血红密孔菌mk528漆酶对刚果红的脱色研究

章淑艳,赵丛波,郭 林,李 伟

（1.河北省微生物研究所,保定071051；2.中国科学院微生物研究所真菌学国家重点实验室，北京100101）

血红密孔菌mk528漆酶对刚果红的脱色研究

章淑艳1,赵丛波1,郭 林2,李 伟2

（1.河北省微生物研究所,保定071051；2.中国科学院微生物研究所真菌学国家重点实验室，北京100101）

刚果红是一种重要的难降解偶氮型染料，研究利用血红密孔菌mk528菌株产生的漆酶粗品在添加介体条件下对其进行脱色降解实验。结果表明：介体阿魏酸对刚果红脱色有抑制作用，而紫脲酸、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（ABTS）和香兰素对刚果红脱色有促进作用；其中氧化还原介体紫脲酸比ABTS和香兰素更有效。通过实验确定刚果红脱色介体系统温度为40℃，pH值5.5，介体紫脲酸用量为0.05mM，漆酶用量为5U/m L，作用时间6 h的最佳条件。刚果红脱色的漆酶-紫脲酸介体系统表明漆酶在偶氮染料脱色降解方面将会有潜在的应用价值。

刚果红；脱色；漆酶；介体；血红密孔菌

Synthetic dyes are w idely applied in the food,cosmetics,plastics and textile industries.In these synthetic dyes,azo dyes have been w idely used in textile industries and dominated the worldw ide market of dyestuffs w ith a share of about70%［1］.Azo dyes can generate radiation amine w ith carcinogenic and induce mutations in organisms［2］.Since azo dyeshave one ormore azo bonds and aromatic rings,they aremore difficult to be degrad-ed than other typesof dyes［3］.

Laccase(EC 1.10.3.2)is an extracellular ligninmodifying enzyme produced by almost all of white-rot fungi,which plays an important role in degradation processes［4］.Laccase can catalyze the oxidation of phenolic and nonphenolic compounds w ith the concurrent reduction ofmolecular oxygen to water［5］.Based on various techniques,laccases or laccase-mediator systems have been applied for decolorization of various dyes and the results were highly prom ising［6］.The range of substrate specificitiesof laccases can be extended to non-phenolic substrates w ith addition of redox mediators.There are two types of mediators proposed:(1)naturalmediators such assyringaldehyde,ferulic acid and vanillin;(2)artificial mediators like violuric acid,1-hydroxybenzotriazole(HBT)and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate)(ABTS).Fungal laccases combined w ith mediators have been reported to decolorize several groups of textile dyes,and differentmediators enhance the action of laccase on the dyesdifferently［7］.

During the past decade,decolorization of various kinds of azo dyes has been described w ith laccase from increasing numbersofwhite-rot fungi.Themediators,including TEMPO,HBT,syringaldehyde,violuric acid and acetosyringone,were proved to be the effectivemediator for synthetic acid dye decolorization by laccase from Trametes villosa［8］,Trametes hirsute,Trametes versicolor［9-10］and Fomes fomentarius［11］,respectively.Congo red，as one of themost importantazo dyes in the textile industry,has been proven to be capable of decolorizing at some degrees decolorized by the laccase from white rot fungi such as Pleurotus pulmonarius［12-13］, Polyporus rubidus［14］and Trametes hirsut a［15］.Pycnoporus sanguineus has been found to be suitable for the decolorization of the azo dyes using the crude enzyme. However,there have been few reports published about degradation of Congo red by the laccase from P.sanguineus w ithmediator conditions.

The aim of thework was to determ ine the ability of crude laccase from P.sanguineus mk528 to decolorize the Congo red w ith mediator conditions.This work developed the processw ith technical parameters thatwere easy to degrade the azo dyesby laccase of P.sanguineus mk528.

1 M aterialsand methods

1.1 Organism

The monokaryotic strain of P.sanguineus mk528 used in this paper was selected from single-spore isolates of P.sanguineus GW 05.Stocks were maintained on PDAmedium w ith periodic transfer.

1.1.1 Chemicalsand reagents

Chemicals and reagents used as buffers and substrates media were commercially available products of analyticalgrade.

1.1.2 Preparation of crude laccase

The culture medium was collected at the maximum laccase activity by centrifugation at 10,000 r/min for 20 m in,and the supernatant fluid was concentrated about 20-fold by ultrafiltration through a cellulose PLGC membrane(molecular mass cut-off of 10 kDa) (M illipore,America).Laccases were further concentrated in turn by the two step ammonium sulfate precipitation,dialysisand lyophilization［16］.

1.2 Laccaseenzymeactivity assays

Laccase activity was determined by monitoring the oxidation of ABTS(0.5 mM)in sodium-tartrate buffer (50 mM,pH 4.0)at 420 nm(∑max=3.6×104M-1cm-1) and 30℃.A ll enzymes assays were carried out using a UV array spectrophotometer(Unico,America).The enzyme activity was expressed in international units(U). One unitof activity leads to the transformation of 1μM substrate in onem inute.

1.3 Effect of temperature and pH on laccase activity and stability

The effect of pH on laccase activity was exam ined w ith the pH range of 2.5-6.5 using ABTS as the substrate in sodium-tartrate buffer(50 mM)or phosphate buffer(50 mM).The optimum temperature of laccase was determined between the range from 20 to 75℃w ith ABTSas the substrate atpH 4.0.The thermal stability of laccase was determ ined in sodium-tartrate buffer(50 mM)atpH 4.0 and 25-75℃for1 h.

1.4 Enzymatic decolorization of Congo red

The decolorization of Congo red was chosen in vitro assays for enzyme degradation.Stock solutions of the dyewas prepared in sterilized distilled water and diluted to the required concentration.To improve the decolorized efficiency of Congo red w ith laccase,the varying pH,enzyme amount,incubation time,mediators, concentration ofmediators and initial dye concentration were investigated.Decolorization reaction mixture containing dye solutionswere adjusted at varying pH,laccase solution(10 U/m L),Congo red(initial concentration 25 mg/L)for 7 h.Decolorization experiments were performed w ith varying enzyme concentration in which the initial dye concentration used was25mg/L,and incubated for7 hw ith pH 5.5.

Themediators including ABTS,ferulic acid,vanil-lin or violuric acid at different concentrationswere tested in the reactionmixture.In order to investigate the tolerance of laccases w ith mediators against the dyestuff, the decolorization was carried outw ith varying concentration solutionsof Congo red.For this,the final laccase activity used was5 U/m L,and incubated for 6 h w ith pH 5.5.

1.5 Decolorization tests

The absorption spectrum of Congo red was measured between 200 and 700 nm w ith a UV-2100 spectrophotometer(Unico,America).Dye decolorization was determ ined by the decrease in absorbance under the maximum wavelength(488 nm)of Congo red and expressed in terms of percentage.The decolorization rate was expressed as the plotaccording to the follow ing expression:

Decolorization(%)=(A0-A)/A0×100, where%is the decolorization percentage obtained, A0=the initialarea,A=the final area.A control test containing the same amountof a heat-denatured laccasewas also performed in parallel.

1.6 Dataanalysis

Soft OriginPro 8 was used in this study.All values reported herewere themean of three replicatesw ith standard error of the mean(SE)represented by error bars. The experiments were performed in triplicate and all of the standard deviationswere lower than 5%of themean valuespresented.

2 Results

2.1 Temperatureand pH dependency

The temperature range,in which the crude enzyme is active,is remarkably w idew ith 25-75℃,but thew ide temperature itshowed considerable activity was over the range of 45-75℃.Themost optimal temperature of the laccase from P.sanguineus mk528 was determined to be approximately 60-65℃ (Fig 1A).The coarse laccase when kept at both 40 and 50℃ for 2 h retained over 80%activity.The laccase activity decreased 35%after incubated at 75℃for 2 h(Fig 1B).So the laccase from P.sanguineus mk528 are thermostable and has much w iderpH profile.

Enzymes assays were carried out at different pH values using ABTS to determine the effectof pH on laccase activity.The pH profileof P.sanguineus mk528 laccase showed activity at pH 2.5-6.5,but the optimum pH of the laccase is about pH 2.5-4.5(Fig 2A).The crude laccase preserved over 75%activity when kept at pH 3-5 for24 h.The laccaseactivity only decreased to 65%after incubated atpH 6 for24 h(Fig 2B).

Fig 1 Effectof temperatureon enzyme activity of laccase from P. sanguineus mk528 atpH 4(A),and temperature stability of the laccase in 2 h(B)

2.2 Effectof pH on Congo red decolorization

The effect of pH on the decolorization of Congo red by laccasew ithoutmediatorwas given in Fig 3.The decolorized percentage exceeded 45%after incubation at 40℃at pH5-6 for 5 h.The decolorized process decreased remarkably along w ith the pH value from 2.5 to 6.5(Fig 3).The percentage of decolorization was not over 30%under pH 5.In combination w ith the results of the optimal pH of laccase activity,itwas likely that the featly pH value of dye decolorization by laccasewas different from the optimal pH of laccase.The optimal pH value for dye decolorization was determ ined as pH5.5, and we chose the pH 5.5 as the conditions in the followingmanipulations of decolorization.No significant color changewas observed in all control flasks containing the dyes in aqueous solutions at different pH w ithout laccase.

2.3 Effectof enzyme concentration on Congo red decolorization

Fig 2Effectof pH on enzymeactivity of laccase from P.sanguineus mk528 at30℃(A),and the pH stability of the laccaseat40℃in 24 h(B).

The effect of enzyme concentration on Congo red was studied using up to 20 U/m L of laccase activity w ithoutmediatorsatpH 5.5.Theefficency of decolorization of Congo red increased along w ith the addition of laccase from 1 U/m L to 20 U/m L in 1 h.However,the decolorization percentage of Congo red w ith laccase activityof 5 U/m L came up to that of w ith both 10 U/m L and 20 U/m L in 2 h.The decolorization percentagew ith 1 U/m L of laccase improved up to about43%,which arrived at the degradation percentage w ith 2 U/m L of laccase in 2 h.Amaximum of 63%dye decolorization was obtained after 7 h in the presence of 5 U/m L of laccase (Fig 4).The results showed thatonly compatible amount of laccasewere necessary for economical decolorization of Congo red and the reaction began to plateau at about 5U/m L of laccase.

Fig 3 Effectof pH on decoloriztion of Congo red(25mg/L-1)by laccase(10U/m L)w ithoutmediator at40℃.

Fig 4 Effectof activity of laccaseon decoloriztion of Congo red (25mg/L)by laccasew ithoutmediator atpH 5.5 and 40℃

2.4 Effectsofmediatorson Congo red decolorization

Theeffectsof four different redoxmediatorson laccase decolorization of Congo red were summarized in Fig 5.Among them,ferulic acid inhibited the decolorization of Congo red.ABTS just improved the decolorization in 2 h distinctively.Vanillin and violuric acid promoted the decolorization of Congo red by the laccase higher than 60%in 5 h.Violuric acid,being themosteffective redoxmediator,improved the decolorization percentagenear70%(Fig 5A).Subsequently,the laccase decolorizing studieswere performed in the presence of the latter three mediators.In this system,25 mg of Congo red is treated w ith 5 U/m L of laccase activity in pH 5.5.

The optimal decolorization percentages of Congo red being about 64.1%(w ith 0.25 mM vanillin),69.2% (w ith 5μM ABTS),and 81.2%(w ith 0.05 mM violuric acid)were obtained,respectively.The ration of concentration beween ABTSand vanillinwas1∶50(Fig 5B and 5C).In contrast,vanillin had lesseffecton Congo red decolorization than ABTS and violuric acid,and violuric acid was considered themore efficientmediator for the decolorization of Congo red by the laccase.For violuric acid,the strongestdecolorization was 54%over the contol in 6 h,and the concentration ration beween ABTS and violuric acid is1∶10(Fig 5B and 5D).

2.5 Effectof the concentration of Congo red on decolorization

Fig 5 Effectofmediatorson Congo red(25mg/L)decolorization Decolorizing effectivenessw ith ABTS(0.05mM),ferulic acid(1mM), vanillin(1mM)and violuric acid(0.5mM)(A),and w ith the varying concentrationsofmediator in the laccasemediator treatment(5U/m L) atpH 5.5 and 40℃(B,C,D).

As shown in Fig 6,initial dye concentration was chosen at4,8,15,25 and 50mg/L of Congo red,respectively.According to the results,the decolorization was dependent on initial dye concentration.Initial dye concentration of 15mg/L of Congo red tolerated laccase activity w ith mediators ABTS or vanillin.However,the rate of dye decolorization gradually always increased w ith the increasing concentration of Congo red ofw ith violuric acid(Fig 6).The color of Congo red turned Pale yellow from red in the optimal laccase violuric acid system. The results demonstrated that the higher concentration of dye could inhibit the decolorization w ith ABTS or vanillin and that the concentration of dye had little effect on the decolorizationw ith violuric acid.

3 Discussion

The advantage of monokaryotic strains over dikaryotic strainsofwhite-rot fungi is that they are genetically more stable as they suffer fewer recombination events［17］.So in this study,themonokary strain of P.sanguineus has been selected for laccase production,which is higher than the other P.sanguineus strains reported. The capability of the white-rot fungus P.sanguineus to decolorize synthetic dyes under different culture conditions has been previously studied.This can be very favorable for some industrial purposes such as industrial dyes decolorization［18］.In consideration of the high levels and enzyme stability of laccase,we think that the P. sanguineus mk528 has the potential value for the application in the industries.

The past studies have proven that the optimal pH for dye decolorization lay in the acidic range.Butsuch a low pH is not suitable for the wastewater treatment and laccase capable of decolorizing dye efficiently at w ide pH ranges are desirable for industial applications［19］. This variationmay be due to the changes in the reaction caused by the substrate,oxygen or the enzyme itself［20］. So the optimal pH of laccases for decolorization is highly dependable on the structure of the substrate.The optimal pH of the laccase of P.sanguineus mk528 was able to efficiently degrade Congo red at pH 5.5,and it was obvious different from the optimal pH value of laccase. Generally,the initial enzyme concentration is an impor-tant parameter that significantly influences on the enzymatic degradation［21］.In thiswork,initial laccase activity of 5U/m Lwas featly and econom icalon Congo red degradation over other enzyme concentrations the decolorization percentage of Congo red was increased by a longer incubation time.The decolorization was exceeded 64%after incubation 7 h.The absolute enzyme activity can be crucial,and the decolorization decreases below a certain levelof laccase activity in the decolorization process.

Fig 6 Decolorizing effectivenessof thevarying dye concentrations in the various laccasemediators treatment(5U/m L)atpH 5.5 and 40℃

The laccasemediator system is generally more effective than laccase alone.It is different to enhance the decolorization efficiency of the laccase for differentmediators.Usually,naturalmediators have significant advances over synthetic mediators.Vanillin has only one methoxy group and thusamore sterically accessible phenolic group.Both compoundswere already tested asmediators in decolourization of dyes show ing promising results［22］.Vanillinwas selected as themosteffectivemediator for dye decolorizaiton laccase from T.versicolor.The decolorization of Reactive Red 198 and Rem Red RR was found to be 62%and 68%,respectively［23］.In the laccase-mediator system,violuric acid forms benzotriazolyl-1-oxide radical,whereas ABTS forms both the radical cation and a dication［24］.The redoxmediator HBT had no effect on the decolorization of dye IV by laccase whereas violuric acid was inhibitory.N-hydroxybenzotriazole had no effecton the laccase catalysiswhereas violuric acid inhibited the decolorization of disazo dye IV. The decolorization of four azo dyes by a bacterial laccase was enhanced by addition of the mediator ABTS, and methylsyringate was the bestmediator for Lac15 to decolorize azo dyes［25］.In thiswork,two artificialmediators(ABTS and violuric acid)and two naturalmediators (vanillin and ferulic acid)were used to promote the decolorization of Congo red by the laccase from P.sanguineus mk528.ABTSand violuric acid increased theefficiency and velocity of dye decolorization,and the highest decolorization rate was 81.2%w ith violuric acid.So violuric acid wasmore effective than both vanillin and ABTS as mediator of laccase.The effectiveness order for mediators at same concentration is violuric acid＞vanillin＞ABTS(Fig 5A).As a naturalmediator for dye decolorization,vanillin was notsuperior to violuric acid. Ferulic acid was showed more resistance to decolorize Congo red,since it partially suppressed the activity of laccase.This resultmay be related to the differentoxidationsmechanisms between ABTSand N-hydroxylmediators(violuric acid)［26］.

The decolorization capacity of laccase mediator system was influenced by the level ofmediator,syntheticmediators generally at concentrations above 1mM inactivate the laccase［27］.Our results showed that concentrationsof both violuric acid and vanillin higher than 0.5 mM were inhibitory for laccase activity.At a concentration of 5μM of ABTS exhibited themostefficientdecolorization,higheror lower concentration reduced the ability.Theneed and/ornatureof redoxmediator for the degradation of a particular dye could depend on the source of the fungal laccase［28］.The rate of dye decolorization was directly proportional to the initial dye concentration. In our study,the concentration of dye had a little effect on the decolorization efficiency w ith violuric acid as a mediator.In addition,increasing concentration of Congo red led to a higher decolorization rate.This could be related to the structure of the mediators and the dyes strongly.The reason may be that violuric acid has been chosen as a prototype of the-NOH type of mediators, whose high redox potentialhas found prom ising application as amediator in laccase biocatalytic processes.The results indicate that the laccase from P.sanguineus mk528 has potential application on the decolorization of dyeeffluentsw ith violuric acid asmediator.

The toxicity of azo dyes ismainly due to some enzymes and bacteria on the skin would converte azo dyes into carcinogenic aromatic am ineswhen the skin stained w ith dyes.The color group of congo red were consisted of conjugated double bonds and which would be decolored by laccase and mediator.We conjecture that the structure of double bonds and aromatic amines of congo red were destroyed and lead to the toxicity of congo red dim inished or disappeared.Further efforts to investigate the toxicity of products are currently underway in our laboratory.

4 Conclusions

In a word,the laccase mediator system reported here appears to be a good candidate and superior to other laccase mediator systems for the potential applications of enzymatic process involved in decolorization of azo dyes.In this study,the optimal operational conditions formaximum decoloration were found to be 4-50 mg/LCongo red,0.05mM violuric acid and 5U/m L laccase atpH 5.5.Thisenzymatic system providesa reasonable basis for development of a biotechnological dye degradation process and cost reduction of recalcitrant compound treatments.Further work is needed to develop a promising tool and validate the suggested rules for thebiodegradation ofazo dyes.

5 Acknow ledgements

This study was supported by the Cooperation Project of Hebei Academy of Sciences and Chinese Academy of Sciesces.The authors are grateful to Dr.Zhengying Li for her valuable assistance and to Dr.Jianqiang Kong for the rewarding discussion.

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Decolorization of Congo red w ith laccase from Pycnoporus sanguineus m k528 undermediator conditions

ZHANG Shu-yan1,ZHAO Cong-bo1,GUO Lin2,LIWei2
(1.InstituteofM icrobiology,HebeiAcademy of Sciences,Baoding 071051；2.State Key Laboratory ofMycology, InstituteofM icrobiology,ChineseAcademy of Sciences,Beijing 100101,China)

Congo red,an important recalcitrantazo-type dye,was decolorized using the crude laccase from amonokaryotic strain of Pycnoporus sanguineus mk528 undermediator conditions.Results showed that the naturalmediator ferulic acid inhibited the decolorization of Congo red and the other three potential laccasemediatorsenhanced decolorization of Congo red including violuric acid,2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate)(ABTS)and vanillin. The artificialmediator violuric acid,being themosteffective redoxmediator,wasmore effective than both ABTS and vanillin.The optimal condition for decolorization of Congo red was6 h incubation at pH 5.5 and 40℃w ith both 5 U/ m L laccase activity and 0.05mM violuric acid.Thisoptimal laccasemediator system appearsa good candidate for the potentialapplicationsof enzymatic process involved in decolorization of azo dyes.

Congo red；decolorization；laccase；mediators；Pycnoporussanguineus

X703·1；TQ925

A

2095－1736（2015）03－0030－07

10.3969/j.issn.2095-1736.2015.03.030

2014-07-14；

2014-09-29

中国科学院与河北省科学院两院合作项目（项目编号:2014058016）

章淑艳，专业方向为微生物对土壤修复、有机污染物降解方面的研究，E-mail:zsy7602@163.com；

李 伟，专业方向为真菌漆酶的基础和应用研究，E-mail:liw@im.ac.cn。