CN102154147B - Bacillus pumilus LC01 laccase and application thereof - Google Patents
Bacillus pumilus LC01 laccase and application thereof Download PDFInfo
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- CN102154147B CN102154147B CN 201010599461 CN201010599461A CN102154147B CN 102154147 B CN102154147 B CN 102154147B CN 201010599461 CN201010599461 CN 201010599461 CN 201010599461 A CN201010599461 A CN 201010599461A CN 102154147 B CN102154147 B CN 102154147B
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- bacillus pumilus
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Abstract
The invention discloses a catalytic property of bacillus pumilus LC01 (CGMCC No.4257) spore laccase and application of the laccase in dye decoloring. The bacillus pumilus LC01 spore laccase has good catalytic activity under the conditions of alkaline and high temperature and can function at the pH of 2.2-10.0 at the temperature of 20-100 DEG C, wherein the optimum reaction temperature is 80 DEG C. The laccase disclosed by the invention has a good decoloring effect on synthesized dye with different structures in the presence of a vector and can be used for treatment of industrial dye waste water.
Description
Technical field
The present invention relates to a kind of bacterial laccase, be specifically related to the catalytic property of the gemma laccase of a bacillus pumilus LC01.In addition, the invention still further relates to this gemma laccase and make the method for synthetic dyestuff decolouring under amboceptor participates in, belong to the using microbe field.
Background technology
Laccase (EC 1.10.3.2) is a kind of polyphenoloxidase of cupric, and it can utilize the aromatic compound of the Copper ioncatalyst oxidation various structures in active centre, simultaneously molecular oxygen is reduced into water.Because the substrate scope of Laccase Catalyzed is very extensive, so the laccase enzyme has been widely used in the fields such as biological restoration of paper industry, textile industry, foodstuffs industry and soil.
The Laccase Catalyzed oxidation substrates generally comprises two kinds of modes of action, the first be substrate molecule directly and bunch reaction of laccase copper be oxidized to corresponding excited state molecule.Yet some substrate can not be by the laccase direct oxidation, and reason is that their molecules can not penetrate into the reactive site of enzyme too greatly, perhaps can not be by the laccase direct oxidation because its redox potential is higher.The second way of laccase oxidation substrates is to be medium by some micromolecular amboceptor materials, laccase first is oxidized to excited state with the small molecules amboceptor, then the amboceptor of these excited state again with substrate reactions macromolecular or that redox potential is higher, this mode is also referred to as laccase-mediator system (LMS).The application of laccase-mediator system helps further to enlarge the substrate scope of laccase, improves the industrial applicability of laccase.
At occurring in nature, laccase is distributed widely in plant, fungus and bacterium, and particularly fungi, be the main producer of present laccase.Although at present the theoretical investigation of bacterial laccase is goed deep into not as fungal laccase, bacterium has the advantages that growth is fast, be easy to cultivate, bacterial laccase has better stability under alkaline environment and high temperature than the fungi laccase.Because the general temperature such as industrial paper waste is higher, alkalescence is stronger, and fungal laccase is subject to the restriction of poor stability in actual applications, so bacterial laccase has a better application prospect industrial.The gemma coat composition CotA of bacillus is a kind of albumen with laccase activity, because bacterial spore has stronger resistance to multiple unfavorable factors such as high temperature, high pressure, extreme pH and high salt, so the gemma laccase can show because of the characteristic of this special tectonic of gemma better temperature stability and pH stability.
Dyestuff is widely used in the industries such as weaving, printing and dyeing, leather, the whole world is annual produce ten thousand kinds of dyestuffs after, the dyestuff that has 10-15% at least, does great damage to the ecotope of water body in physical environment by discharge of wastewater.Mostly the synthetic dyestuff of industrial application are aromatics, complex structure, difficult degradation.The treatment process of waste water from dyestuff mainly contains physico-chemical processes and biological treatment at present, and materialization treatment technology expense is high, has secondary pollution, and biological process becomes because of its economic environmental protection the method for administering preferably.Wherein laccase is owing to having substrate-function scope widely, but the dyestuff of catalyzed degradation various structures has application prospect preferably on industrial dye waste water is processed.Existing much about the research of the laccases decolouring dyestuff of using whiterot fungi or other originated from fungus, but less at the research report aspect dye decolored about bacterial laccase and bacterial laccase-mediator system.By the decolorizing effect of research bacterial laccase to different synthetic dyestuff, can further promote the industrial application of bacterial laccase.
Summary of the invention
The object of the invention is to provide a kind of bacillus pumilus (Bacillus pumilus) LC01, the present invention also aims to simultaneously provide the catalytic property of a kind of bacillus pumilus (Bacillus pumilus) LC01 gemma laccase, and a kind of method of utilizing the gemma laccase decolouring synthetic dyestuff of bacillus pumilus (Bacillus pumilus) LC01 product is provided.
Bacillus pumilus provided by the invention (Bacillus pumilus) LC01 has been preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 22nd, 2010, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.4257.
Above-mentioned strain of i (bacillus) pumilus is to separate from forest farm, Baolong shop, Heilongjiang Province of China Wuchang City forest topsoil to obtain.Bacillus pumilus LC01 bacterial strain Gram-positive is given birth to amphitrichous in gemma.On solid Luria-Bertani (LB) substratum, cultivate 24h for 37 ℃, the little yellow of bacterium colony, flat rounded, bacterium colony surface wettability, smooth, colony edge are lobate, the opaque and positive and negative solid colour of bacterium colony.The optimum growth temperature of bacterial strain is 30 ℃, and the most suitable growth pH is 6.0, can tolerate the NaCl of 1%-13%.
The gemma laccase that bacillus pumilus LC01 of the present invention produces can play a role in pH value 2.2-10.0 scope, and the optimum pH of the different substrates of catalysis is different: during take ABTS as substrate, enzyme is lived the highest when pH3.0; During take syringaldazine as substrate, enzyme is lived the highest when pH6.6; When being substrate with 2,6-syringol, enzyme is lived the highest when pH8.6.The gemma laccase that bacillus pumilus LC01 of the present invention produces has extremely strong stability under alkaline condition, can keep 68.56% of initial enzyme work after 10 days in 30 ℃ of insulations under the condition of pH9.0.These characteristics are that most fungal laccases are not available, because paper industry much adopts alkaline process, make paper waste alkalescence higher, and this gemma laccase fabulous stability under alkaline condition is conducive to it and brings into play useful effect in the processing such as paper waste.
The enzymatic reaction temperature range of the gemma laccase that bacillus pumilus LC01 of the present invention produces is wider, its optimum temperuture is 80 ℃, can keep higher activity between 50-100 ℃, be 90.47% of the highest enzyme work in the time of 90 ℃, even still can keep 86.06% of the highest enzyme work under the high temperature of 100 ℃, fungal laccase completely loses activity substantially at this temperature.Simultaneously, this gemma laccase has thermostability preferably, and after insulation 10h under 50 ℃ and 60 ℃, its enzyme work is respectively 62.43% and 23% of initial activity, and the transformation period under 80 ℃ is more than 1h.This gemma laccase at high temperature fabulous stability has a very large actual application value industrial.Metallic cation has larger impact to the activity of described laccase, as Na
+, K
+, Mg
2+And Ca
2+Its activity there is activation, and Ag
+, Cu
2+, Co
2+, Mn
2+, Al
3+And Hg
2+Work has restraining effect to enzyme, wherein Hg
2+And Mn
2+Can make gemma laccase complete deactivation of the present invention.Gemma laccase activity change under the NaCl of different concns that bacillus pumilus LC01 of the present invention produces is larger, 0.01mol/L NaCl the activity of described gemma laccase is had promoter action, 0.1mol/L the NaCl of above concentration has restraining effect to activity, can keep 21.53% of original activity when the NaCl of 1mol/L exists.
The gemma laccase that bacillus pumilus LC01 of the present invention produces has decolorization preferably to the industrial synthetic dyestuff of different chemical structures.In the presence of amboceptor Syringylethanone, when pH6.6, the percent of decolourization after RBBR, reactive black KN-B, isatin and Viola crystallina 6h is respectively 70.62%, 92.71%, 89.42% and 95.02%, reactive black KN-B and Viola crystallina are almost completely decoloured.When pH9.0, the percent of decolourization after RBBR, reactive black KN-B, isatin and Viola crystallina 6h is respectively 90.57%, 91.60%, 96.83% and 95.15%, illustrates that this gemma laccase has still kept higher decoloring ability to various dyestuffs under alkaline environment.
Bacillus pumilus provided by the present invention (Bacillus pumilus) LC01 is through fermentation culture, prepared gemma laccase has pH and temperature catalysis scope widely, have preferably stablely under alkalescence and hot conditions, have more superior suitability than the laccase of originated from fungus.In the presence of amboceptor material Syringylethanone, this gemma laccase can effectively decolour to the synthetic dyestuff of different chemical structures, still kept decolorizing effect preferably under alkaline condition, therefore gemma laccase provided by the invention has application prospect preferably in the processing of industrial dye waste water.
Description of drawings
Fig. 1 is the impact of pH on bacillus pumilus LC01 gemma laccase activity during take ABTS as substrate;
Fig. 2 is the impact of pH on bacillus pumilus LC01 gemma laccase activity during take syringaldazine as substrate;
Fig. 3 is the impact of pH on bacillus pumilus LC01 gemma laccase activity when being substrate with 2,6-syringol;
Fig. 4 is bacillus pumilus LC01 gemma laccase enzyme when pH9.0 time history plot alive;
Fig. 5 is that differing temps is on the impact of bacillus pumilus LC01 gemma laccase activity;
Fig. 6 is the stability of bacillus pumilus LC01 gemma laccase activity in the time of 50-80 ℃;
Fig. 7 is that the NaCl of different concns is on the impact of bacillus pumilus LC01 gemma laccase activity;
When Fig. 8 is pH6.6 bacillus pumilus LC01 gemma laccase take Syringylethanone as amboceptor to the decolouring result of RBBR, reactive black KN-B, isatin and Viola crystallina
When Fig. 9 is pH9.0 bacillus pumilus LC01 gemma laccase take Syringylethanone as amboceptor to the decolouring result of RBBR, reactive black KN-B, isatin and Viola crystallina
Embodiment
Following embodiment is in order to understand better the present invention.
Test materials used in following examples if no special instructions, is the biochemical reagents that can buy.
The enzyme activity determination method refers to calculate laccase activity with absorbancy after spectrophotometer detection laccase and substrate reactions.Described substrate is syringaldazine, and 2,2-azine-two (3-ethyl benzothiazole-6-sulfonic acid) (ABTS) or 2,6-syringol.
It is that the required enzyme amount of product is an enzyme activity unit that the activity of enzyme is defined as per minute oxidation 1 μ mol substrate.Reaction system is as follows:
(1) ABTS: reaction system contains 1mmol/L ABTS, 100 μ L gemma suspensions, and citric acid-phosphate buffered saline buffer (0.1mol/L, pH 3.0), cumulative volume 3mL, after 30 ℃ of reaction 3min at 420nm place mensuration light absorption value.
(2) syringaldazine: reaction system contains the 0.1mmol/L syringaldazine, 100 μ L gemma suspensions, and citric acid-phosphate buffered saline buffer (0.1mol/L, pH 7.0), cumulative volume 3mL, after 30 ℃ of reaction 3min at 525nm place mensuration light absorption value.
(3) 2,6-syringol: the system of answering contains 2mmol/L 2, the 6-syringol, 100 μ L gemma suspensions, and citric acid-phosphate buffered saline buffer (0.1mol/L, pH 7.0), cumulative volume 3mL, after 30 ℃ of reaction 5min at 468nm place mensuration light absorption value.
The preparation of embodiment 1 bacillus pumilus LC01 gemma laccase
Bacillus pumilus LC01 prepares the gemma laccase by following process after cultivating for some time on product spore substratum.
Above-mentioned product spore culture medium prescription (g/L) is: nutrient broth 8; KCl 1; MgSO
47H
2O 0.25; MnCl
24H
2O 0.002; Agar 17; Transfer autoclave sterilization after pH7.0, add the 0.5mmol/LCaCl of filtration sterilization
2And 0.001mmol/L FeSO
4
The preparation process of gemma laccase is:
1) bacillus pumilus LC01 streak inoculation is produced on the spore substratum in solid, after 5d is cultivated in 37 ℃ of inversions.
2) with aseptic deionized water, the gemma on solid medium is rinsed.
3) add the N,O-Diacetylmuramidase of final concentration 0.1mg/mL, 37 ℃ of water-bath 10min.
4) successively with the 1mol/L NaCl that contains 10mmol/L EDTA and 0.3mg/mL PMSF, 0.14mol/L NaCl and 0.1% (w/v) SDS cleans one time.
5) clean one time with aseptic deionized water.
6) clean three times with aseptic deionized water after 80 ℃ of water-bath thermal shock 10min.
7) get a certain amount of suspension after, with all the other suspensions pack into the sterilization after triangular flask in, 4 ℃ keep in Dark Place.
8) suspension that takes out is placed in 80 ℃ of baking ovens dries to constant weight, calculate gemma suspension concentration (mg/ml), and record.
The character of embodiment 2 bacillus pumilus LC01 gemma laccases
1.pH the impact of value on gemma laccase activity and stability
PH adopts pH2.2-7.8 citric acid-phosphate buffered saline buffer (0.1mol/L), pH7.4-9.0Tris-HCl (0.05mol/L) damping fluid and pH8.6-10.0 glycine-sodium hydrate buffer solution (0.05mol/L) to measure on the impact of laccase activity, pH on the impact of laccase stability by 30 ℃ the time with laccase and pH3.0, citric acid-phosphate buffered saline buffer of 7.0, measure remaining activity after mixing placement 1-10d in the Tris-HCl damping fluid of pH9.0.Fig. 1-3 show, gemma Laccase Catalyzed provided by the invention is in extensive range, but all catalytic substrate reactions in the scope of pH2.2-10.0, and with ABTS, the optimal pH that syringaldazine and 2,6-syringol record when being substrate is respectively 3.0,6.6 and 8.6.Fig. 4 shows, bacillus pumilus LC01 gemma laccase has better stability in the environment of pH9.0, still can keep higher activity after 10d.
2. the impact of temperature on gemma laccase activity and stability
Temperature is being measured in 20-100 ℃ of scope under optimal pH the impact of laccase activity.The thermal stability determination of laccase is measured remaining activity after 50-80 ℃ of placement 0-10h in citric acid-phosphate buffered saline buffer of pH6.6.Fig. 5 shows, the optimal reactive temperature of bacillus pumilus LC01 gemma laccase is 80 ℃, as can be seen from Figure 6, bacillus pumilus LC01 gemma laccase 80 ℃ of high temperature half-life more than 1h.
3. the impact of metal ion on the gemma laccase activity
Add respectively common metal ion in reaction system, making its final concentration is 10mmol/L, at 30 ℃ of insulation 5min, take the reaction system that do not add metal ion as blank, then surveys according to a conventional method enzymic activity take syringaldazine as substrate.
4.NaCl the impact on the gemma laccase activity
Bacillus pumilus LC01 gemma laccase is evenly mixed with the NaCl solution of different concns, measure remaining activity take syringaldazine as substrate after 30 ℃ of incubation 5min.Fig. 7 result shows, the following NaCl of 0.01mol/L has promoter action to bacillus pumilus LC01 gemma laccase activity, and activity descends gradually when concentration increases.When NaCl concentration rose to 1mol/L, activity still can keep 21.53% left and right.
The decolouring of embodiment 3 bacillus pumilus LC01 gemma laccases to synthetic dyestuff
1. the calculating of the reaction system of decolorization experiment and percent of decolourization
The experiment reaction system is 6ml, adds successively pH 6.6 citric acids-phosphate buffered saline buffer (0.1mol/L), dyestuff (kind and final concentration see Table 1), bacillus pumilus LC01 gemma laccase (final concentration is 1mg/ml) and Syringylethanone (final concentration is 0.1mmol/L) in system.Simultaneously take the reaction system that do not add bacillus pumilus LC01 gemma laccase as blank.Timing sampling, 12000rpm surveys the light absorption value under each dyestuff maximum absorption wavelength (seeing Table 1) after centrifugal 2min.All are measured equal triplicate and average.Calculate afterwards dye decolored rate.The percent of decolourization calculation formula of dyestuff is (A
0-A)/A
0* 100%, A wherein
0Be initial dyestuff light absorption value, A is the light absorption value that regularly sampling is surveyed.
Table 1 dye type, final concentration and maximum absorption wavelength
Result shows, bacillus pumilus LC01 gemma laccase demonstrates the ability of stronger decolouring dyestuff.In the presence of amboceptor Syringylethanone, through the percent of decolourization of the synthetic dyestuff of four kinds of different structures after 6h decolouring all more than 70%, wherein to the percent of decolourization of Viola crystallina up to 95.02% (Fig. 8).
2. the decolorization experiment of dyestuff under alkaline condition
Because the advantage of bacterial laccase is to have the catalytic activity higher than fungal laccase under alkaline condition, for further investigate bacterial laccase in alkaline environment to the decoloring ability of dyestuff, select the condition research bacillus pumilus LC01 gemma laccase of pH9.0 to the decolouring of four kinds of synthetic dyestuff.Through after 6h, the percent of decolourization of RBBR, reactive black KN-B, isatin and Viola crystallina is respectively 90.57%, 91.60%, 96.83% and 95.15%, this result is higher than the percent of decolourization of various dyestuffs under pH6.6, show that this gemma laccase has still kept higher decoloring ability to various dyestuffs under alkaline environment, have suitability preferably when processing the waste water from dyestuff of different pH values.
Claims (1)
1. bacillus pumilus (Bacillus pumilus) LC01, the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.4257.
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| CN102757909B (en) * | 2012-06-19 | 2013-05-29 | 南京农业大学 | Death bacillus vallismortis for producing spore laccase and application of death bacillus vallismortis |
| CN103320453A (en) * | 2013-06-25 | 2013-09-25 | 江南大学 | Bacillus pumilus laccase gene as well as expression and application thereof |
| CN104495971B (en) * | 2014-11-27 | 2016-09-07 | 福建农林大学 | After pleurotus eryngii is gathered, planting material processes the method containing aniline blue pollutant effluents |
| CN104830741A (en) * | 2015-05-28 | 2015-08-12 | 陕西科技大学 | Preparing method of compound microorganism agent for papermaking wastewater |
| CN105776567A (en) * | 2015-12-31 | 2016-07-20 | 中国科学院成都生物研究所 | Laccase composite dielectric body and decolorization method |
| CN110642461B (en) * | 2019-09-23 | 2021-11-02 | 西南科技大学 | Alkali-activated leather-immobilized bacillus pumilus/H2O2Method for purifying printing and dyeing wastewater |
| CN114088864B (en) * | 2021-11-22 | 2023-12-15 | 南华大学 | Application of nano sensor with Hg ion triggering catalytic activity in Hg ion detection |
| CN114350554A (en) * | 2021-12-29 | 2022-04-15 | 西南科技大学 | Preparation and application method of radioactive decontamination strippable membrane biodegradation microbial inoculum |
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