CN102443546A - White rot Inonotus hispidus mutant strain T906 for laccase production and preparation method thereof - Google Patents
White rot Inonotus hispidus mutant strain T906 for laccase production and preparation method thereof Download PDFInfo
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- CN102443546A CN102443546A CN201110385810XA CN201110385810A CN102443546A CN 102443546 A CN102443546 A CN 102443546A CN 201110385810X A CN201110385810X A CN 201110385810XA CN 201110385810 A CN201110385810 A CN 201110385810A CN 102443546 A CN102443546 A CN 102443546A
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Abstract
The invention discloses a white rot Inonotus hispidus mutant strain T906 for laccase production and a preparation method thereof. The mutant strain T906 (CoriolopsisgallicaT906) mentioned in the invention is preserved in China Center for Type Culture Collection. The preservation number is CCTCCM2011317, the preservation date is September 15, 2011 and it is preserved in Wuhan University, Wuhan, China. Wild Inonotus hispidus (preservation number being cfcc88387) purchased from China Forestry Culture Collection Center is used as an original strain, and the mutant strain is obtained by ultraviolet and nitrosoguanidine combined mutation screening. Under an optimum condition, the laccase production of the mutant strain reaches 213U/mL. The mutant strain has mycelial morphology and cultural characteristics which are obviously different from the original strain, does not degenerate after serial passages for over 30 times and has genetic stability.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind of white rot coarse wool leather pore fungi mutagenesis bacterium T906 that produces laccase and preparation method thereof.
Background technology
Laccase is a kind of copper bearing polyphenoloxidase; Its effect substrate is extensive; Effectively multiple phenol of lignin degrading and catalyzed oxidation and aromatic amine compounds all have broad application prospects and potential using value in fields such as bioenergy, environment protection, paper industry, foodstuffs industry, biological detection.Major part can cause that the putrescible fungi of woody or herbage all can produce laccase.
Wherein, white-rot fungi is one type of general designation that can cause the septic filamentous fungus of wooden white, has outstanding lignin degradation ability, is mainly basidiomycetes, and few part is an ascomycetes, comprise Coriolus Qu61 (
Coriolus), transverse hole fungus belong to (
Poria), Polyporus (
Polyporus), the flat lead fungi of raw wool belong to (
Phanerochaete), pleurotus (
Pleurotus), the smoke pipe Pseudomonas (
Bjerkandera) and trametes (
Tramete) etc.But the laccase throughput of wild white-rot fungi is generally low, has satisfied not the suitability for industrialized production and the application of laccase far away, makes the research of laccase application facet still rest on laboratory stage.
Coarse wool leather pore fungi (
Coriolopsis gallica) be that a kind of lignocellulose degradation ability is strong, generation ligocellulose degradation enzyme enzyme is complete white rot basidiomycetes; Extensively distribute in China; Mainly growing on the willow, is a kind of main biological degradation person of poplar, and it is one of the strongest wild strain of present known generation laccase ability.Its degradation capability than lignin degradation pattern bacterium Phanerochaete chrysosporium (
Phanerochaete chrysosporium) stronger.
Summary of the invention
The object of the present invention is to provide a strain can produce in a large number laccase white rot coarse wool leather pore fungi (
Coriolopsis gallica) mutagenesis bacterium T906 (preserving number: CCTCC M 2011317); Be a kind of new bacterial strain, with the thalli morphology and the obviously difference of cultural characteristic existence of starting strain, and continuous passage is not degenerated more than 30 times; Have genetic stability, enzyme activity can reach 213 U/mL under optimal conditions.
The present invention also provides the method for cultivation of this mutagenic fungi.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
A kind of white rot coarse wool leather pore fungi mutagenesis bacterium T906 that produces laccase (
Coriolopsis gallicaT906), be preserved in Chinese typical culture collection center, preserving number is CCTCC M 2011317, and preservation date is on September 15th, 2011, and the preservation place is a China. Wuhan. and Wuhan University;
The thalli morphology of this mutagenesis bacterium: the dull and stereotyped cultivation to white or oyster white filamentous fungus seldom or not produces spore; Microscopic features and wild coarse wool leather pore fungi (
Coriolopsis gallica) compare, mycelium is shorter, and mycelia is entwined closeer; Cultural characteristic: shake-flask culture be difficult for to generate the bacterium ball, 30 ℃ of shake-flask culture 6 ~ 20 days, and fermented liquid is khaki color, and color deepens in time gradually.
The white rot coarse wool of a strain mass production laccase provided by the invention leather pore fungi T906, be with available from the wild coarse wool leather pore fungi of China Forest microbial strains preservation administrative center (
Coriolopsis gallica) (preserving number: cfcc 88387) be starting strain, unites the mutagenesis screening gained through ultraviolet ray and nitrosoguanidine.Mutagenesis bacterium T906 cultivates with the PDA liquid nutrient medium; The laccase vigor is higher more than 10 times than wild bacterium; The highest laccase activity can reach 213 U/mL under the culture condition optimizing; And the proterties of mutagenesis bacterium T906 high yield laccase can genetic stability, time does not see degradation phenomena surplus the subculture 30 continuously, has good commercial application prospect and important researching value.
The mutafacient system that the present invention produces the white rot coarse wool leather pore fungi T906 of laccase is:
(1) wild coarse wool leather pore fungi (
Coriolopsis gallica) in the PDA liquid nutrient medium, cultivate and treated that mycelium formed in 2 ~ 6 days, adding aseptic glass sphere, the straight mycelium of shaking culture grinds, and 1 ~ 4 layer of aseptic paper handkerchief filters mycelium, collects filtrating;
(2) filtrating is transferred in the uncovered sterile petri dish, adds aseptic magnetic agitation, place stirring at low speed on the magnetic stirring apparatus;
(3) (2) described whipping appts is placed on apart from uv lamp 20 cm ~ 30 cm places, shone for 10 ~ 90 seconds;
(4) get (3) described bacterium liquid through uv irradiating, dull and stereotyped with the spreading rod coating PDA-RB light blue flat board or the PDA-methyl catechol that are soaked in 1 ~ 10 ng/mL nitrosoguanidine;
(5) get bacterium liquid in addition, use the spreading rod coating PDA-RB light blue flat board or the PDA-methyl catechol that soak without nitroso guanidine solution dull and stereotyped, as contrast without uv irradiating;
(6) (4) and (5) said flat board is put 30 ℃ of incubator lucifuges and be cultured to thalline and grow, the mutagenesis bacterium that the big or dull and stereotyped variable color circle of picking growth change is big, shake-flask culture and measure the laccase vigor behind the dull and stereotyped succeeding transfer culture.
Compared with prior art, the present invention has following beneficial effect:
(1) the white rot coarse wool leather pore fungi mutagenesis bacterium T906 laccase output that is obtained is high, and inheritance stability has a good application prospect and important researching value.
(2) method of employing ultraviolet ray and nitrosoguanidine associating mutagenesis has remedied the shortcoming that simple use ultraviolet mutagenesis is easy to generate reverse mutation, makes the heredity of mutagenesis bacterium excellent characteristic more stable.
(3) step of nitrosoguanidine mutagenesis combines with the step of spread plate, has simplified the operating process of ultraviolet ray and nitrosoguanidine associating mutagenesis.
(4) use the nitroso guanidine solution of ultralow density to soak spreading rod, make operating process safer, and avoid the pollution of nitrosoguanidine other equipment articles for use etc.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Embodiment:
The wild coarse wool leather pore fungi that filters out from the northern China willow (
Coriolopsis gallica) be inoculated in the PDA test tube slant, cultivated 4 ℃ of preservations 5 days for 30 ℃.
2. the wild coarse wool leather pore fungi switching PDA flat board of preserving the test tube slant, 30 ℃ of incubators are inverted and were cultivated 7 days, get 3 bacterium sheets with the punch tool of diameter 0.6 cm, and put into the PDA liquid nutrient medium and cultivated 3 days,
3. aseptic technique adds the sterilization granulated glass sphere down, covers and completely shakes the bottle bottom, and 200rpm cultivated 3 hours.
4. filter mycelium with 3 layers of sterilization paper handkerchief, collect filtrating, and filtrating is transferred in the uncovered sterilization petridish, add sterilization magnetic agitation, place stirring at low speed on the magnetic stirring apparatus, in the darkroom, from 30 or 40 seconds of UV-light 25cm irradiation.
5. collect the bacterium liquid behind uv irradiating, dull and stereotyped with the spreading rod coating PDA-RB light blue flat board or the PDA-methyl catechol that are soaked in 3 ~ 6 ng/mL nitrosoguanidines, put 30 ℃ of incubator lucifuges and cultivated for 1 ~ 2 week.
6. with the bacterium liquid without uv irradiating, dull and stereotyped with cleaning spreading rod coating PDA-RB light blue flat board or PDA-methyl catechol, as contrast.
7. the picking thalli growth changed greatly or dull and stereotyped variable color circle mutagenesis bacterium greatly, in PDA-RB light blue flat board or the dull and stereotyped succeeding transfer culture of PDA-methyl catechol 7 days.
8. be chosen at the mutagenesis bacterium that the variable color circle is big on the subculture flat board, get 3 bacterium sheets, be inoculated in fermention medium with the punch tool of diameter 0.6 cm, 150rpm, 30 ℃ of shaking tables are cultivated, and are contrast with wild coarse wool leather pore fungi.Whenever got fermented liquid at a distance from 1 day from the 3rd day, centrifugal, supernatant uses ABTS to measure the laccase vigor as substrate, chooses the mutagenesis bacterium of laccase vigor greater than wild coarse wool leather pore fungi, and (table 1) for use preserved in the PDA test tube slant.
Wild coarse wool leather pore fungi of table 1 and mutagenesis coarse wool leather pore fungi T906 fermentation result are relatively
Claims (3)
- A white rot coarse wool leather pore fungi mutagenesis bacterium T906 who produces laccase ( Coriolopsis gallicaT906), be preserved in Chinese typical culture collection center, preserving number is CCTCC M 2011317, and preservation date is on September 15th, 2011, and the preservation place is a China. Wuhan. and Wuhan University;The thalli morphology of this mutagenesis bacterium: the dull and stereotyped cultivation to white or oyster white filamentous fungus seldom or not produces spore; Microscopic features and wild coarse wool leather pore fungi ( Coriolopsis gallica) compare, mycelium is shorter, and mycelia is entwined closeer; Cultural characteristic: shake-flask culture be difficult for to generate the bacterium ball, 30 ℃ of shake-flask culture 6 ~ 20 days, and fermented liquid is khaki color, and color deepens in time gradually.
- 2. the preparation method of the white rot coarse wool of the said production laccase of claim 1 leather pore fungi mutagenesis bacterium T906, it is characterized in that be with available from the wild coarse wool leather pore fungi of China Forest microbial strains preservation administrative center ( Coriolopsis gallica) (preserving number: cfcc 88387) be starting strain, unites mutagenic obtained through ultraviolet ray and nitrosoguanidine.
- 3. remove from office the preparation method of pore fungi mutagenesis bacterium T906 according to the white rot coarse wool of the said production laccase of claim 2, it is characterized in that said mutafacient system is:(1) wild coarse wool leather pore fungi ( Coriolopsis gallica) in the PDA liquid nutrient medium, cultivate and treated that mycelium formed in 2 ~ 6 days, adding aseptic glass sphere, the straight mycelium of shaking culture grinds, and 1 ~ 4 layer of aseptic paper handkerchief filters mycelium, collects filtrating;(2) filtrating is transferred in the uncovered sterile petri dish, adds aseptic magnetic agitation, place stirring at low speed on the magnetic stirring apparatus;(3) (2) described whipping appts is placed on apart from uv lamp 20 cm ~ 30 cm places, shone for 10 ~ 90 seconds;(4) get (3) described bacterium liquid through uv irradiating, dull and stereotyped with the spreading rod coating PDA-RB light blue flat board or the PDA-methyl catechol that are soaked in 1 ~ 10 ng/mL nitrosoguanidine;(5) get bacterium liquid in addition, use the spreading rod coating PDA-RB light blue flat board or the PDA-methyl catechol that soak without nitroso guanidine solution dull and stereotyped, as contrast without uv irradiating;(6) (4) and (5) said flat board is put 30 ℃ of incubator lucifuges and be cultured to thalline and grow, the mutagenesis bacterium that the big or dull and stereotyped variable color circle of picking growth change is big, shake-flask culture and measure the laccase vigor behind the dull and stereotyped succeeding transfer culture.
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Cited By (6)
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| CN104711272A (en) * | 2014-12-14 | 2015-06-17 | 上海勤生缘生物科技有限公司 | Laccase protein gene and cloning and sequencing method thereof |
| CN107129935A (en) * | 2017-04-25 | 2017-09-05 | 鲁东大学 | A kind of DSE bacterium and its application in blueberry growth and drought resisting is improved |
| CN108384723A (en) * | 2018-03-13 | 2018-08-10 | 中国林业科学研究院亚热带林业研究所 | A kind of complex micro organism fungicide preparation method and applications |
| CN113564058A (en) * | 2021-09-08 | 2021-10-29 | 南昌大学 | Method for promoting production of laccase from Inonotus hirsutus by using food waste |
| CN113604366A (en) * | 2021-09-08 | 2021-11-05 | 南昌大学 | Cerrena crassa NCULAC F1 for producing laccase and method for preparing high-activity laccase liquid |
| CN114410595A (en) * | 2021-12-30 | 2022-04-29 | 湖南利尔康生物股份有限公司 | Fungal laccase, preparation method and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711272A (en) * | 2014-12-14 | 2015-06-17 | 上海勤生缘生物科技有限公司 | Laccase protein gene and cloning and sequencing method thereof |
| CN107129935A (en) * | 2017-04-25 | 2017-09-05 | 鲁东大学 | A kind of DSE bacterium and its application in blueberry growth and drought resisting is improved |
| CN107129935B (en) * | 2017-04-25 | 2020-06-19 | 鲁东大学 | DSE (Deuterorhizobium-beta) bacterium and application thereof in improving growth and drought resistance of blueberries |
| CN108384723A (en) * | 2018-03-13 | 2018-08-10 | 中国林业科学研究院亚热带林业研究所 | A kind of complex micro organism fungicide preparation method and applications |
| CN108384723B (en) * | 2018-03-13 | 2020-11-10 | 中国林业科学研究院亚热带林业研究所 | Preparation method and application of compound microbial agent |
| CN113564058A (en) * | 2021-09-08 | 2021-10-29 | 南昌大学 | Method for promoting production of laccase from Inonotus hirsutus by using food waste |
| CN113604366A (en) * | 2021-09-08 | 2021-11-05 | 南昌大学 | Cerrena crassa NCULAC F1 for producing laccase and method for preparing high-activity laccase liquid |
| CN113564058B (en) * | 2021-09-08 | 2024-02-09 | 南昌大学 | Method for promoting phellinus lanuginosus to produce laccase by using food waste |
| CN113604366B (en) * | 2021-09-08 | 2024-03-29 | 南昌大学 | Coriolus hirsutus NCULAC F1 for producing laccase and method for preparing high-activity laccase liquid |
| CN114410595A (en) * | 2021-12-30 | 2022-04-29 | 湖南利尔康生物股份有限公司 | Fungal laccase, preparation method and application thereof |
| CN114410595B (en) * | 2021-12-30 | 2022-12-13 | 湖南利尔康生物股份有限公司 | Fungal laccase, preparation method and application thereof |
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