Puri?cation,biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens
Jing Si a ,Feng Peng b ,Baokai Cui a ,?
a Institute of Microbiology,Beijing Forestry University,Beijing 100083,China
b
Institute of Biomass Chemistry and Technology,Beijing Forestry University,Beijing 100083,China
h i g h l i g h t s
"A novel laccase (Tplac)from white rot fungus Trametes pubescens was puri?ed and characterized."Tplac performed better catalytic ef?ciency toward ABTS with k cat /K m at 8.34s à1
l M à1.
"Tplac was highly stable and resistant under alkaline conditions.
"Tplac was intrinsically highly metal-tolerant by enhancing the af?nity toward substrate."Tplac could degrade and detoxify dyes used in textile industries.
a r t i c l e i n f o Article history:
Received 5July 2012
Received in revised form 16October 2012Accepted 19October 2012
Available online 29October 2012Keywords:
Trametes pubescens laccase Puri?cation
Alkali-resistant capacity Metal tolerance
Dye decolorization application
a b s t r a c t
Extracellular laccase (Tplac)from Trametes pubescens was puri?ed to homogeneity by a three-step method,which resulted in a high speci?c activity of 18.543U mg à1,16.016-fold greater than that of crude enzyme at the same level.Tplac is a monomeric protein that has a molecular mass of 68kDa.The enzyme demonstrated high activity toward 1.0mM ABTS at an optimum pH of 5.0and temperature of 50°C,and under these conditions,the catalytic ef?ciency (k cat /K m )is 8.34s à1l M à1.Tplac is highly sta-ble and resistant under alkaline conditions,with pH values ranging from 7.0to 10.0.Interestingly,above 88%of initial enzyme activity was maintained in the presence of metal ions at 25.0mM,leading to an increase in substrate af?nity,which indicated that the laccase is highly metal-tolerant.These unusual properties demonstrated that the new fungal laccase Tplac has potentials for the speci?c industrial or environmental applications.
ó2012Elsevier Ltd.All rights reserved.
1.Introduction
Laccase (benezenediol:oxygen oxidoreductase,EC 1.10.3.2),the most abundant member of the multicopper protein family,is widely distributed in plants,fungi,insects,and bacteria (Claus,2004).This protein contains four histidine-rich copper binding do-mains,which coordinate copper atoms types I–III that differ in their environment and spectral properties (Thurston,1994).The enzyme can catalyze the oxidation of an array of substrates,such as mono-,di-,and polyphenols,aromatic amines,methoxyphenols,and ascorbate through a one-electron transfer.The oxidation is coupled to the reduction of oxygen to H 2O (Thurston,1994).Fur-thermore,laccase is of particular interest with regards to various commercial applications because of its ability to oxidize a wide range of reaction capabilities and relevant substrate speci?cities.Thus,research concerning laccase is being carried out in various ?elds of interest:textile,pulp and paper,food,and cosmetics industries,as well as in bioremediation,biosensor,biofuel,and organic synthesis applications (Arora and Sharma,2010).To date,more than 100laccases have been isolated from different microor-ganisms.However,most of these laccases are ‘common’with a lower yield of enzymatic activity and tolerance to extreme condi-tions (Kim et al.,2012).This reduced performance hampers their large-scale commercial and industrial use for most applications.Therefore,it is necessary to search for novel laccases with higher yields of activity and versatile properties.
Global industrialization has resulted in the release of large amounts of potentially toxic compounds into the biosphere (Gomi et al.,2011).Among these compounds,dye-containing ef?uents represent highly problematic wastewaters due to their higher chemical (COD)and biochemical oxygen demand (BOD),sus-pended solids,and the content of toxic compounds,as well as their color,which makes them easily recognized and poses esthetic
0960-8524/$-see front matter ó2012Elsevier Ltd.All rights reserved.https://www.doczj.com/doc/256422139.html,/10.1016/j.biortech.2012.10.085
?Corresponding author.Address:Institute of Microbiology,Beijing Forestry University,P.O.Box 61,Qinghuadong Road 35,Haidian District,Beijing 100083,China.Tel./fax:+861062336309.
E-mail address:baokaicui@https://www.doczj.com/doc/256422139.html, (B.Cui).
problems(Jonstrup et al.,2011).Cleaning up the environment by removal of hazardous contaminants from textile ef?uents is a cru-cial and challenging problem that requires numerous approaches to reach long-lasting suitable solutions.Among the various types of dyes in the textile processing industry,azo dyes are extensively used,and they dominate the dyestuff market with a share of approximately70%(Enayatizamir et al.,2011).Physical and chem-ical methods have been adopted in the treatment of azo dyes,but they have led to the generation of secondary pollution by releasing hazardous byproducts(Kalpana et al.,2011).Thus,microbial treat-ment of dyes has gained popularity due to its safety,ef?ciency,and ability to transform hazardous chemicals into less toxic com-pounds(Asgher et al.,2008).
White rot basidiomycetes are among the most potent organ-isms to biodegrade and detoxify a wide range of wastes and pollu-tants,as carried out by phenol-targeting redox enzymes,namely, laccases and peroxidases(Mendon?a et al.,2008).However,waste-water discharged from textile industries characterized by neutral or alkaline pHs and high concentrations of metal ions is causing serious threats and severely damaging the natural habitat(Xiao et al.,2012).These conditions limit the functions of fungal laccases and can cause them to lose their activities.Thus,exploring novel laccases that can be directly used under the aforementioned spe-cial conditions is an important job in the area of dye degradation.
Trametes pubescens is a common white-rot fungus,and its crude enzyme,which was previously extracted and acclimatized,was used for dye decolorization(Roriz et al.,2009).Accordingly,the present paper reports on the puri?cation and biochemical charac-terization of a novel alkali-resistant and metal ion-tolerant laccase Tplac from white rot fungus T.pubescens.The enzyme was puri?ed by anionic exchange and Sepharose chromatography and evaluated for its potentials for dye decolorization.
2.Methods
2.1.Dyes and chemicals
The dyes used in this study were prepared by being?ltered through a0.22-l m membrane to remove bacteria before use.For this study,2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS),agar powder,and trypsin were all Sigma–Aldrich products (St.Louis,MO,USA).L-Cysteine,L-3,4-dihydroxyphenylalanine (L-DOPA),2,6-dimethoxyphenol(2,6-DMP),dithiothreitol(DTT), sodium azide(NaN3),and protein marker were purchased from TakaRa(Dalian,China).Other chemicals used were of analytical reagent grade.
2.2.Fungal strain and inocula preparation
T.pubescens Cui7571was collected from Chebaling Nature Re-serve of Guangdong Province in China.This strain was maintained through periodic(monthly)transfer on yeast extract glucose agar (YGA)at4°C.The YGA medium used for the experiment contained (g Là1of distilled water):yeast extract5,glucose20,agar20,KH2PO4 1,MgSO4á7H2O0.5,ZnSO4á7H2O0.05,and vitamin B10.01,and the pH value of the medium was adjusted to5.0before sterilization.
Prior to use,the stored fungal strain was inoculated onto newly prepared YGA plates and grown at28°C.Five mycelial disks(1cm diameter)were removed from the peripheral region of the5-day-old YGA plate and used to inoculate into a250-mL Erlenmeyer ?ask containing100mL of yeast extract glucose medium(YG,iden-tical to YGA without agar).The cultivation was carried out in a dark chamber under150rpm shaking speed at28°C.After6days, mycelia were homogenized using an Ace Homogenizer(Hengao Co.,Tianjin,China)at5000rpm for30s,and the pellet suspensions were later prepared as inocula for the next experiment.
2.3.Production and puri?cation of Tplac
An aliquot of10mL of the inocula(0.045g,dry weight)was inoculated into a250-mL Erlenmeyer?ask containing100mL of YG medium and incubated at28°C in a shaking incubator.After 6days,the cultures were centrifuged at12,000rpm for20min to remove mycelia and medium debris,and the cell-free supernatant was used as a crude enzyme solution.
The supernatant was salt fractionated with75%(w/V)ammo-nium sulfate at4°C overnight and dialyzed with a10kDa cut-off membrane against0.1M citrate–phosphate buffer(pH5.0)and further concentrated by PEG20000.The resulting solution was then loaded onto a DEAE-cellulose DE52anionic exchange chroma-tography column(30?2.6cm;Pharmacia)pre-equilibrated with 0.1M citrate–phosphate buffer(pH5.0)overnight at a?ow rate of 1.0mL minà1.The laccase protein was eluted initially with 0.1M citrate–phosphate buffer(pH5.0)and subsequently with a linear salt gradient of0–1.0M NaCl solution in0.1M citrate–phos-phate buffer(pH5.0)at a?ow rate of2.0mL minà1.Activity frac-tions were assayed for protein contents by the Bradford method using bovine serum albumin as standard protein(Bradford, 1976),and the laccase activity of each fraction was determined at420nm using1.0mM ABTS as substrate(Kalyani et al.,2008). One unit was de?ned as the amount of enzyme that oxidized 1l mol of substrate per minute.Fractions containing the main lac-case activity were collected,pooled,dialyzed,and concentrated by PEG20000.Next,the eluted solution was applied to a Sepharose GL-6B chromatography column(60?2.6cm;Pharmacia)pre-equilibrated with0.1M citrate–phosphate buffer(pH5.0)over-night at a?ow rate of1.0mL minà1.The laccase was re-eluted with 1.0M NaCl in0.1M citrate–phosphate buffer(pH 5.0)at 1.5mL minà1and monitored as mentioned above.Finally,fractions containing the main laccase activity were collected,pooled,dia-lyzed,concentrated by PEG20000,and stored atà20°C until fur-ther use.
2.4.Biochemical characterization of puri?ed Tplac
2.4.1.Gel electrophoresis and the spectral property of puri?ed Tplac
The puri?ed laccase Tplac was subjected to sodium dodecyl sul-fate–polyacrylamide gel electrophoresis(SDS–PAGE)for molecular mass determination.This assay was performed according to a pre-viously described protocol(Eisenman et al.,2007)with a5%(w/V) stacking gel and a12%(w/V)separating gel using a vertical gel electrophoresis system(Bio-Rad).The sample was dissolved in2 volumes of4?loading buffer and denatured by incubating at 100°C for8min.After electrophoresis,the gel was stained with Coomassie Brilliant Blue R-250for2h at room temperature,and the molecular mass of Tplac was measured by comparison with a protein marker.Similarly,protein with laccase activity and its iso-enzyme were evaluated using non-denaturing PAGE(native PAGE) on a5%stacking gel and a12%separating gel.The native gel was stained with0.1M citrate–phosphate buffer(pH5.0)containing 1.0mM ABTS or1.0mM guaiacol.
The UV–visible adsorption spectrum of puri?ed Tplac in0.1M citrate–phosphate buffer(pH5.0)was recorded between200and 800nm with a UV–visible spectrophotometer(UNICO4802,You-nike Co.,Shanghai,China).
2.4.2.Internal amino acid sequence of puri?ed Tplac
The puri?ed Tplac was loaded onto SDS–PAGE.After electro-phoresis and protein visualization,the laccase bands of interest were cut up from the gel and digested overnight using trypsin as
50J.Si et al./Bioresource Technology128(2013)49–57
described earlier(Shevchenko et al.,1996).The cleaved peptides were eluted and analyzed by nano liquid chromatography coupled with tandem mass spectrometry(LC–MS/MS)for interest amino acid sequencing.Amino acid sequences were identi?ed by homol-ogy using an mass spectrometry data analysis program,SEQUEST (Thermo Finnigan,San Jose,CA,USA),against the database of the National Center for Biotechnology Information(NCBI)fungal lac-case sequence database,and aligned across thirteen laccases by ClustalX1.83algorithm and DNAMAN6.0software.
2.4.
3.Effects of pH and temperature on the activity and stability of puri?ed Tplac
The effect of pH value on Tplac activity was determined in the citrate–phosphate buffer within a pH range of1.0–13.0at25°C using1.0mM ABTS as substrate.The pH stability of the enzyme was assessed by pre-incubating the enzyme in citrate–phosphate buffer with pH values ranging from1.0to13.0at25°C for72h, and the residual laccase activities were determined with ABTS as substrate.
The optimum temperature for Tplac was examined in the cit-rate–phosphate buffer with different temperatures from10to 90°C at pH5.0using1.0mM ABTS as substrate.The thermal stabil-ity of the puri?ed laccase was evaluated by pre-incubating the en-zyme in citrate–phosphate buffer(pH 5.0)with different temperatures from10to90°C for2h,and the residual laccase activities were determined with ABTS as substrate.
Aliquots of samples were taken at regular intervals and were centrifuged at12,000rpm for20min and the supernatant was used for laccase activity determination.Experiments were all per-formed in triplicate and laccase activities at the optimum pH or temperature were taken as control(100%).
2.4.4.Substrate speci?city and kinetic property of puri?ed Tplac
Various substrates,i.e.,ABTS,catechol,2,6-DMP,L-DOPA,ferulic acid,guaiacol,hydroquinone,phenol,pyrogallol,syringaldazine, tyrosine,and veratryl alcohol,were used to determine the sub-strate speci?city of Tplac at1.0mM in0.1M citrate–phosphate buffer(pH5.0).The rate of substrate oxidation was determined by measuring the absorbance increase at the respective wave-length,and the molar extinction coef?cient(e m)of each substrate was obtained from the literature(Eisenman et al.,2007;Kalyani et al.,2012;Litthauer et al.,2007).Experiments were all performed in triplicate.
The apparent Michaelis–Menten constant(K m)and catalytic constant(k cat)of Tplac were determined using ABTS as substrate in0.1M citrate–phosphate buffer(pH5.0)at50°C.A Linewe-aver–Burk plot was made from the initial oxidation rates at differ-ent ABTS concentrations ranging from0.1mM to 1.0mM.The catalytic ef?ciency(speci?city constant,k cat/K m)of the puri?ed en-zyme was calculated according to K m and k cat data.
2.4.5.Effects of inhibitors and metal ions on the activity of puri?ed Tplac
The effects of various inhibitors(0.05,0.1,and1.0mM)on puri-?ed Tplac activity were investigated using L-cysteine,DTT,EDTA, and NaN3.The remaining laccase activity was measured by pre-incubating the puri?ed enzyme in the presence of each inhibitor at50°C for15min using ABTS as substrate.Experiments were all performed in triplicate.
The effects of various metal ions,at concentration of25.0mM, on puri?ed Tplac activity were also evaluated by separately adding Cu2+(copper sulfate),K+(potassium chloride),Na+(sodium chlo-ride),Mn2+(manganese sulfate),Ca2+(calcium chloride),Fe2+(fer-rous sulfate),Fe3+(ferric chloride),Mg2+(magnesium chloride), Zn2+(zinc sulfate),Ba2+(barium chloride),or Al3+(aluminum chlo-ride)to the reaction mixture.Similarly,the enzymatic assays were conducted under the aforementioned conditions,and experiments were all performed in https://www.doczj.com/doc/256422139.html,ccase activity determined at the optimum pH and temperature conditions in the absence of any inhibitor or metal ion was taken as control(100%).K m and k cat val-ues of puri?ed Tplac in the presence of metal ions at25.0mM were determined using ABTS as substrate.
2.5.Dye decolorization capacity of puri?ed Tplac
2.5.1.Dye decolorization
The decolorization capacity of the puri?ed laccase Tplac for structurally various dyes was monitored by the decrease in absor-bance at the wavelength of each dye.The10.0mL reaction mix-tures for dye decolorization contained50.0mg Là1dye in0.1M citrate–phosphate buffer(pH5.0)and1.0U mLà1pure enzyme solution.In all the cases,the mixtures were incubated in a dark chamber under150rpm shaking speed at50°C.In parallel,the negative control contained all components except enzyme,and experiments were all performed in triplicate.Aliquots of samples were taken at regular intervals and were centrifuged at 12,000rpm for20min and the supernatant was used for decolor-ization determination.Decolorization rate was expressed in terms of percentage and calculated as follows:
Decolorization ratee%T?
Initial absorbanceàFinal absorbance
Initial absorbance
?100
2.5.2.Effects of heavy metal ions on the dye decolorization capacity of puri?ed Tplac
The effects of heavy metal ions on Congo Red biodegradation capacity of puri?ed Tplac were studied by separately adding Cu2+ (copper sulfate),Zn2+(zinc sulfate),or Fe3+(ferric chloride)into the dye decolorization reaction mixtures and their concentrations were varied in the range of20.0–60.0mM.Similarly,experiments were all performed in triplicate,and the decolorization rate deter-mination was conducted under the aforementioned conditions. Dye decolorization rate determined in the absence of any heavy metal ion was taken as control.
2.5.
3.Degraded metabolites identi?cation
Once complete dye decolorization was achieved,the metabo-lites formed after biodegradation of Congo Red were extracted three times with an equal volume of ethyl acetate with vigorous shaking.The combined organic phase was?ltered over Na2SO4 on?lter paper and concentrated in a rotary vacuum evaporator. GC–MS analysis was carried out using a QP2010mass spectropho-tometer(Shimadzu model No.U-2800).Ionization voltage was 70eV and the temperature of the injection port was280°C.Gas chromatography was conducted in temperature programming mode with a Resteck column(0.25?30mm,XTI-5).Initial column temperature was80°C for2min,which was later increased line-arly at10°C per min up to280°C and held for7min.GC–MS inter-face was maintained at290°C and helium was used as the carrier gas at a?ow rate of1.0mL minà1with a30min run time.
2.5.4.Phytotoxicity test
The ethyl acetate extracted metabolites of Congo Red formed after biodegradation by Tplac were dried and dissolved in50mL of distilled water to a?nal concentration of2.0g Là1.Seeds of Phaseolus mungo,Sorghum vulgare,and Triticum aestivum were used for phytotoxicity tests,and the experiments were carried out at room temperature by placing ten seeds in separate5.0-mL of solutions containing either dye,metabolites,or distilled water.
J.Si et al./Bioresource Technology128(2013)49–5751
Germination (%)and plumule (cm)and radicle recorded after 7days.2.6.Statistical data analysis
The results obtained during experimentation terms of mean values and standard error subjected to statistical analysis of one-way (ANOVA)and Tukey–Kramer comparison test software.Probability (P value)less than 0.05or ??
P <0.01)was considered signi?cant or respectively.
3.Results and discussion 3.1.Puri?cation of laccase Tplac
Since the laccase constitutively produced by basidiomycete fun-gi can be used in many ?elds,it is necessary to develop an effective large-scale,high-purity production process.In the present study,laccase Tplac of T.pubescens obtained from a 6-day culture was used for subsequent puri?cation.The puri?cation of this laccase was performed using a three-step method of ammonium sulfate precipitation,anionic exchange,and Sepharose chromatography.After ammonium sulfate precipitation,a total amount of about 8.390mg mL à1of protein,corresponding to approximately 36.253U mL à1of laccase activity,was loaded onto DEAE-cellulose DE52anionic exchange chromatography column eluted initially with buffer and subsequently with 0–1.0M NaCl solution.Supple-mentary Fig.S1depicts that there were two apparent fractions containing laccase activity during the elution procedure.Interest-ingly,the fraction containing the higher activity was observed when the eluent was 0.1M citrate–phosphate buffer (pH 5.0),amounting to a speci?c activity of 5.798U mg à1,which was 5.008times higher than that of crude enzyme at identical experi-mental conditions.Furthermore,the fraction was pooled and con-centrated and applied to Sepharose GL-6B chromatography column for further puri?cation (Supplementary Fig.S2).Table 1lists the puri?cation data of Tplac.Overall,the three-step procedure re-sulted in a high speci?c activity of 18.543U mg à1,16-fold greater than that of crude enzyme at the same level.
3.2.Gel electrophoresis and the spectral property of puri?ed Tplac As demonstrated in Fig.1a,the homogeneity of the puri?ed Tplac was veri?ed by SDS–PAGE with Coomassie Brilliant Blue R-250staining analysis.A unique protein band was obtained for Tplac,with a mobility corresponding to a molecular mass of 68kDa,which was higher than that of Trametes versicolor ,which had a molecular mass of 60kDa (Zhu et al.,2011).This could be ex-plained that various compositions of subunits exist in different fungal laccases (Fang et al.,2012).Activity staining of Tplac also re-vealed a single band corresponding to the position of laccase activ-ity,as visualized with ABTS or guaiacol as substrate (Fig.1b).These observations suggested that the puri?ed laccase from T.pubescens is a typical fungal laccase in molecular mass and a monomeric pro-tein in composition.
The nature of the catalytic center was determined by spectral property of the puri?ed laccase (data were not shown).Tplac’s UV–vis spectrum exhibited a shoulder at 340nm,typical of a type III binuclear copper center.An absorption peak at 610nm indicated the presence of a type I copper center,which is considered to be responsible for the enzyme’s blue color (Sadhasivam et al.,2008).3.3.Internal amino acid sequence of puri?ed Tplac
Internal peptide sequencing of the puri?ed Tplac exhibited sev-eral fragments,such as HWHG,GTFWYHSHLSTQYCDGLRG,KRYRFRLVS,and NSAILRY,identical to those of published fungal laccases from Coriolopsis gallica ,Dichomitus squalens ,Lentinus tigri-nus ,Polyporus brumalis ,Polyporus ciliatus ,Trametes sp.420,Tra-metes sp.AH28-2,Trametes trogii ,T.versicolor ,and Trametes villosa respectively,which belong to the multicopper oxidase fam-ily (Fig.2).Additionally,results in Fig.2show that Tplac had two copper binding domains (type I and II)and shared three potential N -glycosylation sites (Thurston,1994).A homology search re-vealed that the deduced gene product had 78.01%,76.76%,82.16%,82.16%,80.50%,82.16%,76.35%,79.67%,76.76%,76.35%,77.18%,or 77.18%amino acid identify with PDB:2VDZ (C.gallica ),EJF60081(D.squalens ),AAX07469.1(L.tigrinus ),PDB:2QT6(L.tigrinus ),ABN13591.1(P.brumalis ),AAG09231.1(P.ciliatus ),AAW28936.1(Trametes sp.420),PDB:3KW7(Trametes sp.AH28-2),PDB:2HRG (T.trogii ),CAA77015(T.versicolor ),EIW62366(T.versicolor ),or AAB47735(T.villosa )respectively.These results im-plied that Tplac from T.pubescens is a typical laccase with the con-served copper binding sites and it has some differences from other fungal laccases,but a new protein.
Table 1
Puri?cation of laccase Tplac from the crude culture of Trametes pubescens .Puri?cation step
Total activity (U mL à1)Total protein (mg mL à1)Speci?c activity (U mg à1)Puri?cation fold Yield (%)Crude culture ?ltrate
44.25338.223 1.158 1.000100Ammonium sulfate precipitation
36.2538.390 4.321 3.73281.92DEAE-cellulose DE52anionic exchange chromatography column
27.372 4.721 5.798 5.00861.85Sepharose GL-6B chromatography column
21.836
1.178
18.543
16.016
49.34
Fig.1.Molecular mass determination of puri?ed Tplac from Trametes pubescens through SDS–PAGE (a)(Lane 1crude culture ?ltrate;Lane 2puri?ed laccase from ammonium sulfate precipitation;Lane 3puri?ed laccase from DEAE-cellulose DE52anionic exchange chromatography;Lane 4puri?ed laccase from Sepharose GL-6B chromatography;M protein molecular mass marker)and zymogram analysis (b)with native PAGE (Lane 1ABTS staining;Lane 2guaiacol staining).
52
3.4.Effects of pH and temperature on the activity and stability of puri?ed Tplac
As shown in Fig.3a,puri?ed laccase Tplac from T.pubescens dis-played activity over a broad pH range of 4.5–11.0with an optimum pH at 5.0,and under this condition,the laccase activity was up to 22.157U mL à1.The pH stability pro?le shows that Tplac was highly stable over a broad range of pH values ranging from 4.5–10.0,maintaining 75%of its original activity after incubation at 25°C for 72h.When the pH at 5.0,the laccase activity
was
Multiple amino acid sequences alignments of puri?ed Tplac from Trametes pubescens with other fungal laccases.The accession numbers were:PDB:2VDZ EJF60081(Dichomitus squalens ),AAX07469.1(Lentinus tigrinus ),PDB:2QT6(Lentinus tigrinus ),ABN13591.1(Polyporus brumalis ),AAG09231.1(Polyporus AAW28936.1(Trametes sp.420),PDB:3KW7(Trametes sp.AH28-2),PDB:2HRG (Trametes trogii ),CAA77015(Trametes versicolor ),EIW62366(Trametes versicolor AAB47735(Trametes villosa ).The ClustalX1.83algorithm and DNAMAN6.0software were used for alignment.Residue positions identical in all thirteen sequences with a black background.Conserved copper binding residues were boxed in red.Potential N -glycosylation sites of Tplac were marked with red arrows.suggested the amino acids obtained from nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)sequencing.
20.218U mLà1after reaction for72h.By contrast,the enzyme was unstable at lower pH values,exhibiting approximately5%of its ori-ginal activity after incubation at pH1.0–4.0for72h.Most fungal laccases are functional at acidic or neutral pH values but lose their activities under alkaline conditions(Zhang et al.,2009;Zou et al., 2012).Thus,the high activity,stability,and resistant ability of Tplac under alkaline conditions made this enzyme suitable for spe-cial applications.
As displayed in Fig.3b,Tplac showed its maximal activity at 50°C,amounting to a laccase activity of28.687U mLà1,and dis-played more than60%of the maximal activity at temperature rang-ing from25to75°C.After incubation at50°C for2h,the laccase activity was20.744U mLà1.The enzyme was stable at relatively moderate temperatures,i.e.,25–60°C,which indicated that Tplac has no special requirement for the operational capital and appara-tus.However,the laccase lost almost50%of its original activity after incubation at75°C for2h and was completely inactive at 80°C.The moderate or high temperature-dependent activity of puri?ed laccase Tplac from T.pubescens could be due to the higher temperature environment of southern China,where the fungal strain Cui7571was collected.
3.5.Substrate speci?city and kinetic property of puri?ed Tplac
Puri?ed laccase Tplac from T.pubescens displayed high activity toward a wide range of substrates,including phenolic substrates, such as catechol,2,6-DMP,L-DOPA,ferulic acid,guaiacol,and hydroquinone,as well as non-phenolic substrate,such as ABTS. However,no activity was detected with tyrosine(Table2).Tplac’s activity to the various substrates was ranked as follows: ABTS>2,6-DMP>L-DOPA>guaiacol>syringaldazine>ferulic acid> veratryl alcohol>hydroquinone>catechol>pyrogallol>phenol. Meanwhile,the K m,k cat,and k cat/K m values for ABTS were105.0 l M,876sà1,and8.34sà1l Mà1respectively,which were higher than those of certain fungal laccases(Guo et al.,2011;Zhu et al., 2011).The relatively low K m value for ABTS also indicated a high af?nity of the enzyme toward this substrate.
3.6.Effects of inhibitors on the activity of puri?ed Tplac
Laccase was inhibited to various extents by the usual inhibitors, such as L-cysteine,DTT,EDTA,and NaN3,which indicated the key roles of the thiol groups and metal-binding active sites on laccase activity(Johannes and Majcherczyk,2000;Lorenzo et al.,2005). The effects of various inhibitors on Tplac activity are summarized in Table2.A signi?cant reduction in laccase activity was observed in the presence of0.05mM NaN3,0.1mM DDT,or0.1mM L-cys-teine,whereas no inhibition was assayed with0.1mM metal ion chelator EDTA under the same experimental conditions.Even a higher concentration(1.0mM)of EDTA did not inhibit ABTS oxida-tion by Tplac,which was similar to the results obtained by Shin and Lee(2000).It seems to be that the inhibitory effect of EDTA de-pends on the substrate,and many substrates can alter the activity of an enzyme by in?uencing the binding of substrate and/or its
Table2
Effects of various substrates and inhibitors on the activity of puri?ed Tplac from Trametes pubescens.
Substrate Wavelength(nm)Relative activity a(%)Inhibitor Concentration(mM)Relative activity a(%)
ABTS420100±8.37L-Cysteine0.0589.75±8.54 Catechol40077.36±10.050.178.74±9.02 2,6-DMP47098.36±9.37 1.038.89±9.14
L-DOPA28095.05±9.46Dithiothreitol0.0579.14±8.94 Ferulic acid28787.34±8.860.119.78±7.86 Guaiacol47091.19±9.37 1.0 5.66±8.17 Hydroquinone24882.32±10.35EDTA0.05100
Phenol27060.34±8.370.1100
Pyrogallol45072.23±9.58 1.097.85±8.67 Syringaldazine52589.37±10.12Sodium azide0.0533.27±9.34 Tyrosine28000.1 2.12±9.13 Veratryl alcohol28085.51±8.87 1.00
a The value of100%relative laccase activity toward ABTS was28.687U mLà1obtained at optimum pH and temperature conditions.Each value is the mean value±standard error mean of triplicate.
54J.Si et al./Bioresource Technology128(2013)49–57
turnover number(Johannes and Majcherczyk,2000).Lorenzo et al. (2005)found that the laccase activity was strongly inhibited by EDTA using syringaldazine or2,6-DMP as substrate,whereas EDTA was not an ef?cient inhibitor using ABTS as substrate.
3.7.Effects of metal ions on the activity and ABTS oxidation of puri?ed Tplac
Another remarkable property of Tplac versus other laccases is its higher tolerance toward metal ions.One of the major obstacles retarding the development of the practical application in biotech-nological industries is the restricted ability of laccase in the pres-ence of various metal ions(Murugesan et al.,2009).As displayed in Table3,the effects of common environmental metal ions on lac-case activity were tested by adding Cu2+,K+,Na+,Mn2+,Ca2+,Fe2+, Fe3+,Mg2+,Zn2+,Ba2+or Al3+respectively.Interestingly,above 88%of initial enzyme activity was maintained in the presence of these metal ions at their?nal concentrations of25.0mM after incubation at50°C for15min.Meanwhile,the enzyme activity was enhanced by approximately111.32%with Cu2+,106.93%with Mn2+,104.90%with Na+,104.08%with Zn2+,or100.35%with Mg2+ respectively.These results were much better than those reported by previous studies(Guo et al.,2011;Zhu et al.,2011).It was worth noting that in terms of consideration for potentially industrial application,the highly tolerant activity of Tplac toward metal ions was a highly valued property.
To further understand the effects of metal ions on Tplac activity, the kinetic constants of ABTS oxidation were determined in the presence of various metal ions.Table3shows that the k cat/K m val-ues of Tplac toward ABTS in the presence of metal ions from Cu2+to Mg2+were higher than that of the control at the same level,sug-gesting that these metal ions at25.0mM could enhance the af?nity of Tplac toward substrate,thus stimulating the enzyme activity. However,the k cat/K m values of Tplac toward ABTS gradually de-creased following the separate addition of metal ions from Ca2+ to Al3+.Because the dramatic variations of k cat/K m data were ob-served in the presence of metal ions from Cu2+to Al3+,two theories are proposed to explain the metal effects regarding laccase activity. One theory is that the binding of several metal ions,i.e.,Cu2+,Mn2+, Na+,Zn2+,or Mg2+,induces conformational modi?cation of the enzyme and stimulates decomposition of the trimer complex con-taining substrate,enzyme and metal ion,as evidenced by noncom-petitive inhibition model(Duggleby,1979).It is well-known that the laccase contains three types of copper sites(type I,II,and III), and its catalytic site is a cluster of four copper atoms,which per-forms monoelectronic oxidation of suitable substrates(Frasconi et al.,2010).Therefore,the other theory is that the metal ion,i.e.,Ca2+,K+,Fe2+,Fe3+,Al3+,or Ba2+,binds near the T I site of laccase and acts as a competitive inhibitor of electron donors by blocking the access of substrates to the T I site or inhibiting the electron transfer at the T I active site,thereby leading to inhibition of lac-case activity(Fang et al.,2012).
3.8.Dye decolorization capacity of puri?ed Tplac
3.8.1.Dye decolorization
In textile industry,the decolorization of synthetic textile dyes and ef?uents by physical or chemical methods is?nancially and of-ten not very effective,therefore,enzyme-based decolorization is currently of great interest in this?eld(Arora and Sharma,2010). In this study,?ve representatives of structurally various dyes were used to evaluate the decolorization capacity of puri?ed laccase Tplac.As shown in Table4,the enzyme from T.pubescens per-formed high decolorization capacity toward all of the selective dyes,with80.53%of Congo Red(50.0mg Là1)being decolorized by1.0U mLà1pure enzyme after incubation for72h.It was sug-gested that the Tplac has potentials for use in various dyes biore-mediation.Based on the dye decolorization results,the azo dye Congo Red was selected as the model dye for the following experiments.
3.8.2.Effects of heavy metal ions on the dye decolorization capacity of puri?ed Tplac
Effects of heavy metal ions on the dye decolorization capacity of puri?ed Tplac were analyzed on the basis of time taken for100%
Table3
Effects of metal ions on the activity and ABTS oxidation of puri?ed Tplac from Trametes pubescens.
Metal ion Relative activity a
(%)
K m
(l Mà1)
k cat
(sà1)
k cat/K m
(sà1l Mà1)
Control100105.08768.34
Cu2+111.32±10.2385.5105612.35
Mn2+106.93±9.6388.697811.04
Na+104.90±9.5491.096310.58
Zn2+104.08±9.8499.09249.33
Mg2+100.35±8.31103.09048.78
Ca2+97.34±7.86107.78668.04
K+97.11±9.21107.38427.85
Fe2+95.88±9.05105.28147.74
Fe3+93.40±10.22109.57927.23
Ba2+89.88±9.34117.2763 6.50
Al3+88.42±8.37114.7711 6.20
a The value of100%relative laccase activity toward ABTS was28.687U mLà1 obtained at optimum pH and temperature conditions.Each value is the mean value±standard error mean of triplicate.Table4
Dye decolorization capacity of puri?ed Tplac from Trametes pubescens.
Dye Chemical class Color
index
name
Wavelength
(nm)
Decolorization
rate a(%)
Congo Red Azo Direct
red25
49780.53±9.34
Crystal Violet Triphenylmethane Basic
violet3
59534.21±8.34
Neutral Red Heterocycle Basic red
5
55375.15±7.38
Methylene
Blue
Thiazine Basic
blue9
66150.24±8.63 Reactive
Brilliant
Blue X-BR
Anthraquinone Reactive
Blue4
60366.57±9.57
a Each value is the mean value±standard error mean of triplicate.
20
40
60
80
100
120
140
020253035405060
Concentration (mM)
Cu Zn Fe
Effects of heavy metal ions on the dye decolorization capacity of puri?ed from Trametes pubescens.Time required for100%decolorization rate of azo Congo Red by Tplac was obtained in the presence of various heavy metal ions Zn2+,and Fe3+at different concentrations ranging from20.0to60.0mM.
J.Si et al./Bioresource Technology128(2013)49–5755
decolorization rate of azo dye Congo Red.The values obtained dur-ing experiments conducted in the presence of three metal ions at different concentrations are shown in Fig.4.The time taken for complete decolorization of azo dye Congo Red by puri?ed Tplac from T.pubescens was78h in the absence of any heavy metal ion.Noteworthy was that,the time required for100%decoloriza-tion rate was decreased by approximately21h in the presence of Cu2+at30.0mM and returned to its original level at40.0mM, which could be attributed to the highly valued property of metal tolerance of Tplac,thus stimulating the applicability of the enzyme for metal containing ef?uents removal(Kalpana et al.,2011).For metal ion Zn2+,a slight reduction in the time taken for100%decol-orization rate of Congo Red was observed as its concentration from 20.0to30.0mM.However,a vivid increase in the time required for complete decolorization occurred with continuously increasing concentrations of Zn2+,and the maximum time taken for100% decolorization rate of Congo Red was achieved at60.0mM,which was1.46-fold higher than that of the control in the absence of Zn2+. Meanwhile,the time taken for complete decolorization by puri?ed Tplac in the presence of Fe3+increased dramatically with the in-crease in metal ion concentration even at low levels.When the concentration of metal ion Fe3+was60.0mM,the time required for100%dye decolorization was133h,which indicated that the presence of some heavy metal ions in textile ef?uents may create the problem of low biodegradability which increases the biological treatment time(Lorenzo et al.,2005).In the light of these results it can be concluded that this suggested laccase Tplac is found to be ef?cient for the remediation of heavy metal ions containing dye ef?uents.
3.8.3.Degraded metabolites identi?cation
By organic solvent extraction and GC–MS analysis,the degraded metabolites of Congo Red were identi?ed as naphthalene amine (molecular mass143,m/z143,retention time19.657,area 18.31%),biphenyl amine(molecular mass169,m/z169,retention time10.856,area4.56%),biphenyl(molecular mass154,m/z154, retention time17.753,area9.37%),and naphthalene diazonium (molecular mass156,m/z156,retention time14.287,area 14.22%).According to the identi?ed metabolites,the degraded pathway was proposed and is shown in Supplementary Fig.S3. The?rst step was the reduction of the–N@N–bond,which re-sulted in the formation of two reactive intermediates(A and B), thereby leading to several reactions that lead to the production of stable intermediate.
3.8.
4.Phytotoxicity test
As demonstrated in Table5,the germination percentage and lengths of the plumule and radicle of P.mungo,S.vulgare,and T. aestivum seeds were greater with Congo Red’s degradation metab-olites or with water treatment when compared to untreated Congo Red treatment.Hence,phytotoxicity studies revealed that the puri-?ed laccase Tplac from T.pubescens can detoxify azo dyes used in textile industries.4.Conclusion
In summary,a novel laccase,Tplac,from white rot fungus T. pubescens was puri?ed and characterized.The enzyme performed with greater catalytic ef?ciency toward ABTS than did certain other fungal laccases,with the catalytic ef?ciency(k cat/K m)of8.34sà1-l Mà1being observed.Furthermore,Tplac exhibited alkali-resistant activity,metal tolerance,and dye decolorization capacity at room temperature.These unusual properties demonstrated this laccase’s potential suitability for use in such industries as bioremediation, textile,paper,and pulp.
Acknowledgement
This study was supported by the Program for New Century Excellent Talents in University(NCET-11-0585).
Appendix A.Supplementary data
Supplementary data associated with this article can be found, in the online version,at https://www.doczj.com/doc/256422139.html,/10.1016/j.biortech. 2012.10.085.
References
Arora, D.S.,Sharma,R.K.,2010.Ligninolytic fungal laccases and their biotechnological applications.Appl.Biochem.Biotechnol.160,1760–1768. Asgher,M.,Bhatti,H.N.,Ashraf,M.,Legge,R.L.,2008.Recent developments in biodegradation of industrial pollutants by white rot fungi and their enzyme system.Biodegradation19,771–783.
Bradford,M.M.,1976.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding.
Anal.Biochem.72,248–254.
Claus,H.,https://www.doczj.com/doc/256422139.html,ccases:structure,reactions,distribution.Micron35,93–96. Duggleby,R.G.,1979.Experimental designs for estimating the kinetic parameters for enzyme-catalysed reactions.J.Theor.Biol.81,671–684.
Eisenman,H.C.,Mues,M.,Weber,S.E.,Frases,S.,Chaskes,S.,Gerfen,G.,Casadevall,
A.,2007.Cryptococcus neoformans laccase catalyses melanin synthesis from
both D-and L-DOPA.Microbiology153,3954–3962.
Enayatizamir,N.,Tabandeh,F.,Rodríguez-Couto,S.,Yakhchali,B.,Alikhani,H.A., Mohammadi,L.,2011.Biodegradation pathway and detoxi?cation of the diazo dye Reactive Black5by Phanerochaete chrysosporium.Bioresour.Technol.102, 10359–10362.
Fang,Z.M.,Li,T.L.,Chang,F.,Zhou,P.,Fang,W.,Hong,Y.Z.,Zhang,X.C.,Peng,H.,Xiao, Y.Z.,2012.A new marine bacterial laccase with chloride-enhancing,alkaline-dependent activity and dye decolorization ability.Bioresour.Technol.111,36–
41.
Frasconi,M.,Favero,G.,Boer,H.,Koivula, A.,Mazzei, F.,2010.Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin.Biochim.Biophys.Acta1804,899–908.
Gomi,N.,Yoshida,S.,Matsumoto,K.,Okudomi,M.,Konno,H.,Hisabori,T.,Sugano, Y.,2011.Degradation of the synthetic dye amaranth by the fungus Bjerkandera adusta Dec1:inference of the degradation pathway from an analysis of decolorized products.Biodegradation22,1239–1245.
Guo,L.Q.,Lin,S.X.,Zheng,X.B.,Huang,Z.R.,Lin,J.F.,2011.Production,puri?cation and characterization of a thermostable laccase from a tropical white-rot fungus.
World J.Microbiol.Biotechnol.27,731–735.
Johannes,C.,Majcherczyk,A.,https://www.doczj.com/doc/256422139.html,ccase activity tests and laccase inhibitors.J.
Biotechnol.78,193–199.
Jonstrup,M.,Kumar,N.,Murto,M.,Mattiasson,B.,2011.Sequential anaerobic-aerobic treatment of azo dyes:decolourisation and amine degradability.
Desalination280,339–346.
Table5
Phytotoxicity test of azo dye Congo Red and its metabolites formed after biodegradation by puri?ed Tplac from Trametes pubescens.
Parameters studied Phaseolus mungo Sorghum vulgare Triticum aestivum
Water Congo Red Metabolites Water Congo Red Metabolites Water Congo Red Metabolites
Germination(%)10075100907010010065100 Plumule a(cm)15.63±0.55?? 6.52±0.32??14.52±0.32?15.76±0.32?7.15±0.37??13.87±0.2314.35±0.41?? 6.35±0.45??12.78±0.35?Radicle a(cm) 2.32±0.11??0.68±0.11? 2.20±0.18?8.57±0.31 1.42±0.12??7.18±0.22? 3.70±0.23?? 1.40±0.08?? 2.81±0.23?
Based on the statistical analysis of one-way analysis of variance(ANOVA)and Tukey–Kramer comparison test by using SPSS18.0software,probability(P value)less than0.05 or0.01(?P<0.05or??P<0.01)was considered signi?cant or highly signi?cant respectively.
a Each value is the mean value±standard error mean of triplicate.
56J.Si et al./Bioresource Technology128(2013)49–57
Kalpana,D.,Shim,J.H.,Oh,B.T.,Senthil,K.,Lee,Y.S.,2011.Bioremediation of the heavy metal complex dye Isolan Dark Blue2SGL-01by white rot fungus Irpex lacteus.J.Hazard.Mater.198,198–205.
Kalyani, D.C.,Patil,P.S.,Jadhav,J.P.,Govindwar,S.P.,2008.Biodegradation of reactive textile dye Red BL1by an isolated bacterium Pseudomonas sp.SUK1.
Bioresour.Technol.99,4635–4641.
Kalyani,D.,Dhiman,S.S.,Kim,H.,Jeya,M.,Kim,I.W.,Lee,J.K.,2012.Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxi?cation of biomass.Process Biochem.47,671–678.
Kim,H.,Lee,S.,Ryu,S.,Choi,H.T.,2012.Decolorization of Remazol Brilliant Blue R by a puri?ed laccase of Polyporus brumalis.Appl.Biochem.Biotechnol.166,159–164.
Litthauer,D.,van Vuuren,M.J.,van Tonder,A.,Wolfaardt,F.W.,2007.Puri?cation and kinetics of a thermostable laccase from Pycnoporus sanguineus(SCC108).
Enzyme Microb.Technol.40,563–568.
Lorenzo,M.,Moldes,D.,Rodríguez Couto,S.,Sanromán,M.A.,2005.Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds.Chemosphere60,1124–1128.
Mendon?a,R.T.,Jara,J.F.,González,V.,Elissetche,J.P.,Freer,J.,2008.Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications.J.Ind.Microbiol.Biotechnol.35,1323–1330. Murugesan,K.,Kim,Y.M.,Jeon,J.R.,Chang,Y.S.,2009.Effect of metal ions on reactive dye decolorization by laccase from Ganoderma lucidum.J.Hazard.Mater.168, 523–529.
Roriz,M.S.,Osma,J.F.,Teixeira,J.A.,Rodríguez Couto,S.,2009.Application of response surface methodological approach to optimise Reactive Black5
decolouration by crude laccase from Trametes pubescens.J.Hazard.Mater.
169,691–696.
Sadhasivam,S.,Savitha,S.,Swaminathan,K.,Lin,F.H.,2008.Production,puri?cation and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1.Process Biochem.43,736–742.
Shevchenko,A.,Jensen,O.N.,Podtelejnikov,A.V.,Sagliocco,F.,Wilm,M.,Vorm,O., Mortensen,P.,Shevchenko,A.,Bouvherie,H.,Mann,M.,1996.Linking genome and proteome by mass spectrometry:large-scale identi?cation of yeast proteins from two dimensional https://www.doczj.com/doc/256422139.html,A93,14440–14445. Shin,K.S.,Lee,Y.J.,2000.Puri?cation and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus.Arch.
Biochem.Biophys.384,109–115.
Thurston,C.F.,1994.The structure and function of fungal laccase.Microbiology140, 19–26.
Xiao,X.,Xu, C.C.,Wu,Y.M.,Cai,P.J.,Li,W.W.,Du, D.L.,Yu,H.Q.,2012.
Biodecolorization of Naphthol Green B dye by Shewanella oneidensis MR-1 under anaerobic conditions.Bioresour.Technol.110,86–90.
Zhang,H.,Zhang,Y.,Huang, F.,Gao,P.,Chen,J.,2009.Puri?cation and characterization of a thermostable laccase with unique oxidative characteristics from Trametes hirsuta.Biotechnol.Lett.31,837–843.
Zhu,Y.,Zhang,H.,Cao,M.,Wei,Z.,Huang, F.,Gao,P.,2011.Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization.Biotechnol.Bioprocess Eng.16,1027–1035.
Zou,Y.J.,Wang,H.X.,Ng,T.B.,Huang, C.Y.,Zhang,J.X.,2012.Puri?cation and characterization of a novel laccase from the edible mushroom Hericium coralloides.J.Microbiol.50,72–78.
J.Si et al./Bioresource Technology128(2013)49–5757
Biochemical Weapon 生化武器（Biochemical Weapon）旧称细菌武器。是指以细菌、病毒、毒素等使人、动物、植物致病或死亡的物质材料制成的武器。作为一种大规模杀伤性武器，至今仍然对人类构成重大威胁。生化武器是利用生物或化学制剂达到杀伤敌人的武器，它包括生物武器和化学武器。 生物武器是生物战剂及其施放装置的总称，它的杀伤破坏作用靠的是生物战剂。生物武器的施放装置包括炮弹、航空炸弹、火箭弹、导弹弹头和航空布撒器、喷雾器等。以生物战剂杀死有生力量和毁坏植物的武器统称为生物武器. 生物战剂是军事行动中用以杀死人、牲畜和破坏农作物的致命微生物、毒素和其他生物活性物质的统称。旧称细菌战剂。生物战剂是构成生物武器杀伤威力的决定因素。致病微生物一旦进入机体（人、牲畜等）便能大量繁殖，导致破坏机体功能、发病甚至死亡。它还能大面积毁坏植物和农作物等。由于它伤害太严重，性质极其恶劣，所以已被定为禁用武器。 非人手段 活体解剖：一个代号为“马路大”的特别项目进行人体试验：受试验者从中国的住民中抓来，也被称为“圆木”(丸太）。此项作业的要点是：必须保证解剖对象是绝对清醒的状态，也就是说，绝对不能麻醉。因为日本军医认为麻醉后的研究数据是不真实的。其解剖场景惨绝人寰。解剖时，那凄厉的惨叫声是常人所无法想象的。此项工作也是731部队所有医师所必备的基础技能。[7] 手榴弹试验：用人在不同的距离和位置进行手榴弹试验。[7] 冻伤试验：用来测试人在不同温度下抗寒程度。试验资料为北支那防疫给水部专业人员与驻蒙军团联合进行的一次野外冻伤试验的资料，题目为《极秘·驻蒙军冬季卫生研究成绩》，资料编成者为冬季卫生研究班，形成年代为昭和十六年三月（1941年3月）。[7] 该资料是侵华日军细菌与毒气战研究所所长金成民，应日本横滨市立大学校长加藤佑三先生邀请，受平房区政府委派赴日讲学访问期间，多次单独或与日本友人一同赴东京神田等地的图书馆及资料馆查阅资料，在一资料室内，发现这部保存较好的资料。[7] 火焰喷射器实验：731部队将试验者关在废弃装甲车内，用火焰喷射器烤之，以测验火焰喷射器威力。但这 实验是毫无意义的，没有装甲车会呆在原地让你烤，纯粹是“娱乐”罢了。[7] 鼠疫实验：将鼠疫杆菌注入试验者体内，观察其反应。这种方法也应用在了被日本军队在边境抓住的苏联战俘身上。开发落叶剂和细菌弹：其中最突出的“成果”是石井炸弹，美军后来在越南使用的菠萝弹也是该弹的改进型。石井炸弹为陶瓷外壳，内装携细菌的跳瘙。石井四郎还有一项发明是石井滤水器。以解决士兵在野外作战的污水处理为饮水的问题，算是731部队唯一有用处的发明了。[7] 无麻醉拔牙：目的是测试伤员在未麻醉的情况下是否可以忍受拔牙手术，实验结果是无人能够忍受，但如果已经严重松动的牙齿则另当别论。 人与马血互换：将身体强壮的马路大血液抽去大部，此时马路大全身痉挛，几名军医都无法完全按住。立即输入马匹血液，并观察马路大的表现，结果身体排异性明显，马路大全部死亡。 病菌对胎儿的影响：让女马路大怀孕后感染病菌，待胎儿成形后进行活体解剖，观察胎儿的状态。
水的净化(Water Purification) 水的净化(Water Purification)水的净化(Water Purification) 水的净化(water purification) the provision of safe water necessitates one of the rnajor expenditures of manpower and revenue in our modern cities. the purification of water is basically a two-step or three-step process carried out under the strict supervision of public health scientists and engineers. as the first step, natural water from the least contaminated source is allowed to stand in large reservoirs, where most of the mud, clay, and silt settle out; this is called sedimentation . often in water with high mud content, lime and aluminum sulfate are added to the water in the settling reservoirs. these chemicals react in the water to form aluminum hydroxide, which settles slowly and carries much of the suspended material, including most of carries much of the suspended material, including most of the bacteria, to the bottom of the reservoirs. as the second step, the water is filtered through beds of sand and grovel, which remove other impurities and chemicals in it. during or after filtration, chemicals are ordinarily added to the water to kill
Molecular mechanisms of DNA double- strand break repair Roland Kanaar, Jan H. J. Hoeijmakers and Dik C. van Gen DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair. The integrity of DNA inside cells is constantly being challenged by endogenous and exogenous DNA- damaging agents. Because a large variety of lesions can occur in DNA, it is not surprising that multiple pathways have evolved that each repair a subset of these lesions 1 . The significance of DNA repair is illustrated by the phenotypes of xeroderma pigmen tosum, Cockayne’s syndrome, trich othiodystrophy and hereditary nonpolyposis colorectal cancer pa2,3 . These disorders are caused by mutations in DNA repair genes that predispose the patients to cancer, neurological abnormalities or both. In addi- tion to efficient DNA repair, correct activation of cell-cycle checkpoints upon induction of DNA damage is of crucial importance for the maintenance of genomic integrity. Checkpoints allow actively dividing cells to pause and repair DNA damage before segregation of the replicated genome into daughter cells. Their importance is underscored by inherited disorders associated with defects in activating cellcycle checkpoints such as ataxia telangiectasia and Nijmegen breakage syndrome 4,5. These disorders cause hypersensitivity to DNA-damaging agents and spontaneous chromosomal instability. In this review, we focus on mechanisms of DNA double-strand break (DSB) repair. DSBs are generated by endogenously produced radicals and exogenous agents such as ionizing radiation (IR), whichis often used in anti-cancer therapy. Repair of DSBs
Protein Purification Handbook 18-1132-29 Edition AC Protein Purification – Handbook
Antibody Purification Handbook 18-1037-46 The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75 Protein Purification Handbook 18-1132-29 Ion Exchange Chromatography Principles and Methods 18-1114-21Affinity Chromatography Principles and Methods 18-1022-29Hydrophobic Interaction Chromatography Principles and Methods 18-1020-90Gel Filtration Principles and Methods 18-1022-18Handbooks from Amersham Biosciences Reversed Phase Chromatography Principles and Methods 18-1134-16 Expanded Bed Adsorption Principles and Methods 18-1124-26 Chromatofocusing with Polybuffer and PBE 18-1009-07 Microcarrier cell culture Principles and Methods 18-1140-62
污水处理中的常用术语 BOD（Biochemical Oxygen Demand）——生化需氧量 在有氧条件下，由于微生物的作用，水中可以分解的有机物完全氧化分解时所需要的溶解氧量，叫生化需氧量，用mg/L表示。由于有机物的种类很多，欲测出其中各自的含量是办不到的，故常用BOD这个综合指标来表示。微生物分解有机物所消耗的氧量与有机物的浓度密切相关，有机物含量愈高，消耗的氧也就愈多，这就是用BOD值来间接反映有机物含量多少的根据。 完全氧化分解污水中的有机物约需100天左右，而20天的BOD值十分接近完全的BOD值（相差1%左右）。因此，常把20日BOD值（即BOD20）当作完全BOD值。但20日仍嫌太长，实际上采用5日BOD值，即BOD5。 BOD5与BOD20相差较大，但就一般污水而言，二者存在比较固定的比值，如生活污水BOD5：BOD20=0.7。 COD（Chemical Oxygen Demand）——化学需氧量 在一定条件下，水中能被强氧化剂氧化的所有污染物质（包括有机物和无机物）的量，以氧的mg/L表示，叫化学需氧量。 有机物基本上属于还原性物质，能被化学氧化剂氧化。有机物愈多，消耗的氧化剂量也愈多，因此可以用消耗的氧化剂量（换算成O2的mg/L）来间接反映有机物的含量。但有机物不是全部能被氧化的，如以醋酸为主的低级脂肪酸就几乎不能被氧化。此外，被氧化的污染物质还包括还原性的无机物——Fe2＋、N O2－等。
COD的测定方法分铬法（以重铬酸钾做氧化剂）和锰法（以高锰酸钾做氧化剂）两种，分别记为COD Cr和COD Mn。 高锰酸钾法测定的结果受操作条件影响较大，且高锰酸钾溶液不稳定，对氧化程度也有影响，因而测定结果不能代表水中污染物质的确切含量。而重铬酸钾法则克服了上述缺陷，它具有更强的氧化能力，能将污水中绝大部分有机物和还原性无机物氧化。其溶液非常稳定。该法已被广泛采用。其与猛法之间的比值一般为：COD Cr：COD Mn=3：2。 由于BOD5的测定比较麻烦，可以找出其与COD之间的相关关系，做出二者的相关曲线，这样，测出COD便可由相关曲线查出BOD5值。但这种做法有一定局限性，因为BOD5和COD的比值是随水质成份的变化而变化的。有些有毒物质BOD5测不出来，COD却能测出；而某些羧基化合物易于在BOD5中反映出来，而在COD中又反映不出，故对水质复杂，进水负荷波动频繁的生产工艺，其BOD5与COD的相关关系不是固定不变的。但对试验用的配制污水和生产工艺稳定、进水负荷波动很小的污水，在一定时间内利用二者的相关曲线还是可行的。 TOC（Total Organic Carbon）——总有机碳 污水中有机物含量的总和。它还包括了强氧化剂重铬酸钾难以氧化的有机物质，因此，它比COD Cr在某种意义上更准确、全面。国外多采用TOC这个指标。其测定方法是将水样在高温下燃烧，有机碳则氧化为CO2，测出所产生的CO2量，便可求得水样的总有机碳（TOC）值，单位以碳的mg/L表示。在作该项分
RPAS Purification Module: A New Tool for the Purification of Recombinant E-tagged ScFv Antibodies Edition AA Introduction The Recombinant Phage Antibody System (RP AS) is an integrated modu-lar system designed for the cloning and expression of recombinant mouse antibody fragments in bacteria, and the purification of the expressed single chain fragments variable (ScFv) antibodies. The technology, developed in collaboration with Cambridge Antibody T echnology Ltd., UK, is based on a phage-display system in which anti-body fragments are expressed as fusion proteins displayed on the phage surface. When antibody genes are cloned in the phagemid vector pCANTAB 5 E,soluble recombinant antibodies can be produced for use as immuno-logical reagents. The ScFv carries a 13 amino acid peptide tag (E-tag)which is recognized by a mouse monoclonal Anti-E tag antibody.The system is comprised of four integrated modules:- Mouse ScFv Module - Expression Module - Detection Module - Purification Module The work presented here will show the performance of the Purification Module. Affinity Chromatography using mouse monoclonal Anti-E tag as a ligand,coupled to Sepharose High Performance, offers a very specific purification method for E-tagged ScFv antibodies. This convenient, one-step purification method results in > 95% pure and active E-tagged ScFv antibodies in 10-15 minutes using pre-packed HiTrap Anti-E T ag column, a syringe and the pre-made buffers. No other special equipment is needed. Karin Andersson, Peder Bergvall, Birgitta Formgren, Anna Heijbel, Ann-Christine H?ggqvist and Ray Mernaugh*.Amersham Biosciences AB, S-751 82, Uppsala, Sweden.*Amersham Biosciences Inc., Milwaukee, WI53202, USA. 18-1113-78 Fig. Antibody model showing subunit composition. Fragments generated by proteolytic cleavage and/or recombinant technology appear in the shaded area. Abstract Selection of recombinant antibodies by the phage display technique and expression of soluble functional fragments in E.coli has become a new tool for many scientists. Purification of the expressed protein can be achieved by a number of methods, affinity chromatography being especially powerful. The RP AS Purification Module is a part of Recombinant Phage Antibody System (RP AS). Soluble, functional mouse single chain fragments variable (ScFv) antibodies are produced in Escherichia coli using the pCANT AB 5 E expression vector. The ScFv carries a 13 amino acid peptide tag (E-tag) which is recognized by an Anti-E T ag affinity column. The ScFv binds to the Anti-E Tag column at neutral pH and is easily eluted from the column by a decrease in pH. The work presented shows the purification of two different recombinant E-tag ScFv antibodies directly from E. coli periplasmic extracts. The purified material was characterised by SDS-P AGE, ELISA and Western Blot.All purified ScFv fragments were immunologically active and >95% pure. The purifications were performed within 10-15 minutes without using complicated instrumentation, just a simple syringe.The influence of flow rate, sample concentration and sample volume on the yield of ScFv is presented. The results show that the binding capacity for the column, HiTrap ?Anti-E Tag (Anti E-Tag Sepharose ? High Performance) included in RP AS Purification Module, is approximately constant, even under different loading conditions. The stability of the HiT rap Anti-E T ag column is high, giving a lifetime of over 20 purification cycles.
QIAquick? PCR Purification Kit The QIAquick PCR Purification Kit (cat. nos. 28104 and 28106) can be stored at room temperature (15–25°C) for up to 12 months. For more information, please refer to the QIAquick Spin Handbook, March 2008, which can be found at: https://www.doczj.com/doc/256422139.html,/handbooks. For technical assistance, please call toll-free 00800-22-44-6000, or find regional phone numbers at https://www.doczj.com/doc/256422139.html,/contact. Notes before starting Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume). All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature. Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots. 1.Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow. 2.Place a QIAquick column in S a provided 2 ml collection tube or into z a vacuum manifold. For details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook. 3.To bind DNA, apply the sample to the QIAquick column and S centrifuge for 30–60 s or z apply vacuum to the manifold until all the samples have passed through the column. S Discard flow-through and place the QIAquick column back in the same tube. October 2010
Appl Microbiol Biotechnol(2003)60:523–533 DOI10.1007/s00253-002-1158-6 M I N I-R E V I E W K.Terpe Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems Received:8July2002/Revised:25September2002/Accepted:27September2002/Published online:7November2002 Springer-Verlag2002 Abstract In response to the rapidly growing field of proteomics,the use of recombinant proteins has increased greatly in recent years.Recombinant hybrids containing a polypeptide fusion partner,termed affinity tag,to facil-itate the purification of the target polypeptides are widely used.Many different proteins,domains,or peptides can be fused with the target protein.The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized.Nevertheless, it is difficult to choose the right purification system for a specific protein of interest.This review gives an overview of the most frequently used and interesting systems:Arg-tag,calmodulin-binding peptide,cellulose-binding do-main,DsbA,c-myc-tag,glutathione S-transferase,FLAG-tag,HAT-tag,His-tag,maltose-binding protein,NusA,S-tag,SBP-tag,Strep-tag,and thioredoxin. The production of recombinant proteins in a highly purified and well-characterized form has become a major task for the protein chemist working in the pharmaceu-tical industry.In recent years,several epitope peptides and proteins have been developed to over-produce recombinant proteins.These affinity-tag systems share the following features:(a)one-step adsorption purifica-tion;(b)a minimal effect on tertiary structure and biological activity;(c)easy and specific removal to produce the native protein;(d)simple and accurate assay of the recombinant protein during purification;(e) applicability to a number of different proteins.Neverthe-less,each affinity tag is purified under its specific buffer conditions,which could affect the protein of interest (Table1).Thus,several different strategies have been developed to produce recombinant proteins on a large scale.One approach is to use a very small peptide tag that should not interfere with the fused protein.The most commonly used small peptide tags are poly-Arg-,FLAG-, poly-His-,c-myc-,S-,and Strep II-tag.For some appli-cations,small tags may not need to be removed.The tags are not as immunogenic as large tags and can often be used directly as an antigen in antibody production.The effect on tertiary structure and biological activity of fusion proteins with small tags depends on the location and on the amino acids composition of the tag(Bucher et al.2002).Another approach is to use large peptides or proteins as the fusion partner.The use of a large partner can increase the solubility of the target protein.The disadvantage is that the tag must be removed for several applications e.g.crystallization or antibody production. In general,it is difficult to decide on the best fusion system for a specific protein of interest.This depends on the target protein itself(e.g.stability,hydrophobicity),the expression system,and the application of the purified protein.This review provides an overview on the most frequently used and interesting tag-protein fusion systems (Table2). The Arg-tag was first described in1984(Sassenfeld and Brewer1984)and usually consists of five or six arginines. It has been successfully applied as C-terminal tag in bacteria,resulting inrecombinant protein with up to95% purity and a44%yield.Arginine is the most basic amino acid.Arg5-tagged proteins can be purified by cation exchange resin SP-Sephadex,and most of the contami-nating proteins do not bind.After binding,the tagged proteins are eluted with a linear NaCl gradient at alkaline pH.Polyarginine might affect the tertiary structure of proteins whose C-terminal region is hydrophobic(Sassen-feld and Brewer1984).The Arg-tagged maltodextrin-binding protein of Pyrococcus furiosus has been crystal- K.Terpe()) Technical Consultant of the IBA GmbH, Protein expression/purification and nucleic acids,37079G?ttingen, Germany e-mail:terpe@iba-go.de Tel.:+49-551-50672121 Fax: +49-551-50672181
Ni-NTA Purification System For purification of polyhistidine-containing recombinant proteins Catalog nos. K950-01, K951-01, K952-01, K953-01, K954-01, R901-01, R901-10, R901-15
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Table of Contents Kit Contents and Storage (iv) Accessory Products (vi) Introduction (1) Overview (1) Methods (2) Preparing Cell Lysates (2) Purification Procedure—Native Conditions (7) Purification Procedure—Denaturing Conditions (11) Purification Procedure—Hybrid Conditions (13) Troubleshooting (15) Appendix (17) Additional Protocols (17) Recipes (18) Frequently Asked Questions (21) Technical Service (22) References (23) iii
Kit Contents and Storage Types of Products This manual is supplied with the following products: Kit Name Catalog No. Ni-NTA Purification System K950-01 Ni-NTA Purification System with Antibody with Anti-Xpress? Antibody K951-01 with Anti-myc-HRP Antibody K952-01 with Anti-His(C-term)-HRP Antibody K953-01 with Anti-V5-HRP Antibody K954-01 Ni-NTA Agarose (10 ml) R901-01 Ni-NTA Agarose (25 ml) R901-15 Ni-NTA Agarose (100 ml) R901-10 System Components The Ni-NTA Purification System components are listed below and include enough resin, reagents, and columns for six purifications. Component Composition Quantity Ni-NTA Agarose 50% slurry in 30% ethanol 10 ml 5X Native Purification Buffer 250 mM NaH2PO4, pH 8.0 2.5 M NaCl 1 × 125 ml bottle Guanidinium Lysis Buffer 6 M Guanidine HCl 20 mM sodium phosphate, pH 7.8 500 mM NaCl 1 × 60 ml bottle Denaturing Binding Buffer 8 M Urea 20 mM sodium phosphate, pH 7.8 500 mM NaCl 2 × 125 ml bottles Denaturing Wash Buffer 8 M Urea 20 mM sodium phosphate, pH 6.0 500 mM NaCl 2 × 125 ml bottles Denaturing Elution Buffer 8 M Urea 20 mM NaH2PO4, pH 4.0 500 mM NaCl 1 × 60 ml bottle Imidazole 3 M Imidazole 20 mM sodium phosphate, pH 6.0 500 mM NaCl 1 × 8 ml bottle Purification columns 10 ml columns 6 iv
Purification and Biochemical Characterization of Extracellular β-Glucosidases from the Hypercellulolytic Pol6Mutant of Penicillium occitanis Fatma Bhiri &Semia Ellouz Chaabouni &Ferid Limam & Rachid Ghrir &Nejib Marzouki Received:13August 2007/Accepted:19December 2007/ Published online:4March 2008#Humana Press 2008 Abstract The Pol6mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and β-glucosidase production with high cellulose degradation efficiency (Jain et al.,Enzyme Microb Technol ,12:691–696,1990;Hadj-Taieb et al.,Appl Microbiol Biotechnol ,37:197–201,1992;Ellouz Chaabouni et al.,Enzyme Microb Technol ,16:538–542,1994;Ellouz Chaabouni et al.,Appl Microbiol Biotechnol ,43:267–269,1995).In this work,two forms of β-glucosidase (β-glu 1and β-glu 2)were purified from the culture supernatant of the Pol6strain by gel filtration,ion exchange chromatography,and preparative anionic native electrophoresis.These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis.However,both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW,98kDa),shared the same amino acid composition,carbohydrate content (8%),and kinetic properties.Moreover,they strongly cross-reacted immunologically.They were active on cellobiose and p NPG with Km values of 1.43and 0.37mM,respectively.β-glu 1and β-glu 2were competitively inhibited by 1mM of glucose and 0.03mM of δ-gluconolactone.They were also significantly inhibited by Hg 2+and Cu 2at 2mM.The addition of purified enzymes to the poor β-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H 3P04swollen cellulose but had no effect with P .occitanis crude extract.Besides their hydrolytic activities,β-glu 1and β-glu 2were endowed with trans-glycosidase activity at high concentration of glucose. Appl Biochem Biotechnol (2008)149:169–182 DOI 10.1007/s12010-008-8146-y F.Bhiri :S.E.Chaabouni (*) Unité?Enzymes et Bioconversion ?,National School of Engineers of Sfax, BP W 3038,Sfax Cedex,Tunisia e-mail:semia.chaabouni@enis.rnu.tn F.Limam :R.Ghrir National Institute of Scientific and Technical Research,BP 95,2050,Hammam-Lif,Tunisia N.Marzouki National Institute of Applied Sciences and Technology,BP 676,1080Tunis Cedex,Tunisia