CN1308435C - Laccase, preparing method thereof and strain special for same - Google Patents

Laccase, preparing method thereof and strain special for same Download PDF

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Publication number
CN1308435C
CN1308435C CNB2004100340160A CN200410034016A CN1308435C CN 1308435 C CN1308435 C CN 1308435C CN B2004100340160 A CNB2004100340160 A CN B2004100340160A CN 200410034016 A CN200410034016 A CN 200410034016A CN 1308435 C CN1308435 C CN 1308435C
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laccase
pycnoporus
cgmcc
fermentation
ultrafiltration
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CN1690186A (en
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郭林
王文惠
张虎成
李伟
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Institute of Microbiology of CAS
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Abstract

The present invention discloses laccase, a method for producing laccase and a special bacterial strain for producing laccase, which relates to the technical field of microorganism fermenting industry. The bacterial strain which is provided by the present invention is pycnoporus sanguineus mk528 with CGMCC No. 1124; the laccase which is provided by the present invention is obtained by fermenting pycnoporus sanguineus mk528 with CGMCC No. 1124. The method for producing laccase comprises the following steps: the pycnoporus sanguineus mk528 with CGMCC No. 1124 is cultivated by fermentation, and the laccase is extracted from a fermentation medium. The activity of the laccase in the fermentation liquid obtained by fermentation of pycnoporus sanguineus mk528 with CGMCC No. 1124 provided by the present invention reaches 63 U/ml; moreover, lignin peroxidase and manganese peroxidase can not be generated in the process of cultivation of the bacterial strain, which is convenient for extraction of laccase. The laccase which is obtained by extraction can be widely used for replacing chemicals, such as formaldehyde, etc., in paper making industry, waste water treatment industry and wood processing industry.

Description

A kind of laccase and production method thereof and special preparing strain
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 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.
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 10000 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 L50mM 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 (6)

1, pycnoporus samguineus (Pycnoporus sanguineus) bacterial strain mk528, its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC № 1124.
2, a kind of method of producing laccase is included in the step that aerobic and lucifuge condition bottom fermentation are cultivated the described pycnoporus samguineus mk528 of claim 1 and extract laccase from fermention medium; The substratum of described fermentation culture comprises maltose, ammonium tartrate and inorganic component, adds 2 in substratum after fermentation culture 3-5 days, the 5-xylidine; The described laccase that extracts from fermention medium may further comprise the steps: centrifugation, ultrafiltration and concentration, ammonium sulfate precipitation, ion exchange column separate and separate with gel-filtration column.
3, method according to claim 2 is characterized in that: the inorganic component in the described substratum comprises ferrous sulfate, sal epsom, manganous sulfate, zinc sulfate, copper sulfate, calcium chloride and cobalt chloride.
4, according to claim 2 or 3 described methods, it 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.
5, according to claim 2 or 3 described methods, it is characterized in that: described adding 2, the amount of 5-xylidine are 10 μ M.
6, according to claim 2 or 3 described methods, it is characterized in that: described ultrafiltration and concentration molecular weight cut-off is 10000 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%.
CNB2004100340160A 2004-04-21 2004-04-21 Laccase, preparing method thereof and strain special for same Expired - Fee Related CN1308435C (en)

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DK2059587T3 (en) * 2006-09-01 2012-03-19 Verenium Corp Laccases for bio-bleaching of pulp
CN101955952B (en) * 2010-08-02 2012-05-09 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
CN102676550B (en) * 2012-05-17 2013-08-07 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
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
CN102719410B (en) * 2012-06-29 2016-01-20 浙江农林大学 A kind of culture medium prescription being exclusively used in laccase and preparation method thereof
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
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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