, pH7.2～7.4 add water 1000ml sterilization;
Or 2. the thermophilic carbon monoxide streptomycete Str430 inoculation that is separated in improvement Cha Shi substratum, in 30 ℃ of incubators, cultivated 3～4 days, said improvement Cha Shi substratum is: CMC-Na 15g, NaNO
32g, NaCl 2g, MgSO
40.5g, FeSO
40.1g, Congo red 0.2g, agar 20g, zero(ppm) water 1000mL, pH nature.
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| CN102061270B true CN102061270B (en) | 2012-05-02 |
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| CN103103151B (en) * | 2013-01-18 | 2014-12-24 | 南京林业大学 | Streptomyces thermocarboxydus NL-1 as actinomycete capable of efficiently eroding limestone and application of Streptomyces thermocarboxydus NL-1 |
| CN103484396B (en) * | 2013-08-15 | 2014-12-17 | 北京农学院 | New strain of streptomyces thermocarboxydus and application thereof |
| CN103484410B (en) * | 2013-09-29 | 2014-12-17 | 北京农学院 | Biological agent for compost treatment of livestock manure |
| CN104988095B (en) * | 2015-07-03 | 2019-01-22 | 北京农学院 | One plant of thermophilic carbon monoxide streptomycete low temperature subspecies Dstr3-3 and its application |
| CN105567607B (en) * | 2016-03-03 | 2019-11-01 | 北京林业大学 | High-temperature-resistant garden waste decomposing bacteria ST3 and application thereof |
| CN106191012A (en) * | 2016-07-25 | 2016-12-07 | 怀化学院 | A kind of method utilizing thermophilic carbon monoxide streptomycete to produce cellulase and liquid fermentation medium |
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| CN101294141A (en) * | 2007-04-28 | 2008-10-29 | 上海四季生物科技有限公司 | A set of living body microorganism preparations for preparing composite microorganism fertilizer and preparation method thereof |
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