CN1690186A - Laccase, preparing method thereof and strain special for same - Google Patents
Laccase, preparing method thereof and strain special for same Download PDFInfo
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- CN1690186A CN1690186A CNA2004100340160A CN200410034016A CN1690186A CN 1690186 A CN1690186 A CN 1690186A CN A2004100340160 A CNA2004100340160 A CN A2004100340160A CN 200410034016 A CN200410034016 A CN 200410034016A CN 1690186 A CN1690186 A CN 1690186A
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Abstract
The invention disclosing a laccase and its production process and the special production strain, relates to the microbe-fermenting industrial sphere. The provided strain in the invention is Pycnoporus sanguineus mk528 CGMCC No. 1124. The provided strain in the invention is Pycnoporus sanguineus mk528 CGMCC No. 1124. The method for production of laccase contains fermenting and culturing Pycnoporus sanguineus mk528 CGMCC No. 1124 and extracting laccase from fermentation and culture medium. The enzyme activity of fermentation liquor laccase can reach as high as 65 U/ml by fermenting and culturing the provided Pycnoporus sanguineus mk528 CGMCC No. 1124; neither lignin peroxidase nor manganese peroxidase produced in the culture process of strain, which is convenient to extract laccase, and the extraction-acquired laccase can be largely used in paper mill wastewater disposal and substitute chemicals such as formaldehyde in wood processing industry.
Description
Technical field
The present invention relates to industrial field of microorganism fermentation, particularly relate to a kind of method and special strain therefore thereof of producing laccase.
Background technology
Laccase (p-diphenol oxygen oxidoreductase, EC 1.10.3.2.) be a kind of blueness, contain the phenol oxidase of a plurality of cupric ions, the redox reaction of energy catalysis aldehydes matter, its reaction substrate comprises: phenols and derivative thereof, arylamine and derivative thereof, aromatic carboxylic acid and derivative thereof etc.By constantly research, the oxidable non-phenol substrate scope of laccase is also in continuous expansion at present.Phenols in the laccase degradable xylogen and non-phenols structure play an important role in the biological degradation of xylogen, also can be used to the aromatics of degrading.Laccase can also make lignin class polyphenol substance carry out oxypolymerization in the radical mode, can substitute the traditional timber applied adhesive made of synthetic resin of industry or other chemical, replace with serious pollution chemical process with this free of contamination Enzymology method, can be abatement of pollution in the source.Therefore, laccase is having huge application potential aspect biotechnology and the environment protection.
Whiterot fungi can produce laccase, lignin peroxidase (LiP) and Mn oxide enzyme (MnP) etc., is unique biology that xylogen thoroughly can be degraded to carbonic acid gas and water at present.The laccase of its generation is better than lignin peroxidase and manganese peroxidase enzyme heat stability.Therefore, this biological group has caused world biologist's very big interest.Pycnoporus samguineus in the whiterot fungi is considered to study the desirable strain of laccase, yet the laccase activity power of existing domestic known bacterial strain is all lower, is up to 21U/ml.
Summary of the invention
The purpose of this invention is to provide a strain and produce the pycnoporus samguineus of laccase.
Pycnoporus samguineus provided by the present invention (Pycnoporus sanguineus) mk528 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 03 25th, 2004, preserving number is CGMCC № 1124.
This pycnoporus samguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 separate to obtain from pycnoporus samguineus G5, its upgrowth situation on solid medium as shown in Figure 1, the Giemsa of hyphal cell dyes as shown in Figure 2.The morphology difference of this bacterial strain and its parent strain G5 is as shown in table 1.
Table 1 mycelia morphology difference
| Morphological characteristic | Pycnoporus samguineus | |
| G5 (parent strain) | MK528 (bacterial strain of the present invention) | |
| The mycelia form | Clamp connexion is arranged | No clamp connexion |
| Hyphal cell nuclear | Double-core | Monokaryon |
Pycnoporus samguineus provided by the present invention (Pycnoporus sanguineus) mk528 CGMCC № 1124 is a kind of aerophils, and optimum growth temperature is 30 ℃, and secretion laccase and is accompanied by a large amount of pigment and produces in substratum during fermentation culture.
The method that another object of the present invention provides a kind of laccase and produces laccase.
Laccase provided by the present invention, fermentation pycnoporus samguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 obtains.
Produce the method for laccase, comprise fermentation culture pycnoporus samguineus (Pycnoporus sanguineus) mk528CGMCC № 1124 and from fermention medium, extract laccase.
The fermentation culture process need carries out under aerobic and lucifuge condition, is cultured to the 4th day needs and adds inductor 2,5 xylidines in substratum, and the amount that adds 2,5 xylidines is advisable with 10 μ M.Used fermention medium can use maltose, sucrose or glucose etc. as carbon source, and is little for the influence of the speed of growth of mycelia, but laccase output is wanted height during with maltose; Substratum usually with ammonium tartrate as nitrogenous source, also include multiple inorganic component, as ferrous sulfate, sal epsom, manganous sulfate, zinc sulfate, copper sulfate, calcium chloride, cobalt chloride etc. and some nutritive ingredients, as vitamins B
1, B
2, B
6Deng.Add Testa Tritici in substratum, laccase output can improve 2-3 doubly.
In actual applications, described fermention medium preferably is made up of following material, maltose: 1.0-2.0%; Ammonium tartrate: 0.1-0.3%, KH
2PO
4: 0.132-0.134%; NaH
2PO
412H
2O:0.038-0.040%; MgSO
47H
2O:0.04-0.06%; Sodium succinate (CH
2COONa)
26H
2O:0.117-0.119%; FeSO
47H
2O:0.00314-0.00316%; CaCl
22H
2O:0.009-0.011%; MnSO
4H
2O:0.0034-0.0036%; CH
3COONa3H
2O:0.0407-0.0409%; CoCl
26H
2O:0.005-0.007%; ZnSO
47H
2O:0.0027-0.0029%; CuSO
45H
20:0.0167-0.0169%; Testa Tritici 5-7 gram; Tween-80:0.9-1.1ml; VitaminB1:10 μ g; VitaminB
2: 5 μ g; VitaminB
6: 5 μ g, add water to 1 liter, wherein, described per-cent is to be the per-cent of the solid weight of matrix to volume of water with water.
The described laccase that extracts from fermention medium may further comprise the steps: centrifugation, ultrafiltration and concentration, ammonium sulfate precipitation, ion exchange column and gel-filtration column.
The centrifugation fermented liquid is 10,000 ten thousand daltonian ultrafiltration post ultrafiltration gained supernatant liquors with molecular weight cut-off, and with two step ammonium sulfate precipitation ultrafiltration and concentration liquid, the ammonium sulfate saturation ratio of described two step ammonium sulfate precipitations is respectively 30%-40% and 70%-90%.
Pycnoporus samguineus provided by the present invention (Pycnoporus sanguineus) mk528 CGMCC № 1124 is through fermentation culture, the laccase activity that obtains can be up to 63U/ml, and this bacterial strain does not produce lignin peroxidase and manganese peroxidase in culturing process, be convenient to extract laccase, simplified extraction process, the laccase activity height that extraction obtains, can be widely used in the biological degradation of phenols, arylamine, aromatic carboxylic acid and derivative thereof, in paper-making pulping bio-bleaching and the wastewater treatment, and can be in wood working the instead of chemical cementing agent.
Description of drawings
Fig. 1 is the colonial morphology photo of pycnoporus samguineus mk528 CGMCC № 1124
Fig. 2 is Giema dyeing (* 1100) microphotograph of pycnoporus samguineus mk528 CGMCC № 1124
Embodiment
The separation of embodiment 1, pycnoporus samguineus mk528
Pycnoporus samguineus mk528 separates from pycnoporus samguineus G5 nucleated mycelium to obtain, and pycnoporus samguineus G5 is the nucleated mycelium (Tang Shun that obtains from the pycnoporus samguineus sporophore, Lomascolo A, Guo Lin etc. (2001) the Isolationof white-rot fungi from China for screening laccase-hyperproducing strains. fungus 20:520-525 of system).
Pycnoporus samguineus G5 is containing agar 20g/l
-1With Fructus Hordei Germinatus extract 20g/l
-1Culture dish in 30 ℃ of cultivations, cultivate after 7 days, culture dish be inverted and placed room temperature to continue to cultivate 3-4 week.From the culture dish lid, collect sporidium with the distilled water of high-temperature sterilization then, with the suitable multiple of these sporidium suspension dilutions, be applied to and contain agar 20g/l again
-1With Fructus Hordei Germinatus extract 20g/l
-1Culture dish in, incubated at room temperature at second day or the 3rd day, is separated visible sporidium small colonies monospore from culture dish again, with the method screening laccase high reactivity bacterial strain of syringaldazine, obtains pycnoporus samguineus mk528 again.
Embodiment 2, fermentation culture pycnoporus samguineus mk528 produce laccase
One, the fermentation of pycnoporus samguineus mk528
Solid seed culture medium: 2% Fructus Hordei Germinatus extract, 2% agar.
Fermention medium: maltose 15g, ammonium tartrate 2g, KH
2PO
41.333g, NaH
2PO
412H
2O 0.39g, MgSO
47H
2O 0.5g, sodium succinate (CH
2COONa)
26H
2O 1.18g, FeSO
47H
2O 0.0315g, CaCl
22H
2O0.1g, MnSO
4H
2O 0.035g, CH
3COONa3H
2O 0.408g, CoCl
26H
2O 0.06g, ZnSO
47H
2O 0.028g, CuSO
45H
2O 0.168g, Testa Tritici 6g, Tween-80 1ml, vitamins B
110 μ g, vitamins B
25 μ g, vitamins B
65 μ g added water to 1L, 8 pounds of high pressure moist heat sterilizations 30 minutes.
(Pycnoporus sanguineus (L:Fr) Murr. (MK528)) is seeded on the solid seed culture medium with pycnoporus samguineus.Cultivated 6-8 days down for 30 ℃.Get the bacterium piece (diameter 1cm, 10) at the edge of bacterium colony, be inoculated into be equipped with 100ml in the triangular flask of the fermention medium of high pressure moist heat sterilization, lucifuge is cultivated in 30 ℃ and 200rpm shaking table.(2,5-Dimethylaniline), making its final concentration is 10 μ M, cultivates 8 days again, collects fermented liquid, surveys its enzyme 63U/ml of being alive to be cultured to the 4th day adding inductor 2,5 xylidines.
Two, extract laccase
Get fermented liquid, the centrifugal fermentation throw out of removing of 5000rpm is got supernatant liquor through vacuum filtration, and the suction filtration liquid of acquisition is collected ultrafiltration and concentration liquid through PLGC film (10000D) ultrafiltration.Adopt two step ammonium sulfate precipitation method precipitation concentrated solutions subsequently: the first step stirs precipitation with the saturated ammonium sulphate of 35% (W/V) down at 4 ℃, and centrifugal 1 hour of 10000rpm abandons precipitation, stays supernatant; Second goes on foot the saturated ammonium sulphate that adds 80% (W/V) to the supernatant liquor of the first step gained, stirs precipitation down at 4 ℃, and 4 ℃ quiet to 2 hours, and 10000rpm is centrifugal 1 hour then, abandons supernatant, stays precipitation.Use the 100mM potassium phosphate buffer (pH 5.7) of two volumes to dissolve the precipitation that second step obtained, in the potassium phosphate buffer of 10mM, carry out dialysed overnight, obtain thick laccase preparation.Wherein the ultrafiltration step yield is 81.82%, and ammonium sulfate precipitation step yield is 88.89%, and potassium phosphate buffer dialysis yield is 96.46%.
Get the thick laccase preparation of 10ml gained and be used for the ion-exchange chromatography purifying.Ion Exchange Medium is selected DEAESepharose F.F. for use, and level pad is the histidine buffering liquid (pH6.0) of 20mM.Elution process adopts gradient elution, and gradient is the Histidine elutriant of 0.1-0.6M NaCl, and flow velocity is 1mL/ minute.The laccase of collecting is partly passed through Sephadex G-100, and used elutriant is a water, and flow velocity is 0.5mL/ minute.It is dry then laccase to be collected the liquid cooling freeze-drying, gets blue laccase.
In the present embodiment, it is international 2 that the mensuration of laccase vigor adopts, and 2 '-azine-two-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) (ABTS) { 2,2'-azino-bis-[3-ethylthiazoline-6-sulphonate] } method measured.
The calculation formula of enzyme activity is U=Δ AV/ (ε LT)
In above-mentioned formula, Δ A represents the variation of ultraviolet spectrophotometer absorption value, V represent reaction system volume (unit: ml), ε=3.6 * 10
4Mcm-1=36 (μ mol/ml)
-1Cm
-1, being the molar extinction coefficient of oxidized form ABTS, L is that (unit: cm), T is the time (unit: minute) of reaction for the optical path of cuvette.
The enzyme activity determination condition is among the present invention: reaction volume V=1mL, the optical path L=0.5cm of cuvette, reaction times T=30 second.The tartrate damping fluid (pH 4) of the 500 μ L 50mM that contain of the volume 1mL of reaction system wherein, the water of 390 μ L, the ABTS of 100 μ L500 μ M and the fermented liquid of 10 μ L, selecting wavelength for use is 420nm.If the laccase vigor is too high in the fermented liquid, can suitably dilute.
Claims (9)
1, pycnoporus samguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124.
2, a kind of laccase, fermentation pycnoporus samguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 obtains.
3, a kind of method of producing laccase comprises fermentation culture pycnoporus samguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 and extract laccase from fermention medium.
4, the method for production laccase according to claim 3 is characterized in that: described fermentation culture is carried out under aerobic and lucifuge condition; The substratum of fermentation culture comprises maltose, ammonium tartrate and inorganic component, and described inorganic component comprises ferrous sulfate, sal epsom, manganous sulfate, zinc sulfate, copper sulfate, calcium chloride and cobalt chloride.
5, the method for production laccase according to claim 4 is characterized in that: described fermention medium is made up of following material, maltose: 1.0-2.0%; Ammonium tartrate: 0.1-0.3%, KH
2PO
4: 0.132-0.134%; NaH
2PO
412H
2O:0.038-0.040%; MgSO
47H
2O:0.04-0.06%; Sodium succinate (CH
2COONa)
26H
2O:0.117-0.119%; FeSO
47H
2O:0.00314-0.00316%; CaCl
22H
2O:0.009-0.011%; MnSO
4H
2O:0.0034-0.0036%; CH
3COONa3H
2O:0.0407-0.0409%; CoCl
26H
2O:0.005-0.007%; ZnSO
47H
2O:0.0027-0.0029%; CuSO
45H
2O:0.0167-0.0169%; Testa Tritici 5-7 gram; Tween-80:0.9-1.1ml; VitaminB
1: 10 μ g; VitaminB
2: 5 μ g; VitaminB
6: 5 μ g, add water to 1 liter, wherein, described per-cent is to be the per-cent of the solid weight of matrix to volume of water with water.
6, according to the method for claim 3 or 4 or 5 described production laccases, it is characterized in that: in substratum, add 2 after described fermentation culture 3-5 days, the 5-xylidine.
7, the method for production laccase according to claim 6 is characterized in that: the amount of described adding 2,5 xylidines is 10 μ M.
8, the method for production laccase according to claim 3 is characterized in that, the described laccase that extracts from fermention medium may further comprise the steps: centrifugation, ultrafiltration and concentration, ammonium sulfate precipitation, ion exchange column and gel-filtration column.
9, the method for production laccase according to claim 8 is characterized in that: described ultrafiltration and concentration molecular weight cut-off is 10,000 ten thousand daltonian ultrafiltration post ultrafiltration; Described ammonium sulfate precipitation adopts the ammonium sulfate saturation ratio of two step ammonium sulfate precipitations to be respectively 30%～40% and 70%～90%.
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| CN101955952A (en) * | 2010-08-02 | 2011-01-26 | 安徽大学 | Bacterial laccase gene and expression and application thereof |
| CN102154152A (en) * | 2010-12-22 | 2011-08-17 | 东北林业大学 | Laccase from Bacillus amyloliquefaciens LS05 and use thereof |
| CN102634491A (en) * | 2006-09-01 | 2012-08-15 | 维莱尼姆公司 | Laccase for pulp bio-bleaching |
| CN102676550A (en) * | 2012-05-17 | 2012-09-19 | 浙江商达环保有限公司 | Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene |
| CN102719410A (en) * | 2012-06-29 | 2012-10-10 | 浙江农林大学 | Culture medium formula and preparation method special for laccase |
| CN103420488A (en) * | 2012-05-22 | 2013-12-04 | 上海医药工业研究院 | Compound microorganism live bacteria preparation for degrading papermaking wastewater COD, preparation method and applications thereof |
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| BR9508113A (en) * | 1994-06-24 | 1998-07-14 | Novo Nordisk Biotech Inc | Construction of recombinant vector enzyme recombinant host cell processes to obtain a laccase enzyme to polymerize a lingnin or lingosulfate substrate in solution to depolymerize in situ kraft paste to oxidize dyes or dye precursors to dye hair and to polymerize or oxidize a phenolic compound or aniline dye and container composition |
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| CN102634491A (en) * | 2006-09-01 | 2012-08-15 | 维莱尼姆公司 | Laccase for pulp bio-bleaching |
| CN101955952A (en) * | 2010-08-02 | 2011-01-26 | 安徽大学 | Bacterial laccase gene and expression and application thereof |
| CN102154152B (en) * | 2010-12-22 | 2012-12-12 | 东北林业大学 | Laccase from Bacillus amyloliquefaciens LS05 and use thereof |
| CN102154152A (en) * | 2010-12-22 | 2011-08-17 | 东北林业大学 | Laccase from Bacillus amyloliquefaciens LS05 and use thereof |
| CN102676550A (en) * | 2012-05-17 | 2012-09-19 | 浙江商达环保有限公司 | Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene |
| CN103420488A (en) * | 2012-05-22 | 2013-12-04 | 上海医药工业研究院 | Compound microorganism live bacteria preparation for degrading papermaking wastewater COD, preparation method and applications thereof |
| CN103420488B (en) * | 2012-05-22 | 2014-09-17 | 上海医药工业研究院 | Compound microorganism live bacteria preparation for degrading papermaking wastewater COD, preparation method and applications thereof |
| CN102719410A (en) * | 2012-06-29 | 2012-10-10 | 浙江农林大学 | Culture medium formula and preparation method special for laccase |
| CN104561105A (en) * | 2014-12-15 | 2015-04-29 | 华南理工大学 | Method for synthesizing gold nanoparticles by using pycnoporus sanguineu organisms and application of method for synthesizing gold nanoparticles by using pycnoporus sanguineu organism |
| CN104561105B (en) * | 2014-12-15 | 2018-01-16 | 华南理工大学 | A kind of method and its application of pycnoporus samguineus biosynthesis gold nano grain |
| CN106867956A (en) * | 2017-04-28 | 2017-06-20 | 福建农林大学 | A kind of culture medium for promoting phellinus liteus to grow |
| CN109619658A (en) * | 2019-01-22 | 2019-04-16 | 云南中烟工业有限责任公司 | A kind of method digesting offal silk and the offal silk after enzymatic hydrolysis are for the purposes in tobacco |
| CN112852637A (en) * | 2019-11-28 | 2021-05-28 | 中国科学院微生物研究所 | Korea haemolytica WYS377 and application thereof in laccase production |
| CN112852637B (en) * | 2019-11-28 | 2022-04-26 | 中国科学院微生物研究所 | Korea haemolytica WYS377 and application thereof in laccase production |
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