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On-line version ISSN 1678-4405
Braz. J. Microbiol. vol.38 no.2 São Paulo Apr./June 2007
Produção e caracterização de um complexo enzimático de uma nova linhagem de Clostridium thermocellum com enfase em sua atividade de xilanase
Werner Bessa Vieira; Leonora Rios de Souza Moreira; Amadeu Monteiro Neto; Edivaldo Ximenes Ferreira Filho*
Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brasil
A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S300. Zymogram analysis after SDSPAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under nondenaturing conditions. The hydrolysis of xylan was optimal at temperature range of 5575ºC and pH 6.0. Xylanase activity was quite stable at 65ºC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, Ltryptophan and Lcysteine and strongly inhibited by Nbromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65ºC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65ºC with halflife of 2.5 h and inhibited by dithiothreitol and Ca2+.
Keywords: Clostridium thermocellum, banana stem, xylanase
Uma nova linhagem de bactéria (ISO II) foi isolada de esterco bovino e identificada como filogeneticamente próxima à bactéria termofílica Clostridium thermocellum. A nova linhagem produziu atividades de xilanase, mananase, pectinase e celulase quando cultivada em meio de cultura líquido contendo engaço de bananeira como fonte de carbono. O perfil de produção enzimática após crescimento em engaço de bananeira mostrou que as atividades de xilanase e celulase foram detectadas em diferentes períodos de incubação. Um complexo enzimático, contendo atividades de xilanase, celulase e mananase, foi isolado de amostras de sobrenadante do meio de cultura da linhagem ISO II crescida em engaço de bananeira. O complexo foi parcialmente purificado por ultrafiltração e cromatografia de filtração em gel em coluna de Sephacryl S300. Análise de zimograma mostrou 05 subunidades com atividade de xilanase. A amostra enzimática apresentou bandas únicas de proteína e atividade de xilanase após eletroforese sob condições nãodesnaturantes. A hidrólise de xilana foi ótima no intervalo de temperatura de 5575ºC e pH 6,0. A xilanase foi estável a 65ºC, mantendo 80% de sua atividade original após 12 h de incubação. Os valores de Km aparente, usando arabinoxilanas insolúveis e solúveis como substratos, foram 1,54 and 11,53 mg/mL, respectivamente. A xilanase foi ativada por ditiotreitol, Ltriptofano and Lcisteina e fortemente inibida por Nbromosuccinamida e CoCl2. A caracterização da mananase do complexo mostrou Km e temperatura ótima de 0,846 mg/mL e 65ºC, respectivamente e pH 8,0. Ao contrário da xilanase, a mananase foi menos estável a 65ºC com meia vida de 2,5 h e inibida por ditiotreitol e Ca2+.
Palavraschave: Clostridium thermocellum, engaço de bananeira, xilanase
The banana plant produces a residual component named stem. The stem in natura presents 93% of humidity and parenchymatic cells in abundance (20). In terms of chemical composition, total extractives, holocellulose and lignin account for as much as 47%, 45.6% and 7.4% of its dry weight, respectively (20). For this reason, stem can be considered an alternative carbon source for enzyme production. The banana stem, grain stalk that supports the banana fruits readily available in tropical and subtropical countries, is normally discarded after the fruit harvesting, either in the "packing houses" or in the delivering centers, contributing to serious environmental problems (12,17). Due to its heterogeneity and complex chemical nature, the lignocellulose biodegradation of banana stem requires the coordinated action of several enzymes, including cellulase, xylanase and mannanase with different specificities to effect extensive hydrolysis to its monomeric components.
Some microorganisms are reported to produce enzyme systems containing multiactivity (1,3,18). For example, C. thermocellum and C. cellulolyticum, grampositive, thermophilic and anaerobic bacteria, produce a multienzyme complex (cellulosome) when grown on cellulose as the substrate. In this paper, we describe the isolation, partial purification and characterization of an enzyme complex, containing xylanase, cellulase and mannanase activities from the culture supernatant of a new strain of C. thermocellum ISO II grown on banana stem.
MATERIALS AND METHODS
Oat spelt xylan, locust bean gum (galactomannan), carboxymethyl cellulose (CMC), pnitrophenylbDglucopyranoside (PNPG), pnitrophenylbDxylopyranoside (PNPX), pnitrophenylaDarabinofuranoside (PNPA), pnitrophenylbDmannopyranoside (PNPM), pectin from citrus fruits, dithiothreitol (DTT), Nbromosuccinimide (NBS), 1ethyl3(3dimethylaminopropyl) carbodiimide (EDC) and diethyl pyrocarbonate (DEPC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Banana stem was kindly provided by Francides Gomes da Silva Jr (University of São Paulo, Brazil). Sephacryl S300 was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Each experiment below was repeated at least three times. The standard deviation for enzyme assays was less than ±20% of the mean.
Microorganism and enzyme production
Clostridium thermocellum strain ISO II was isolated from the manure of cow and identified at the Fundação André Tosello (Campinas, Brazil). Fragments of 16 S rDNA were PCR amplified and submitted to sequence in an automated sequencer (ALFexpress, Amersham Pharmacia). C. thermocellum strain JW20 was kindly provided by Dr. Lars G. Ljungdahl (University of Georgia, USA). For mannanase, cellulase and xylanase production, 2,0 mL of spore suspensions of C. thermocellum strains ISO II and JW20, obtained from a 7dayculture, were cultured at 60ºC for 7 days (early stationary phase) in a prereduced liquidstate medium (9) under anaerobic conditions containing 2% (w/v) of powdered banana stem. After the growth procedure, the resulting culture supernatant was centrifuged for 20 min at 10,400 x g and stored at 4ºC prior enzymatic assays. For enzyme induction, aliquots were harvested every 24 h during 08 days, and used to estimate the enzyme activity.
The hydrolysis of polysaccharides (oat spelt xylan, pectin, galactomannan and CMC) was determined by mixing 50 µL de enzyme solution with 100 mL of substrate (1%, w/v) in 50 mM sodium acetate buffer, pH 5.0 at 50ºC for 30 min. The reducing sugars released was measured using the DNS (dinitrosalicylic) method (4,12). Enzyme activities were expressed as µmol product formed min1ml1 of enzyme solution, i.e., as IU mL1. The enzyme activities against PNPX, PNPA, PNPG, PNPM and filter paper were carried out as described elsewhere (6,21). Protein concentration was measured by the method of Bradford (2), using bovine serum albumin as standard. For the kinetic experiments, substrates were used in concentration ranges of 0.56,0 mg mL1 (insoluble oat spelt xylan), 0.515 mg mL1 (soluble oat spelt xylan) and 0.56.66 mg mL1 (galactomannan). Km and Vmax Values were estimated from MichaelisMenten equation with a nonlinear regression data analysis program (19). The determination of optimum temperature was carried out in the temperature range of 30 to 90ºC. The optimum pH was determined by measuring the activity at 50ºC at various pH values between 3.0 and 9.0. All buffers were adjusted to the same ionic strength with NaCl. The enzyme stability was carried out by preincubating the enzyme solution at 55, 65 and 70ºC and removing aliquots at intervals to measure its activity as described above. The activity of the enzyme was also performed in the presence of amino acid modifying (NBS, iodoacetamide, EDC, DEPC and DTT), metals (CuSO4, ZnSO4, CaCl2, FeCl3, AlCl3 and CoCl2) and aminoacids (Ltryptophan and Lcysteine). The reaction mixtures contained individual reagents at a final concentration of 10 mM (Ltryptophan, NBS, DTT, iodoacetamide, EDC, DEPC and Lcysteine) and 5.6 mM (CuSO4, ZnSO4, CaCl2, FeCl3, AlCl3 and CoCl2). Appropriate controls were included in all cases.
All the purification steps were carried out at 10ºC unless otherwise specified. The culture supernatant was concentrated by ultrafiltration using an Amicon system (Amicon Inc., Beverly, MA 01915, USA) with a 300 kDa cutoff point membrane (PM 300). Aliquots (4 ml) of the concentrate were fractionated by gel filtration on Sephacryl S300 (2.4 x 67 cm) column preequilibrated with 50 mM sodium phosphate buffer, pH 7.0. Fractions of 5.0 ml were collected at a flow rate of 30 ml/h. Fractions (4149) with bmannanase, bxylanase and cellulase activities were pooled, concentrated by freezedrying and stored for later use at 4ºC. The void volume of Sephacryl S300 column was determined by using thyroglobulin (669 kDa) as molecular weight marker under the same conditions as described above.
Enzyme preparations were submitted to denaturing (SDSPAGE) and nondenaturing electrophoresis on 7.5% gels by the method of Laemmli (10). After electrophoresis, the gels were stained for protein with silver nitrate (4). Molecular mass standards from Sigma (USA) were used as markers. Replicate denaturing and nondenaturing electrophoretic gels, containing 1% oat spelt xylan, were submitted to zymogram analysis (16). They were stained for xylanase activity in a Congo red solution (0.1%) for 30 min at room temperature and washed with 1 M NaCl to remove excess dye and fixed with 0.5% acetic acid.
RESULTS AND DISCUSSION
The partial 16S rDNA sequence of ISO II was compared with the 16S rDNA sequences of organisms available in Ribosomal Database Project (www.cme.msu.edu/RDP/html/index.html) and Genbank (www.ncbi.nlm.nih.gov). Phylogenetic analysis of the partial 16S rDNA sequence of the present strain showed that the highest similarity (93%) was obtained with the 16S rDNA of C. thermocellum. Zhilina et al. (22) described a new strain of Clostridium (Z7026) showing 94.8, 94.9 and 95.5% of similarity with 16S rDNA sequences of Acetivibrio cellulolyticus, C. aldrichii and C. thermocellum, respectively.
C. thermocellum strain ISO II was grown on banana stem for determination of the effect of this substrate on xylanase and cellulase production by the bacterium. The enzymes production of C. thermocellum strain ISO II was also compared with C. thermocellum strain JW20. For convenience, culture conditions (the amount of substrate, temperature, pH, inoculum and incubation period) and enzyme assays were the same for both strains. At different time intervals, the samples were taken and assayed for enzyme activity.
Both strains were able to grow on banana stem. The growth profile of strain ISO II on banana stem was accompanied by a highest peak of xylanase activity (2.94 IU/mL) at cultivation interval of 140170 h, while the production of xylanase activity by strain JW20 reached its maximum (5.14 IU/mL) at 168 h cultivation. For both strains, CMCase activity was expressed at 20 h of cultivation in banana stem containing medium. For the growth of strain JW20, an activity peak of 2.4 IU/mL was observed. CMCase activity remained constant after 72 h cultivation and was detectable in strain ISO II in much lower level. The highest activity was obtained after 48 h cultivation with a rapid decrease after this period.
An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from C. thermocellum strain ISO II when grown in banana stem and purified by a combination of ultrafiltration and gel filtration procedures. Accordingly, the summary of the purification procedure refers to the purification of xylanase activity. The culture supernatant was concentrated by ultrafiltration with a 300 kDa cutoff point membrane (PM 300). The ultrafiltrate and concentrate were assayed for activity as a matter of course. Mannanase, cellulase and xylanase activities were found in the concentrate, while pectinase and a small molecular mass xylanase activities permeated the ultrafiltration membrane. For further purification, the concentrate was subjected to gel filtration chromatography on Sephacryl S300 (Fig. 1). One peak of protein coeluted with xylanase, cellulase and mannanase activities in the void volume. The simple twostep purification procedure provided purification fold and yield of 2.66 and 6.35%, respectively. The low yield value was mainly due to loss of enzyme activity in the ultrafiltration step. Xylanase activity (total activity of 7.16 IU) was found in the ultrafiltrate. According to Filho et al. (5), comparison of these values with those reported for the relevant enzyme systems from other sources is not very meaningful because of the interlaboratory variability in xylanase assays and because xylanases differ from one another with respect to whether their actions require or are hindered by substituents on the substrates used. Furthermore, we can not discard the fact that a high amount of pigment present in culture supernatant, frequently reported after fungi growth on liquid medium containing banana stem as the carbon source (12), interfered in the protein assay. MohandOussaid et al. (13) reported three major fractions containing xylanase activity from Clostridium cellulolyticum after chromatography on a FPLC Superose column, being the first one eluted with the void volume and associated with cellulase activity (avicelase and carboxymethyl cellulase). In comparison with the present enzyme of strain ISO II, C. thermocellum strain JW20 also produced an enzyme system with high molecular mass. The ultrafiltration (PM 300) and chromatography in Sephacryl S300 also showed xylanase, cellulase and mannanase activities eluted with the void volume.
The partially purified enzyme complex of strain ISO II migrated on SDSPAGE as several bands varying from 25 to 116 kDa (Fig. 2). The zymogram analysis was performed by renaturing the enzyme after electrophoresis and visualized by staining with Congo red. In this case, five bands staining for xylanase activity (X1, X2, X3, X4 and X5) were coincident with those staining for protein. A clear hydrolysis activity zone was formed against a dark background. X1 migrated with molecular mass above 116 kDa. The molecular mass values of X2, X3, X4 and X5 were estimated to be 97.4, 66, 48.5 and 34 kDa, respectively. X3 showed a molecular mass value close to xylanase XynC, one of the major component of C. thermocellum F1 cellulosome (8). The identification of the proteins responsible for cellulase and mannanase activities will help to establish if these activities are displayed by the same or different enzymes. The native PAGE showed a prominent protein band at the top of the gel with a corresponding xylanase activity band (result not shown). It was reported that the cellulosome of C. thermocellum YS was composed of a number of different proteins, most of them having enzymatic activity, including cellulase and xylanases (11,18), with four protein bands, being two with molecular mass values above 600 kDa and two around 170 and 240 kDa, were detected by the zymogram.
The partially purified enzyme complex exhibited maximal mannanase and xylanase activities at 65ºC and temperature range of 5575ºC, respectively. Mannanase activity was stable under alkaline conditions with optimum pH of 8.0, while xylanase displayed a higher activity at pH 6.0 and maintained more than 60% of its activity at pH range of 3.58.5. It retained 85% of xylanase activity, after incubation at 65ºC for 12 h. At the same temperature, mannanase activity showed halflife of 2.5 h. Xylanase from C. acetobutylicum was stable at 60ºC for 1 h at pH 5.06.5 (14). The optimum temperature and pH of the isolated cellulosometype enzyme of Bacteroides sp. strain P1 were 50ºC and 6.0, respectively (15).
The xylan breakdown is dependent on several factors, including enzyme synergism, the interaction with different subsites on the heterogeneous substrate, the interaction of the subunits within the xylandegrading enzyme system and the probable presence of binding molecules in addition to the catalytic modules (which have different affinities for soluble and insoluble xylan). Despite the difficulties to determine kinetic parameters with a polymeric and rather undefined substrate (in which each molecule has a different number of attacking points), the apparent Km and Vmax values on soluble and insoluble xylans from oat spelt were measured. The xylanase activity from the partially purified enzyme complex of strain ISO II was most active on insoluble xylan (Table 1). The Km value for soluble xylan was much higher than the insoluble one. The same result was found for the enzyme complex of strain JW20. This might suggest a steric hindrance due to the presence of sidechains groups in soluble xylan (19). On the other hand, the xylanase activity from the partially purified enzyme complex of strain JW20 displayed more affinity against soluble and insoluble xylans with Km values of 7.6 mg/mL and 1.48 mg/mL, respectively. In case of the enzyme complex from strain ISO II, the Km value for galactomannan as the substrate was 0.846 mg/mL.
The influence of various reagents on xylanase and manannase activities from the partially purified enzyme complex of strain ISO II was investigated (Table 2). A significant negative effect on xylanase and mannanase activities was observed with Co2+. Xylanase was highly activated by DTT, Lcysteine and Ltryptophan, suggesting an influence of Lcysteine in the catalysis of xylan. Ca2+ did not affect the xylanase activity. On the contrary to xylanase, DTT, iodoacetamide, Fe3+ and Ca2+ inhibited the mannanase activity. In contrast to above, Ca2+ ions were reported to stimulate the activity of soluble components of cellulosomes (18). Evidence for the involvement of the Ltryptophan residue at the active site is given by the strong inhibition of xylanase and mannanase by NBS (4). The inactivation of mannanase by DEPC and EDC indicates that histidine and carboxyl groups may be involved, respectively in catalysis. In comparison to the above results, xylanase activity from the enzyme of strain JW20 was also activated, to a large extent, by DTT, Ltryptophan and Lcysteine. It was slightly activated by EDC and iodoacetamide.
The partially purified enzyme complex of strain ISO II exhibited no action against PNPG, PNPA and PNPM. It displayed activity towards some polymeric substrate (xylan, filter paper, CMC and galactomannan), but it was not active against pectin. Among them, the highest activity was on xylan from oat spelt (Table 3). The enzyme exhibited a slight activity against PNPX. In opposite to the present enzyme complex, XynCII of cellulosome from C. thermocellum F1 and cellulosometype enzymes of Bacteroides succinogenes S85 showed bglucosidase activity (7,8). In addition, the cellulosome of Bacteroides succinogenes S85 consisted of eight endoglucanases and two xylanases. Most of the cellulosomes are composed of different types of enzymes, including endo and exoacting glucanases, xylanase, arabinofuranosidase, mannanase and pectin lyase (1). Twelve cellulosomal enzymes have been identified in C. cellulolyticum, including cellulase, hemicellulase and pectinase (3).
In conclusion, Clostridium thermocellum strain ISO II produced a enzyme complex with xylanase, cellulase and mannanase activities and stability at 65ºC. It showed, at least, 05 subunits with xylanase activity. Further work will be concentrated on the of nature of this enzyme complex (by western blots using C. thermocellum cohesin or dockerin probes), whether it is organized in cellulosomes or not and the characterization of its cellulase activity. The specific activity on CMC and filter paper should be quantified and compared to the xylanase and mannanase activities.
E.X.F.F. acknowledges the receipt of research fellowship from CNPq. L.R.S.M., A.M.N. and W.B.V. acknowledge the receipt of maintenance research scholarship from CNPq, PIBIC and CAPES, respectively. The authors thank Dr. Eduardo de Aquino Ximenes for proof reading the manuscript.
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Submitted: August 07, 2006; Returned to authors for corrections: October 19, 2006 Approved: February 23, 2007.
* Correponding Author. Mailing address: Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, 70910900, Brasília, DF, Brasil. Tel.: (61) 33072152 ou (61) 32734608. Email: firstname.lastname@example.org