CN103820407A - Method for producing high activity laccase by solid fermentation of funalia trogii - Google Patents

Method for producing high activity laccase by solid fermentation of funalia trogii Download PDF

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Publication number
CN103820407A
CN103820407A CN201410058426.2A CN201410058426A CN103820407A CN 103820407 A CN103820407 A CN 103820407A CN 201410058426 A CN201410058426 A CN 201410058426A CN 103820407 A CN103820407 A CN 103820407A
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China
Prior art keywords
laccase
fermentation
solid
solid fermentation
nutrient medium
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CN201410058426.2A
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Chinese (zh)
Inventor
陈洪章
李冠华
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Institute of Process Engineering of CAS
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Institute of Process Engineering of CAS
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Priority to CN201410058426.2A priority Critical patent/CN103820407A/en
Publication of CN103820407A publication Critical patent/CN103820407A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)

Abstract

The invention discloses a method for producing high activity laccase by solid fermentation of funalia trogii. Laccase is called as blue enzyme in green chemistry, and has significant applications in the fields of energy, environment and foods. However, laccase yield in a present laccase solid fermentation process is low; and imperfection of fermentation technology restricts research and applications of the laccase. The invention provides a method for producing high activity laccase by of gas phase double dynamic solid fermentation. A solid matrix is smashed and added into a liquid culture medium containing a nitrogen source and copper sulfate, and the liquid culture medium is put into a gas phase double dynamic solid fermentation reactor after inoculation, to prepare the laccase by fermentation. Compared with a conventional solid fermentation method, the method has laccase yield increased by 30% and a fermentation period shortened by 25%. The invention provides a novel method for laccase by solid fermentation, and lays foundation for large-scale production of laccase by solid fermentation.

Description

A kind of method that bristle slightly covers pore fungi solid state fermentation produces high reactivity laccase
Technical field
Present method belongs to solid state fermentation field, and particularly a kind of bristle slightly covers the method for pore fungi solid state fermentation production high reactivity laccase.
Background technology
Laccase is a kind of polyphenoloxidase of cupric, the oxidation of the materials such as laccase energy catalysis polyphenol, polyamino benzene, make it to generate corresponding benzoquinones and water, belong to blue many copper oxydase (blue multi – copper oxidase) family (Leonowicz et al., 2001).Laccase is called as the blue enzyme in Green Chemistry, removes aldehydes matter in aspect foodstuffs industry; Papermaking aspect, deviates from xylogen, bleached pulp, Processing Paper Wastewater; Be used for dye decolored degradation, degradation of pesticide etc. at field of Environment Protection; Aspect biomass energy, can work in coordination with cellulase hydrolysis diastatic fiber element, remove fermentation inhibitor producing in biomass preprocessing process etc., aspect information science, realize biological monitoring etc., can say that laccase range of application is more and more wider, along with the Shen of research also will play an important role as laccase in human lives.
The fermentation mode of laccase mainly comprises liquid state fermentation and two kinds of modes of solid state fermentation at present, liquid state fermentation is at present at the more fermentation mode of industrial application, but liquid state fermentation energy consumption is high, complicated operation, the more important thing is in fermenting process and can produce bulk fermentation waste water.The existing a lot of reports of technology of preparing laccase by solid-state fermentation, as generally lower in laccase output, and also owing to there is no suitable solid-state fermentation reactor, current great majority all carry out about the research of laccase solid state fermentation in the Erlenmeyer flask of laboratory.The research report that gas-phase double-dynamic solid fermentation produces enzyme is a lot, but different from cellulase fermentations process, the product laccase fungi fermentation cycle is generally longer, and it is significant for solving laccase fermentation that gas-phase double-dynamic solid fermentation reactor is realized laccase solid state fermentation.
Bristle slightly covers pore fungi and belongs to Aphyllophorales polyporaceae bristle and slightly cover pore fungi and belong to, and mainly grows in willow, and the rotten wood of willow, to fall wood, storage wood etc. upper, extensively distribute in China, and be the common white-rot fungi of nature.Bristle slightly covers pore fungi fast growth, and biomass is large, produces enzyme activity high, degraded cellulose and xylogen simultaneously, but stronger to lignin degradation selectivity.But need further reinforcement for its laccase production mode, particularly large-scale production at present, the present invention has set up a kind of method that novel bristle slightly covers pore fungi solid state fermentation and produces high reactivity laccase.
Summary of the invention
The object of the present invention is to provide a kind of bristle slightly to cover the method for pore fungi solid state fermentation production high reactivity laccase, technical scheme of the present invention is as follows:
(1) in fermentation pallet, add solid state substrate, add liquid nutrient medium, sterilizing after stirring;
(2) after sterilizing, access bristle and slightly cover pore fungi seed liquor;
(3) postvaccinal fermented substrate is put into solid-state fermentation reactor enzymatic production.
The described solid state substrate of step (1) is straw, wheat straw, and maize straw, Pericarppium arachidis hypogaeae, wheat bran is pulverized.
The described liquid nutrient medium of step (1) comprises nitrogenous source and copper sulfate, and wherein nitrogenous source is urea, yeast powder, and peptone, ammonium sulfate, one of SODIUMNITRATE or arbitrary combination, its concentration is 1～15g/L; It is 0.5～10g/L that liquid nutrient medium contains concentration of copper sulfate; The pH value of liquid nutrient medium is 4～7.
The described seed liquor of step (2) is that bristle slightly covers 30 ℃ of aerobic shake flask fermentation 4-5 days gained fermented liquids of pore fungi spore suspension access potato liquid nutrient medium, and inoculum size is 5%～10% of solid state substrate total mass.
The described solid-state fermentation reactor of step (3) is gas-phase double-dynamic solid fermentation reactor;
The described fermentating enzyme-producing condition of step (3) is that, in gas-phase double-dynamic solid fermentation process, solid state fermentation temperature is 25～35 ℃, pulsating pressure is 0.1～0.4MPa, the high pressure dimension pressure time is 30～120s, and the normal pressure dimension pressure time is 60～120min, fermentation period 10～18 days.
The method tool that a kind of bristle slightly covers pore fungi solid state fermentation production high reactivity laccase has the following advantages and advantage:
(1) fermentation period is shorter, and laccase output is higher.
(2), using cheap agricultural wastes as solid-state fermentation substrate, reduced its enzymic fermentation cost.
(3 gas-phase double-dynamic solid fermentation technologies are easy to realize industrialization.
Embodiment
Embodiment 1
Straw is crossed 20 mesh sieves after pulverizing, and adds liquid culture medium, and regulating water content is 75%, contains 2g/L yeast powder, 1g/LCuSO in liquid culture medium 4, pH value is 4.5; After above-mentioned medium sterilization, access 8% bristle and slightly cover pore fungi seed liquor; Bristle slightly covers pore fungi seed liquor for bristle slightly being covered to 5 days gained of pore fungi spore suspension access 30 ℃ of aerobic shake flask fermentations of potato liquid nutrient medium; Postvaccinal fermented substrate is put into gas-phase double-dynamic solid fermentation reactor, and fermentation condition is that temperature is 28 ℃, and pulsating pressure is 0.1MPa, and the high pressure dimension pressure time is 60s, and the normal pressure dimension pressure time is 60min, and the laccase output after 12 days of fermenting is 240U/g.
Embodiment 2
Wheat straw crosses 20 mesh sieves after pulverizing, and adds liquid culture medium, and regulating water content is 70%, contains 3g/L urea, 0.5g/LCuSO in liquid culture medium 4, pH value is 5; After above-mentioned medium sterilization, access 7% bristle and slightly cover pore fungi seed liquor; Bristle slightly covers pore fungi seed liquor for bristle slightly being covered to 4 days gained of pore fungi spore suspension access 30 ℃ of aerobic shake flask fermentations of potato liquid nutrient medium; Postvaccinal fermented substrate is put into gas-phase double-dynamic solid fermentation reactor, and fermentation condition is that temperature is 30 ℃, and pulsating pressure is 0.1MPa, and the high pressure dimension pressure time is 60s, and the normal pressure dimension pressure time is 60min, and the laccase output after 12 days of fermenting is 350U/g.
Embodiment 3
After corn straw smashing, cross 20 mesh sieves, and add liquid culture medium, regulating water content is 75%, contains 2g/L ammonium sulfate, 1.5g/LCuSO in liquid culture medium 4, pH value is 5; After above-mentioned medium sterilization, access 5% bristle and slightly cover pore fungi seed liquor; Bristle slightly covers pore fungi seed liquor for bristle slightly being covered to 4 days gained of pore fungi spore suspension access 30 ℃ of aerobic shake flask fermentations of potato liquid nutrient medium; Postvaccinal fermented substrate is put into gas-phase double-dynamic solid fermentation reactor, and fermentation condition is that temperature is 28 ℃, and pulsating pressure is 0.2MPa, and the high pressure dimension pressure time is 90s, and the normal pressure dimension pressure time is 60min, and the laccase output after 18 days of fermenting is 440U/g.
Embodiment 4
Pericarppium arachidis hypogaeae is crossed 20 mesh sieves after pulverizing, and adds liquid culture medium, and regulating water content is 70%, contains 2g/L urea, 3g/LCuSO in liquid culture medium 4, pH value is 4; After above-mentioned medium sterilization, access 8% bristle and slightly cover pore fungi seed liquor; Bristle slightly covers pore fungi seed liquor for bristle slightly being covered to 5 days gained of pore fungi spore suspension access 30 ℃ of aerobic shake flask fermentations of potato liquid nutrient medium; Postvaccinal fermented substrate is put into gas-phase double-dynamic solid fermentation reactor, and fermentation condition is that temperature is 28 ℃, and pulsating pressure is 0.2MPa, and the high pressure dimension pressure time is 60s, and the normal pressure dimension pressure time is 60min, and the laccase output after 18 days of fermenting is 650U/g.

Claims (6)

1. bristle slightly covers a method for pore fungi solid state fermentation production high reactivity laccase, it is characterized in that following steps:
(1) in fermentation pallet, add solid state substrate, add liquid nutrient medium, sterilizing after stirring;
(2) after sterilizing, access bristle and slightly cover pore fungi seed liquor;
(3) postvaccinal fermented substrate is put into solid-state fermentation reactor enzymatic production.
2. method according to claim 1, is characterised in that the solid state substrate in described step (1) is straw, wheat straw, maize straw, Pericarppium arachidis hypogaeae or wheat bran.
3. method according to claim 1, is characterised in that the liquid nutrient medium of described step (1) comprises nitrogenous source and copper sulfate, and wherein nitrogenous source is urea, yeast powder, and peptone and ammonium sulfate, one of SODIUMNITRATE or arbitrary combination, its concentration is 1～15g/L; It is 0.5～10g/L that liquid nutrient medium contains concentration of copper sulfate; The pH value of liquid nutrient medium is 4～7.
4. method according to claim 1, the seed liquor that is characterised in that described step (2) is that bristle slightly covers 30 ℃ of aerobic shake flask fermentation 4-5 days gained fermented liquids of pore fungi spore suspension access potato liquid nutrient medium, and inoculum size is 5%～10% of solid state substrate total mass.
5. method according to claim 1, the solid-state fermentation reactor that is characterised in that described step (3) is gas-phase double-dynamic solid fermentation reactor.
6. method according to claim 1, is characterised in that the fermentating enzyme-producing condition of described step (3) is, solid state fermentation temperature is 25～35 ℃, pulsating pressure is 0.1～0.4MPa, the high pressure dimension pressure time is 30～120s, and the normal pressure dimension pressure time is 60～120min, fermentation period 10～18 days.
CN201410058426.2A 2014-02-20 2014-02-20 Method for producing high activity laccase by solid fermentation of funalia trogii Pending CN103820407A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108450935A (en) * 2017-02-17 2018-08-28 北京中科百瑞能工程技术有限责任公司 A kind of method that medicine-food two-purpose class raw material makes red yeast rice
CN111172199A (en) * 2018-11-12 2020-05-19 中国科学院过程工程研究所 Novel method for solid state fermentation of ethanol reinforced by inert gas
CN116814440A (en) * 2023-02-23 2023-09-29 江苏省农垦农业发展股份有限公司现代农业研究院 Podosporium crannorum MD2 strain and application thereof
CN116814440B (en) * 2023-02-23 2024-05-10 江苏省农垦农业发展股份有限公司现代农业研究院 Podosporium crannorum MD2 strain and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103173422A (en) * 2011-12-22 2013-06-26 中国科学院沈阳应用生态研究所 Solid fermentation medium for producing laccase through utilizing funalia trogii and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103173422A (en) * 2011-12-22 2013-06-26 中国科学院沈阳应用生态研究所 Solid fermentation medium for producing laccase through utilizing funalia trogii and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
魏玉莲等: "白腐菌Funalia trogii 以稻草进行固体发酵产漆酶的研究", 《纤维素科学与技术》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108450935A (en) * 2017-02-17 2018-08-28 北京中科百瑞能工程技术有限责任公司 A kind of method that medicine-food two-purpose class raw material makes red yeast rice
CN111172199A (en) * 2018-11-12 2020-05-19 中国科学院过程工程研究所 Novel method for solid state fermentation of ethanol reinforced by inert gas
CN116814440A (en) * 2023-02-23 2023-09-29 江苏省农垦农业发展股份有限公司现代农业研究院 Podosporium crannorum MD2 strain and application thereof
CN116814440B (en) * 2023-02-23 2024-05-10 江苏省农垦农业发展股份有限公司现代农业研究院 Podosporium crannorum MD2 strain and application thereof

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Application publication date: 20140528