CN103205402A - Fermentation condition of needle mushroom laccase production - Google Patents
Fermentation condition of needle mushroom laccase production Download PDFInfo
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- CN103205402A CN103205402A CN 201310128476 CN201310128476A CN103205402A CN 103205402 A CN103205402 A CN 103205402A CN 201310128476 CN201310128476 CN 201310128476 CN 201310128476 A CN201310128476 A CN 201310128476A CN 103205402 A CN103205402 A CN 103205402A
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Abstract
The invention belongs to the technical field of microbial fermentation and relates to fermentation condition of needle mushroom hybridized 9 laccase production. A preparation method of the needle mushroom hybridized 9 laccase production disclosed by the invention comprises the following steps of: transferring saved needle mushroom hybridized 9 protospecies to a PDA (Potato Dextrose Agar) slant medium, culturing for 7 days at the constant temperature of 28 DEG C, then transferring to a PDA flat plate and culturing for 7 days at the constant temperature of 28 DEG C till the flat is completely covered by the mycelium; manufacturing the cultured mycelium as mycelium tablets with a diameter of 10mm by using a hole puncher and inoculating 4 mycelium tablets to a 250ml triangular flask filled with 50ml of fermentation medium to ferment and culture. The culture conditions are as follows: shaking the flask and culturing for 7 days at the temperature of 28 DEG C and the rotation speed of 180r/min, in which the pH of the fermentation medium is 5.5; the fermentation medium of the needle mushroom hybridized 9 laccase production consists of the following substances in g/l: 20g/l of glucose, 2g/l of carbamide, 3.0g/l of KH2PO3, 1.5g/l of MgSO, 0.05g/l of ZnSO and 0.05g/l of MnSo; and the fermentation medium contains 60 micro mol/l of copper ion and 10 micro mol/l of dimethoxy benzoic acid.
Description
Technical field
Microbial fermentation technology field under the present invention.The present invention relates to the Fermentation Conditions that needle mushroom produces laccase, be fit to industrial applications.
Background technology
Laccase (laccase, ECl.10.3.2) be a kind of cupric polyphenoloxidase (polyphenol oxidases, PPO), belong to blue copper oxidase family together with the Vitamin C oxidase in the plant, mammiferous ceruloplasmin, have many similarities at structure and function.At occurring in nature, laccase is distributed in various plants, the fungus body, and in minority insect and the bacterium.It can the catalyzed oxidation phenolic compound sloughs electronics or proton on the hydroxyl, forms free radical, causes phenols and the cracking of gelation mechanism of lignin grouting compound, and molecular oxygen is reduced to water simultaneously.
1883, Japanese scholar Yoshida first from Japanese lac lacquer tree (Rhus verniciflua) but find a kind of protein of catalysis lacquer solidification process the lacquer liquid; Bertrand was with its called after laccase in 1894.It is found that subsequently some higher fungies also can secretion laccase, laccase extensively is present in the pore fungus (Polyporus) of Basidiomycotina (Basidiomycotina), the neurospora (Neurospora) of Ascomycotina (Ascomycotina) and the aspergillus tubigensis (Aspergillus) of Podospora (Podospora) and Deuteromycotina (Deuteromycotina) etc., and wherein the most important producer is the whiterot fungi in the basidiomycetes.
Recent study shows, laccase has and acts on substrate widely.Because its energy lignin degrading; can with poisonous aldehydes matter effect; make phenoxy herbicide, oil industry wastewater remove toxicity; all right some organophosphorus poisons of oxidative degradation; and form and the function of aspect such as pathogenic at the microbial bacteria volume morphing; make it to have very big researching value and application potential in foodstuffs industry, environment protection, paper industry and other field, have the wide development prospect.
Needle mushroom formal name used at school hair handle money bacterium is commonly called as structure bacterium, plain mushroom, dried mushroom etc., belongs to Agaricales Tricholomataceae genus flammulina.Needle mushroom widely distributes at nature, and has characteristics cheap, that be easy to cultivate.Be proved as a kind of common edible whiterot fungi and can have produced laccase.Many microorganisms all can produce laccase, and in view of whiterot fungi can faster and more extensive lignocellulose degradation than other microorganism, and its laccase that produces is extracellular enzyme, is convenient to separation and the purification of enzyme, therefore has prospects for commercial application widely.Yet the research that current relevant needle mushroom produces laccase is less, particularly aspect the optimization cultivation.Though done about fermentation condition also someone and to have studied (Wang Hongbo, 2009; Rao Yupeng, 2007; Deng), but fermented liquid gained enzyme is alive all not really high, has limited the production of this enzyme.
Because the activity of laccase is subjected to all multifactor restrictions such as culture condition, the bacterial strain secretion laccase is subjected to the influence of the multiple factor, therefore the present invention is according to this reason, one strain needle mushroom (gold assorted 19) product laccase condition and substratum are optimized, probe into substratum and the optimum of needle mushroom fermentation culture, for long-pending abundant materials is established in suitability for industrialized production and the application of laccase.
Summary of the invention
1. the present invention relates to the optimization that needle mushroom produces the laccase condition, its feature is produced the optimization method of laccase condition at needle mushroom, comprises following process:
Step (one): the needle mushroom original seed of preserving transferred brings back to life in the PDA slant medium, 28 ℃ constant temperature culture 6～8d days, 4 ℃ of preservations;
Step (two): get the bacterial classification that step () preserves and receive on the PDA flat board, 28 ℃ of constant temperature culture 6～8 days cover with flat board to mycelia;
Step (three): the mycelia that step (two) is cultivated is made the bacterium sheet of 10 millimeters of diameters with punch tool, inoculate 4 bacterium sheets in 250 milliliters of triangular flasks that 50 milliliters of fermention mediums (including 60 micromoles per liter cupric ions and 10 micromoles per liter dimethoxybenzoic acids) is housed, 28 ℃, 180r/min shake-flask culture 7 days;
Step (four): the fermented liquid after step (three) fermentation centrifugal 15 minutes at 4000r/min, supernatant liquor is crude enzyme liquid;
Step (five): the crude enzyme liquid in the step (four) is substrate with the o-tolidine, measures its enzyme and live under the acetate buffer system, and increasing by 0.01 with the per minute optical density(OD) is an enzyme activity unit (U/mL).
2. according to of the present invention, the best incubation time of fermentation is 7 days, and the pH of fermention medium is 5.5, and liquid amount is 50 milliliters/250 milliliters triangular flasks, and culture temperature is 28 ℃, shaking table speed 180r/min.
3. according to of the present invention, the fermention medium that needle mushroom gold assorted 9 produces laccase is (grams per liter): glucose 20, urea 2, KH
2PO 3.0, and MgSO 1.5, and ZnSO 0.05, and MnSO 0.05.
4. according to of the present invention, the inductor that needle mushroom gold assorted 9 produces laccase is bivalent cupric ion and dimethoxybenzoic acid, and optimal addn is respectively 60 micromoles per liter, 10 micromoles per liter.
Embodiment
The fermentation condition that relates to the assorted 9 product laccases of needle mushroom gold of the present invention comprises following examples, and the following examples can further specify the present invention, but do not limit the present invention in any way.
Embodiment 1: needle mushroom gold assorted 9 produces the experiment of laccase fermentation condition single-factor
1 materials and methods
1.1 material
1.1.1 bacterial classification needle mushroom gold assorted No. 19 available from academy of agricultural sciences, Shandong Province agricultural resource institute, mycelia preserves standby under 4 ℃ of conditions on the PDA substratum.
1.1.2 reagent o-tolidine (Shanghai chemical reagent head factory analytical pure); Other analytical reagents are homemade analytical pure.
1.1.3 instrument 722E type visible spectrophotometer (Shanghai the 3rd analytical instrument factory).
1.1.4 substratum
PDA substratum (g/L): potato 200, glucose 20, agar 20, pH value nature, 121 ℃ of sterilization 20min.
Liquid nutrient medium (g/L): potato 200, glucose 20, yeast extract paste 5.0, KH
2PO
43.0, MgSO
41.5, ZnSO
40.05, MnSO
40.05, pH nature, 121 ℃ of sterilization 20min.
1.2 method
1.2.1 actication of culture
The bacterial classification of preserving is transferred on the PDA slant medium, and 28 ℃ of constant temperature culture 7～8d put and preserve standbyly in 4 ℃ of refrigerators, and activation once about one month.
1.2.2 cultural method
The bacterial classification of preserving is received on the PDA flat board, and 28 ℃ of constant temperature culture 8～10d treat that mycelia covers with flat board, make the bacterium plug of diameter 10mm with punch tool, inoculate 4 bacterium and fill in the 250mL triangular flask that the 70mL liquid nutrient medium is housed, and 28 ℃, the 180r/min shake-flask culture.
1.2.3 the centrifugal 15min of crude enzyme liquid preparative centrifugation machine 4000r/min, supernatant liquor is crude enzyme liquid.
1.2.4 enzyme activity determination
0.2mol/LpH4.6 acetate buffer solution 3.5ml, add 3.36mol/L o-tolidine 0.5mL, add the suitably crude enzyme liquid 0.5mL of dilution again, behind 25 ℃ of insulation 5min, survey A595nm place's optical density value (OD value) in the 722E visible spectrophotometer immediately, enzyme activity with sample and substrate reactions 5min after the change value representation of optical density(OD), increasing by 0.01 with the per minute optical density(OD) is an enzyme activity unit (U/mL).
2. result and discussion
2.1 the time of secretion laccase
With the dull and stereotyped bacterial classification inoculation of cultivating 8d to liquid fermentation medium, 28 ℃, the 180r/min shake-flask culture, from 3d, every 24h sampling and measuring laccase vigor found that: with the prolongation of fermentation time, enzyme is lived and is raise gradually, living to the 7d enzyme, the highest (the enzyme work of 4-8d is respectively: 23,28,30,36,30U/mL), so select 7d to produce the enzyme time as the best.
2.2 carbon source is to producing the influence of laccase
Replace glucose in the liquid fermentation medium with sucrose, Zulkovsky starch, maltose, citric acid, Xylo-Mucine as carbon source respectively, 28 ℃, measure laccase activity behind the 180r/min shaking table constant temperature culture 7d, found that bacterial strain to citric acid, sucrose utilize ability a little less than, it is lower to produce laccase activity.To glucose, Zulkovsky starch, maltose, Xylo-Mucine utilize ability strong (be respectively: 101,79,60,69U/mL), laccase activity is the highest when being carbon source with glucose.
2.3 nitrogenous source is to producing the influence of laccase
Replace yeast extract paste to carry out fermentation culture as the nitrogenous source in the liquid state fermentation substratum with extractum carnis, urea, peptone, ammonium sulfate, ammonium tartrate respectively, measure laccase activity behind the 7d, when found that with extractum carnis, peptone, ammonium sulfate as nitrogenous source, it is lower that bacterial strain produces laccase activity.With urea, yeast extract paste, ammonium tartrate as nitrogenous source, produce laccase activity better (be respectively: 100,59,71U/mL), and be that the nitrogenous source activity is best with urea.
2.4 initial pH value of medium is produced the influence of laccase to needle mushroom
With acetate buffer solution the initial p H value of liquid state fermentation substratum is adjusted to 4.0,4.5,5.0,5.5,6.0,6.5,7.0 respectively, measure laccase activity behind the shake-flask culture 7d, found that, the activity of needle mushroom product laccase is higher relatively in the scope of pH5.0-6.0 (is respectively 79,98, the laccase vigor is the highest during 71U/mL), with pH5.5
2.5 liquid amount is to producing the influence of laccase
In the 250ml triangular flask, be respectively charged into the 30-90ml liquid nutrient medium, 28 ℃, measure laccase activity behind the 180r/min shaking table constant temperature culture 7d.The result is respectively: 60,102,70,30U/mL, and when liquid amount was 50mL, needle mushroom produced laccase activity and reaches the highest,, reach 102U/mL.This may and produce laccase with bacterial strain self growth, and need to consume a large amount of oxygen relevant.
Embodiment 2 orthogonal tests
According to the experiment of example 1 single-factor, choose the Several Factors to enzyme secretion amount maximum: carbon nitrogen source, liquid amount and pH carry out orthogonal experiment.
Adopt L9 (3
4) orthogonal test level of factor table carries out orthogonal test.Carry out 9 groups of experiments according to experimental design table 1, the orthogonal experiments variance analysis sees Table 2.
Table 1L
9(3
4) orthogonal experimental design
Tab.1Analysis?of?the?L
9(3
4)orthogonal?array?design
The variance analysis of table 2 orthogonal experiments
Tab.2Result?of?the?L
9(3
4)orthogonal?array?design
Table 2 explanation: carbon source and nitrogenous source are bigger to the influence of enzyme activity, especially have the greatest impact with nitrogenous source, and pH value are relative less to the enzyme activity influence with liquid amount.This optimization of orthogonal test is combined as: B
1A
3C
3D
2, i.e. urea 2g/L, glucose 20g/L, pH value are that 5.5,250mL triangular flask liquid amount is 50ml.Range analysis is R relatively
B＞R
A＞R
c＞R
D, namely urea quality concentration is to producing having the greatest impact of laccase, and liquid amount is minimum to its influence.
The experiment of embodiment 3 inductors
1. cupric ion
In substratum, add Cu
2+, make Cu in the liquid nutrient medium
2+Final concentration is respectively 0,20 μ mol/L, 40 μ mol/L, 60 μ mol/L, 80 μ mol/L, 100 μ mol/L, and 28 ℃, measure laccase activity behind the 180r/min shaking table constant temperature culture 7d, be respectively 70,80,89,99,85,79U/mL.With do not add Cu
2+Compare, add Cu
2+Laccase activity all has the increase of certain amplitude afterwards, because laccase is the zymoprotein of cupric, may be because laccase gene is transcribed synthetic lacquer enzyme require Cu
2+, therefore lacking Cu
2+Situation under, laccase synthetic influenced is so the vigor of laccase is relatively low.Along with Cu
2+The increase gradually of concentration, laccase activity also progressively increase, and enzyme work reaches the highest when 60 μ mol/L, afterwards active slowly decline.
2. dimethoxybenzoic acid
In substratum, add dimethoxybenzoic acid, make that the dimethoxybenzoic acid final concentration is respectively 0,5 μ mol/L, 10 μ mol/L, 20 μ mol/L, 30 μ mol/L, 40 μ mol/L in the liquid nutrient medium, 28 ℃, measure laccase activity behind the 180r/min shaking table constant temperature culture 7d, be respectively 60,98,123,94,85,80U/mL.Compare with not adding dimethoxybenzoic acid, enzyme live to increase a lot, with 10 μ mol/L for the highest.This is consistent with relevant report, infers that dimethoxybenzoic acid is the synthetic inductor of fungal laccase gene, so be conducive to the secretion of laccase.
Claims (4)
1. the present invention relates to the optimization that needle mushroom produces the laccase condition, its feature is produced the optimization method of laccase condition at needle mushroom, comprises following process:
Step (one): the needle mushroom original seed of preserving transferred brings back to life in the PDA slant medium, 28 ℃ constant temperature culture 6～8d days, 4 ℃ of preservations;
Step (two): get the bacterial classification that step () preserves and receive on the PDA flat board, 28 ℃ of constant temperature culture 6～8 days cover with flat board to mycelia;
Step (three): the mycelia that step (two) is cultivated is made the bacterium sheet of 10 millimeters of diameters with punch tool, inoculate 4 bacterium sheets in 250 milliliters of triangular flasks that 50 milliliters of fermention mediums (including 60 micromoles per liter cupric ions and 10 micromoles per liter dimethoxybenzoic acids) is housed, 28 ℃, 180r/min shake-flask culture 7 days;
Step (four): the fermented liquid after step (three) fermentation centrifugal 15 minutes at 4000r/min, supernatant liquor is crude enzyme liquid;
Step (five): the crude enzyme liquid in the step (four) is substrate with the o-tolidine, measures its enzyme and live under the acetate buffer system, and increasing by 0.01 with the per minute optical density(OD) is an enzyme activity unit (U/mL).
2. according to of the present invention 1, the best incubation time of fermentation is 7 days, and the pH of fermention medium is 5.5, and liquid amount is 50 milliliters/250 milliliters triangular flasks, and culture temperature is 28 ℃, shaking table speed 180r/min.
3. according to of the present invention 1, the fermention mediums that needle mushroom gold assorted 9 produces laccases are (grams per liter): glucose 20, urea 2, KH
2PO 3.0, and MgSO 1.5, and ZnSO 0.05, and MnSO 0.05.
4. according to of the present invention 1, the inductors that needle mushroom gold assorted 9 produces laccases are bivalent cupric ion and dimethoxybenzoic acid, and optimal addn is respectively 60 micromoles per liter, 10 micromoles per liter.
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| CN117887596A (en) * | 2024-03-14 | 2024-04-16 | 云南省农业科学院药用植物研究所 | Preparation method of tricholoma giganteum liquid strain |
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| CN117887596A (en) * | 2024-03-14 | 2024-04-16 | 云南省农业科学院药用植物研究所 | Preparation method of tricholoma giganteum liquid strain |
| CN117887596B (en) * | 2024-03-14 | 2024-05-07 | 云南省农业科学院药用植物研究所 | Preparation method of tricholoma giganteum liquid strain |
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Application publication date: 20130717 |