caracteristicas de la xilanasa producida por aspergillus versicolor

7/27/2019 Caracteristicas de La Xilanasa Producida Por Aspergillus Versicolor

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J. Basic Microbiol. 37 (1997) 6, 387 -394

(Biochemistry and Microbiology Department, Bioscience Institute, UNESP, 13506-900, Rio Claro,SP, Brazil)

Xylanase production by Aspergillus versicolor

ELEONORA ANOCARMO NA, LINEA. PIZZIRANI-KLEINER,EGINA. ROSIM MO NTEIROand JoAo ATILIO ORGE

(Received24 March 1997/Accepted24 June 1997)

The xylanolytic system of Aspergillus versicolor is controlled by induction and carbon cataboliterepression. Carboxymethylcellulose and wheat bran were the best inducers of xylanolytic activity.When the fungus was grown for 5 days on VOGEL'S iquid medium with w heat bran, the optimal pH

and temperature for xylanase production were 6.5 and 30 "C, respectively. Optimal conditions for thexylanolytic activity assay were at pH 6.0 and 55 "C. The half-life at 60 "C of the crude enzyme was6.5 and 21 minutes, i n the a bsen ce or presence of substrate, respectively.

Xylan is the main hem icellulosic component of plant biomass being present in appreciablequantities in agricultural and several agroindustrial wastes. From the products of xylanenzymatic hydrolysis it is possible to obtain cell protein, fuels and other chemicals. Xylana-ses combined with cellulase could have applications in food processing. Cellulase-freexylanases can be also utilized for preparation of cellulose pulps and liberation of textilefibres (WOODWARD 984; BIELY 1985, WONG et al. 1988). In view of the po tentialapplications of xylanases, a study of these enzymes from various sources and their multi-

plicity is desirab le.Among xylanolytic microorganisms, filamentous fungi have been more extensively

studied and the genus Aspergillus has been shown to be an efficient producer of xy lanases.Preliminary observations from our laboratory have demonstrated that a strain of Aspergillusversicolor, isolated from Brazilian soil, produced high xylanase and low cellulase levels,which is an interesting characteristic for some industrial applications. In this report wedescribe the production and some properties of xylanase obtained from this fungus.

Materials and methods

Organism and growth: Aspergillus versicolor isolated from soil was maintained on slants of solidVOGEL'Smedium (VOGEL 9 56). Liquid cultures were prepared on the same medium containing 1%

(w/v) of the carbon source indicated, at a p H adjusted or buffered for each experiment. ERLENMEYERflasks (125 ml) containing 25 ml of medium were inoculated with 1.0 ml of spore suspension( lo7spores/ml) and when not specified they were incubated for 60 h at 30 "C under standing condi-tion. All experiments were conducted in triplicate.

Enzyme preparations and assays: Cultures were harvested by filtration. The filtrate was assayed forextracellular activity. The mycelium was washed with water, frozen and ground with sand in50 m~ sodium acetate buffer, pH 5.0. The slurry was centrifuged at 6,000 x g and the supernatant

was the source of crude extract.Xylanase activity was assayed at 55 "C using 0.6% (w/v) oat spelt xylan (SIGM A)n sodium phos-phate buffer, pH 6.0. Reducing sugars release was measured using the dinitrosalicylic acid method(MILLER1959). One unit of enzyme activity was defined as the amount of enzyme which releases1 pmol xy lose equivalent per m in.

26%



388 ELEONORAANOCARMONAt al.

Avicelase and carboxymethylcellulase (CMCase) activities were determined by the same methodusing avicel (RIEDEL E HAEN) nd carboxymethylcellulose (CMC, RUKA.G.) at 1.0% (w/v) assubstrates. Standard cellulolytic assay conditions were 40 "C and pH 5.0. One unit of cellulase acti-vity was defined as the amount of enzyme which releases 1 pmol glucose equivalent per min. In allcas es specific activities w ere expressed as activity units per m illigram of protein.

Protein determination:Protein content was m easured by the method of LOWRY sing bovine serumalbumin as standard.

Results and discussion

Influence of the carbon source o n xylanase production

CMC, avicel, xylan and xylose, in that order, were good inducers of extracellular xylanaseactivity (Table 1). The highest level of intracellular xylanase activity was detected in mediumsupplemented with xylan, and in all cases the intracellular levels were much lower than the

extracellular ones. G lucose, maltose, lactose, sucrose, cellobiose, starch, pectin, chitin, andsalycin were totally ineffective of inducing extracellular xylanase activity. The fact thatcellulose induced xylanase better than xylan, suggested that xylanase regulation in A . versi-color may be linked with that of cellulase, as is reported for Trichoderma longibrachiatum(ROYERand NAKAS1990). The levels of xylanase activities were not significantly in-fluenced by agitation of the cultures (Table 1).

Extracellular xylano lytic activity was not detec ted in cultures con taining glucose or mix-tures of glucose and xylan. This fact indicates that glucose acts as a catabo lite repressor ofxylanase in A. versicolor.

Table 1

Influence of some carbon sources on xylanase production by A . versicolor

Carbon Specific activity (U/mg prot.)

sourceAgitated culture Standing culture

Extracellular Intracellular Extracellular Intracellular

CMC 22.75 f 2.13 0.48f .03 21.42k 2.02 0.70 f 0.01Avicel 15.58f 1.21 0.38 f 0.02 14.86k 1.02 0.48 f .01Xylan 12.85k 1.15 1.04f 0.24 15.97k 0.96 1.53f 0.01Xylose 6.20 k 0.43 0.70f .06 4.27 k 0.91 0.61 f .04

Average and standard deviation of three cultures.

Table 2

Extracellular celluloly tic activity of A. versicolorgrown o n various substrates

Carbon source Specific activity (U/mg prot.)

CMCase Avicelase

CMC 0.34 k 0.04 0.22 f 0.02

Avicel 0.63 f 0.06 0.38 f 0.04Xylan 1.15f0.19 0.67 f 0.07Wheat bran 0.32 f 0.04 0.05 f 0.01




Xylanase production by A. versicolor

Extracellular cellulolytic activity

Considering that cellulose induced the highest xylanase activity, the effect of xylan as asubstrate was compared for induction of cellulolytic activity. Table 2 shows that xylan wasthe best substrate to induce CM Case and avicelase activities, suggesting that substrate re-gulation of xylanase and cellulase synthesis were linked.

In order to investigate in detail the participation of cellulose on the production ofxylanase by A. versicolor, we verified the levels of xylanase in media containing aviceland xylan, or CMC and xylan, in different ratios. In these experimen ts the total amount ofcarbon source was maintained constant, being altered only the cellulose-to-xylan ratio. T heresults showed that xylanase activity decreased when the amoun t of cellulose in the m ediumwas reduced.

389

Induction of xylanolytic activity by raw agricultural residues

Since cellulose induced the highest xylanase activities, lignocellulosic materials w ith differ-ent cellulose content from various sources, were compared as substrates for the production

of xylanase activities (Table 3). Among the agricultural residues tested, wheat bran was the

Table 3Effect of different natural substrates on xylanase production byA. versicolor

Carbon source Specific activity ( U h g prot.)

Extracellular Intracellular

Wheat bran 12.34f .20 0.94 f .01

Pinus bark 4.60 f .50 ND

Rice straw 1.47f 0.18 ND

Sugar-cane bagasse 1.09f

.100.05 f

.01Potato plant 0.54 f .01 0.03f 0.01

Maize pith 0.43 f 0.06 0.05 f 0.01Sawdust 0.29 f .01 NDEucalyptus bark 0.03 f .01 ND


ND =Not detectable.

Fig. 1Influence of cultural pHon xylanase production

by A. versicolor grownduring 5 days in me-

dium prepared with

MCILVAINEuffer con-

taining wheat bran. (o),extracellular activity.

(A), intracellular activ-

total protein).ity. (9,rowth (mg

CulturepH



390 ELEONORAANO ARMONAt al.

100 . 5

80

20

0

-.-c

0Q Fig. 2

Effect of cultural tem-perature on xylanaseproduction by A. versi-

color cultivated during5 days in medium pH6.5 with wheat bran.(o), extracellular act-ivity. (A), intracellularactivity. (*), growth(mg total protein).

Culture Temperature C C)

best substrate for induction of xylanase activity, exhibiting a level of production very closeto xylan (see Table 1). Pinus bark also was a good xy lanase inducer.

Effect of culture conditions on xylanase production

Since the presence of wheat bran as carbon source produced xylanolytic activity comparable

to media containing pure xylan, we established the optimal conditions for production ofxylanase using this agricultural residue. The replacement of pure xylan by wheat bran ma-kes the enzyme production process economically more feasible. The growth yield of

A . versicolor cultures in wheat bran was pH independent between 4.0 and 7.5, but the opti-mum initial pH for xy lanase production was 6.5 (Fig. 1). The optimum production pH for

150

0 0.0

Fig. 3

Time-course of xy-lanase production byA. versicolor grown inmedium containing 1%(w/v) wheat bran as thesole carbon source.(o), extracellular activ-ity. (A), intracellularactivity. (*), growth(mg total protein).

2 4 6 8 10

Time (day)



Xylanase production by A. versicolor 391

other Aspergillus species was observed at 6.8 for A. niduluns (FERNANDEZ-ESPINARt al.1992), 4.0 for A. awamori (SMITH nd WOOD 1991) and A. sydowii (GHOSH t al. 1993),but it was not significan tly changed from 6.0 to 10.0 pH for A. fischeri (RAJnd CHANDRA1995).

The optimum production temperature was observed at 30 "C, but m aximal growth wasobserved at 20 "C due to the mesoph ilic character of this strain (Fig. 2). At 40 "C the pro-duction of ex tracellular xylanase was drastically diminished. This data is in good agreem entwith those reported for other Aspergillus species (BISWAS t ul. 1988, SMITH nd WOOD1991, GHOSH t al. 1993), with exception of A. niduluns for which temperature optimumwas 37 "C FERN~DEZ-ESPINARt al. 1992).

The time-course of xylanase production by A . versicolor grown in wheat bran showedthat the highest enzymatic levels occured in five day-old cultures (Fig . 3). In these condi-tions, the most of xylanase activity (92.4%) produced by the induced m ycelium is secretedinto the medium. A very low level of the enzyme appeared bound to the cell wall (l.l%),and about 6.5% of the total activity was retained intracellularly .

Properties of the extracellular crude xylanase

The optima of temperature and pH were 55 "C and 6.0, respectively (Fig. 4). This pHoptimum falls w ithin the acid region described for most fungal xylanases. The value of the

100

80

f-' 0

20

3 4 5 6 7PH

Fig. 4

pH-Dependence (a) and temperature-dependence (b) of extracellular xylana-se from A. versicolor cultivated during5 days in medium pH 6.5 with wheatbran. In (a) the enzymatic activity was

determined at 40 "C in MCILVAWEuf-fer, and (b) it was determined in so-dium phosphate buffer 100m pH 6.0.

Temperature&)



392 ELEONORAANO ARMONAt al.

Fig. 5Thermal denaturation of

crude xylanase activity. Anaqueous solution of the crud eenzyme was heated in awater bath at 60 "C. Sampleswere withdrawn at the in-dicated times, chilled andtested for residual xylanaseactivity. (o), sample incu-bated without substrate. (a),samp le heated in presence of1%(w/v) substrate.

Time (min)

A . vers ico lor temperature optimum is very close to that described for crude preparationfrom A . niduluns (FERNANDEZ-ESPINARt ul.1992) and o ther xylanases purified from otherAsperg i l lus species (FREDERICKt al. 1981, I T 0 et ul. 1992, KORMELINKt ul. 1993).

Thermal stability of the extracellular crude xylanase from A . vers ico lor was studied be-tween 40 "C and 70 "C at pH 6.0, without substrate. The half-life at 60 "C was 6.5 min.,which does not differ markedly from that reported for A . fischeri crude xylanase (RAJ and

CHANDRA 995). However, when the A . ver s i co l o r xylanase crude enzym e was incubated at60 "C in the presence of substrate, the half-life increased about 3-fold (Fig. 5).

References

BIELY , ., 1985. Microbial xylanolytic systems. Trends Biotechnol., 3, 286-290.BISWAS, . R., MISHRA, . K. and NA NDA , ., 1988. Xylanase and P-xylosidase production by As-

pergillus ochraceus during growth on lignocelluloses. Biotechnol. Bioeng., 31 ,61 3 -616.FERNANDEZ-ESPINAR,. T., R A M ~ N ,., PINAGA,F. and VALLfiS, S., 1992. Xylanase production by

Aspergillus nidulans. E M S Microbiol. Lett., 91, 91 -96.FREDERICK,. M., FREDERICK,. R., FRATZKE, . R. and REILLY, . J., 1981. Purification and cha-

racterization of a xylobiose- and xylose-producing endo-x ylanase from Aspergillus niger. Car-bohydr. Res., 97,87-10 3.

GHOSH,M., DAS,A., MISHRA, . K. and NANDA ,G., 1993. Aspergillus sydowii MG 49 is a strongproducer of thermostable xylanolytic enzymes. Enzyme M icrob. Technol., 1 5, 70 3 709.

ITO,K., OGASAWARA,., SUGIMOTO,. and ISHIKAWA,., 1992 . Purification and properties of acidstable xylanases from Aspergillus kawachii. Biosci. Biotech. Biochem., 56,547-550 .

KORMELINK,. J. M., SEARLE-VAN EEU WE N, . J. F., WOOD,T. M. and VORAGEN,. G. J., 1993.Purification and characterization of three en do-( 1,4)-P-xy lanases and o ne P-xylosidase from A s-pergillus awamori. J. Biotechnol., 27,249 -265.

MILLER, . H., 1959 . Use of d initrosalicylic acid reagent fo r determination of reduc ing sug ar. Anal.

Chem., 31,4 26-4 28.RAJ, K. C. and CHANDRA,. S., 1995. A cellulase-free xylanase from alkali-tolerant Aspergillusfischeri Fxn l. Biotechnol. Lett., 17, 309-314.

ROYER,. C. and NAKAS,. P., 1990 . Interrelationship of xylanase induction and cellulase inductionof Trichodenna longibrachiatum. Appl. Environ. Microbiol., 56 ,25 35 -2539.



Xylanase production by A. versicolor 393

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Mailing address: ELEONORAANOCARMONA,epartamento de Bioquimica e Microbiologia, I.B.,UNESP, Av. 24-A, 1515, 13506-900-Rio Claro, SP, Brazil

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