Production of xylanases from a newly isolated ... - Springer Link

Biotechnology Letters, Vol 20, No 11, November 1998, pp. 1067–1071

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp. M. Bataillon1, A.-P. Nunes Cardinali2 and F. Duchiron1 1

Laboratoire de Microbiologie Industrielle, Université de Reims-Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France (Fax: 133 3 26 05 38 87) 2 Instituto Nacional de Investigação Agrária, Lisboa, Portugal A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. Keywords: Xylanase, Ferulic acid esterase, Bacillus

Introduction Large amounts of wheat bran are produced annually from the milling processes of many countries around the world. A major proportion of these cereal byproducts are used as animal feed, but in recent years, decreasing cereal prices have stimulated research on the valorization of these products (Voragen et al., 1997). The major constituents of wheat bran are polysaccharides. Many papers report on the enzymatic degradation of cereal arabinoxylan. Conversion of xylans into monomeric compounds gives rise to precursors of the sweetener xylitol, flavours, non-ionic surfactants or fermentation substrates. Recently the interest in thermostable enzymes has increased and thermostability has become a desirable property of enzymes used in many industrial applications. The advantages of thermostable enzymes in industrial processes include reduced risk of contamination, increased solubility of the substrate, increased diffusion rates and higher stability of enzymes against denaturing agents and proteolytic enzymes. Our aim was to find thermostable enzymes that could hydrolyze arabinoxylan from destarched wheat bran to produce pentoses (xylose and arabinose). Industrial destarched wheat bran is obtained by washing with hot water (Naudin and De Baynast, 1990). This paper reports on the isolation of a thermophilic Bacillus sp. that produces thermostable extracellular xylanase and b-xylosidase. The production, and main properties of the crude enzymes and the hydrolysis of destarched wheat bran are discussed. We also show that the presence of a ferulic acid esterase during © 1998 Chapman & Hall

hydrolysis increases the amount of xylose in the soluble fraction. Materials and methods All analytical and media chemicals were purchased from Sigma. Wheat bran arabinoxylan was extracted from destarched wheat bran as previously described (Bataillon et al., 1998). Aspergillus niger ferulic acid esterase (FAE-III) was obtained from Institute of Food Research, UK (Faulds and Willianson, 1994). Bacterial strain isolation conditions The basal medium used for screening was Thermus medium basal salts (Williams and Da Costa, 1992) with (g/l): wheat bran arabinoxylan, 2; ammonium phosphate, 2; pH 8.2. The strain used in this study (SPS-0) was isolated from the hot spring at São-Pedro do Sul in Northern Portugal. Water samples were filtered through membrane filters (pore size: 0.45 mm); the filters were placed on the surfaces of a agar plates medium. The plates were wrapped in plastic bags and incubated at 60°C over 7 days. Colonies were picked and transferred in agar plates medium; colonies with extracellular xylanolytic activity were identified by clear zones produced around the colonies (Subramaniyan et al., 1997). Morphological properties and taxonomic characteristics of the bacteria were studied according to methods in Bergey’s Manual of Determinative Bacteriology. Culture conditions and enzyme production The medium used was Thermus medium basal salts with (g/l): wheat bran arabinoxylan, 5; yeast extract, 4; tryptones, 1; pH 8.2. Enzyme production was carried out in 500 ml Biotechnology Letters ⋅ Vol 20 ⋅ No 11 ⋅ 1998

1067

M. Bataillon et al. baffled flasks containing 50 ml medium. Flasks were shaken continuously on an orbital shaker at 130 rpm and the temperature maintained at 60°C. Large-scale production of batch fermenter cultures were undertaken. Xylanase production in the fermentor was comparable with that in culture flasks, thereby indicating that scaling up does not result in a reduction of xylanase production. Fermenter cultivations were carried out in a 40 l (working volume) stirred bioreactor (Chemap, France). During culture fermentation, the pH remained in the range of 7.6–8.1. Dissolved O2 concentration was monitored and kept above 20% saturation by automatic control of air flow and stirring rates. Cell growth into liquid cultures was monitored by measuring the optical density at 610 nm. After 24 h culture, the cells were removed by centrifugation at 4000 g for 15 min. The cell free supernatant was concentrated by ultrafiltration with a PM 10 membrane (10 kDa cut-off; Amicon) and stored at 4°C. Xylanase and b-xylosidase activities in the supernatant were respectively 1000 and 60 nkat/ml and this preparation was used for hydrolysis experiments. Influence of temperature and pH The xylanase activity was measured at temperatures ranging from 35 to 85°C. Enzyme activities were also tested in buffer at pH values from 4 to 11. In order to determine the temperature stability of the enzymes, the ultrafiltered crude enzyme solution was incubated at 60°C, 70°C, 85°C and 100°C and pH 6.0 in the absence of substrate. Samples were removed at different time intervals and immediately cooled. Residual enzyme activity was determined under standard assay conditions. Enzymes activity assays Xylanase activity was measured by a modification of the method of Bailey et al. (1992) by incubating the enzyme solution with a substrate solution of 10g birchwood xylan/l in phosphate buffer (pH 6.0, 0.05 M) at 70°C for 10 min. The release of reducing sugars was measured as xylose by the dinitrosalicylic acid reducing sugar test (Miller, 1959). b-Xylosidase activity was conducted according to the method of Herr et al. (1978) using pnitrophenyl-b-Dxyloside. One nkat (0.0599 IU) of the enzyme activity is defined as the amount of enzyme which catalyses the release of 1 nmol of xylose per s. One unit of activity ferulic acid esterase was defined as the amount of enzyme releasing 1 mmol ferulic acid per min at pH 6.0 and 37°C. Hydrolysis of wheat bran Hydrolysis was carried out in 300 ml reactors with birchwood xylan or wheat bran in 5% (w/v) distilled water, in a

1068

Biotechnology Letters ⋅ Vol 20 ⋅ No 11 ⋅ 1998

reaction mixture volume of 50 ml at 60°C. The aliquots were removed at different time intervals and filtered (0.2 mm). Controls without substrate (enzyme control) and controls without enzyme (substrate control) were made. The hydrolysis of destarched wheat bran complemented with FAE-III esterase was performed under the same experimental conditions and under the following steps : a 5% solution of destarched wheat bran with SPS-0 xylanase (3000 nkat/g of substrate); after 5h hydrolysis, the temperature was decreased from 60°C to 37°C and 100 ml of FAE-III esterase (0.077 nkat/g of substrate) was added; after a further 2 h incubation, the temperature was increased from 37°C to 60°C and the sample was further hydrolysed with SPS-0 xylanase. Analytical methods Monomeric and oligomeric reaction products were analyzed by HPLC using an Aminex HPX-42A column. The column temperature was maintained at 85°C and water was used as eluent at a flow rate of 0.5 ml/min. The sugars were detected with a refractive index detector. The soluble proteins present in the enzyme preparation was determined using the method of Bradford (1976) with the Bio-Rad Coomassie Blue protein reagent assay. Bovine serum albumin was used as standard. Results and discussion Isolation and characterization of Bacillus SPS-0 The thermophilic strain SPS-0 was isolated from an enrichment culture containing wheat bran arabinoxylan. The strain was selected among other isolates for producing high xylanase activity on xylan plates. The strain grew in the pH range from 6.0 to 9.0 and in the temperature range from 40 to 65°C. Its maximum growth rate was reached at pH 7.0 and 60°C. The isolate was an aerobic, Gramvariable rod (3–8 3 0.5 mm), forming endospores (ellipsoidal, terminal). The membrane fatty acids profile of the aerobic isolate SPS-0 was almost identical to that of Bacillus thermoleovorans (Sunna et al., 1997) with the main fatty acids being represented by iso-C15 (iso-branched pentadecanoic acid), iso-C16, and iso-C17. Those fatty acids account for 76% of the total membrane fatty acids. In addition, the results of the 16S-rDNA sequencing of isolate SPS-0 share 99.2% sequence similarity to Bacillus flavothermus. These results confirmed that the strain SPS-0 belonged to the genus Bacillus and was probably most closely related to Bacillus flavothermus. Effect of carbon source on enzyme production The induction experiments were studied by growing the organism in the presence of different carbon sources at a concentration of 2 g/l (Table 1).

Xylanases from a new Bacillus Table 1 Effect of carbon sources on extracellular xylanases production of strain SPS-0 determined after a 24 h cultures on medium containing yeast extract (1 g/l) and tryptones (1 g/l). Carbon sources [2.0 g.l21] None (control) Arabinose Cellobiose Cellulose Galactose Glucose Maltose Sorbitol Starch Wheat bran arabinoxylan Xylan (birchwood) Xylose

Xylanase activity (nkat/ml) nd 0.6 nd nd nd nd nd nd nd 12.6 3.2 3.5

nd: not detectable

When cells grew on cellobiose, cellulose, galactose, glucose, maltose, sorbitol or starch, no xylanase activity was detected, suggesting that the strain SPS-0 produce an induced endoxylanase. The levels of inducibility varied from arabinose (endoxylanase activity: 0.6 nkat/ml) to wheat bran arabinoxylan (endoxylanase activity: 12.6 nkat/ ml; specific activity: 30.2 nkat/mg). The optimum concentration of wheat bran arabinoxylan was in the range of 4 to 6 g/l. Under those conditions, the endoxylanase production was maximal and equal to 53.4 nkat/ml (specific activity: 101.2 nkat/mg), and bxylosidase activity was 3.2 nkat/ml (specific activity: 6.1 nkat/mg). Production of Bacillus SPS-0 xylanase was induced by xylose, xylan, wheat bran arabinoxylan and arabinose; wheat bran arabinoxylan was the most effective. The results found are close to those reported for many Bacillus strains where xylan showed a strong inducing effect (Dimitrov et al., 1997; Sunna et al., 1997), rather than that found for Bacillus sp. BP-7, where xylose was an important inducer of the xylanase (López et al., 1998). Bacillus SPS-0 did not produce xylanase in glucose supplemented cultures. Similar results have been reported for Bacillus sp. BP-7 and would suggest that expression of the xylanase of Bacillus SPS-0 is catabolite repressed. b-Xylosidase and a-Larabinofuranosidase activity was also detected in the culture supernatant (results not shown). Properties of the xylanases The optimal temperature for xylanase activity of isolate SPS0 was 75°C, when incubated for 10 min at pH 6.0. These values are in agreement with those values reported

Figure 1 Determination of temperature and pH optimum for xylanase activity. Culture filtrates samples were incubated at temperatures ranging from 35 to 85°C (pH 6.0) and at pH ranging from 4.0 to 11.0 (70°C) for 10 min. The release of reducing sugars was measured as xylose by the dinitrosalicylic acid method.

for xylanases produced by Bacillus thermoleovorans and Bacillus flavothermus (Sunna et al., 1997). The optimum pH for xylanase activity (at 70°C) was found to be about 6.0. The enzyme was active between pH 6.0 and pH 9.0. At pH 9.0, 80% activity remained, while at pH 4.0 only 20% of the activity was evident (Fig. 1). The thermal stability of the crude enzyme was measured, in the absence of substrate, at 60°C, 70°C, 85°C and 100°C. At 70°C, 85°C and 100°C, the xylanases from SPS0 showed half-life values of 90 min, 30 min and 10 min Biotechnology Letters ⋅ Vol 20 ⋅ No 11 ⋅ 1998

1069

M. Bataillon et al. with wheat bran, the main sugars were xylose and arabinose (Fig. 4a). Xylo-oligosaccharides of DP 2–4 were also detected in both experiences. Reduction of xylobiose when wheat bran was used as substrate, could be in part due to the presence of b-xylosidase in the substrate (Beldman et al., 1996). The hydrolysis product pattern found indicates that the xylanases contained in crude enzyme are endoxylanases. Aspergillus niger ferulic acid esterase (FAE-III) enhanced the hydrolysis of destarched wheat bran by xylanases from different sources (Bartolomé et al., 1995).

Figure 2 Effect of temperature on the stability of the xylanase of SPS-0. The enzyme was incubated in buffer at pH 6.0 at different temperatures (d : 60 °C; j : 70°C; r : 85°C; m : 100°C) and the residual activity was assayed with birchwood xylan at 70°C for 10 min.

respectively. At 60°C, the enzyme still retained 100% of its activity after 6 h incubation (Fig. 2). Hydrolysis of wheat bran by crude xylanases Hydrolysis experiments with birchwood xylan and wheat bran were carried out at 60 °C for 24h. The substrate solution (50 g/l) was incubated with 7.5 ml of the concentrated enzyme solution (1000 nkat/ml) to obtain 3000 nkat/g of substrate.

Fig. 4 shows HPLC profiles of the soluble samples obtained from 24 h incubation of destarched wheat bran with SPS-0 xylanases in absence and presence of FAE-III. In the absence of esterase (Fig. 4a), SPS-0 crude xylanase released mainly xylose (2.5 mg/ml) and arabinose (1.0 mg/ml). Production of free arabinose agrees with other authors reporting a purified arabinofuranosidase activity from Bacillus polymyxa (Morales et al., 1995), Bacillus stearothermophilus (Gilead and Shoham, 1995) and Bacillus subtilis (Weinstein and Albershein, 1979). In the presence of esterase (Fig. 4b), the profile of the products was qualitatively the same but the total amount of each saccharide (arabinose 1.2 mg/ml), in particular xylose was higher (4.3 mg/ml), showing that the presence of the esterase increased the initial rate of xylanase hydrolysis by 40% (calculated only as xylose detected). These results show that the production of xylose from destarched wheat bran by SPS-0 xylanase was more complete in the presence of FAE-III

Analysis of the hydrolytic products of the xylanasebirchwood xylan reaction (Fig. 3) showed that, after 24 h incubation time, xylose and xylobiose were the predominant end-products. When this xylanase was incubated

Figure 3 HPLC hydrolysis patterns of birchwood xylan obtained with crude xylanase SPS-0 (X: xylose; X2: xylobiose; X3: xylotriose; X4: xylotetraose).

1070


Figure 4 HPLC profiles destarched wheat bran obtained with crude xylanase SPS-0 in the absence (a: ) and presence (b: ) of a pure ferulic acid esterase (FAE-III) (A: arabinose; X: xylose; X2: xylobiose; X3: xylotriose; X4: xylotetraose).

Xylanases from a new Bacillus than that found with Trichoderma viride and Aspergillus niger xylanases (Bartolomé et al., 1995). Conclusion From preliminary study, it can be observed that a crude xylanase preparation from Bacillus strain SPS-0 hydrolyzed destarched wheat bran into monomeric C5 sugars xylose and arabinose. The ability of SPS-0 xylanases to hydrolyze that substrate increased by 40% in presence of a feruloyl esterase (FAE-III), showing a reciprocal cooperation between the enzymes. Supplementation of the growth media with wheat bran arabinoxylan increased both endoxylanase and b-xylosidase. The xylanolytic system produced by Bacillus SPS-0 had a high thermostability with 100% retained activity at 60°C after 6h. Acknowledgments This work was supported by a grant from the Europol’Agro of Reims, France. We thank Prof. M. Da Costa, University of Coimbra, Portugal, for his hospitality and assistance with respect to the screening of micro-organisms, and Prof. F. Rainey of DSMZ, Braunschweig, Germany, for 16S-rDNA sequence analysis. We thank Dr Craig B. Faulds for the gift of Aspergillus niger ferulic acid esterase (FAE-III) and also for the revision of this manuscript. References Bailey M.J., Biely P. and Poutanen K. (1992). J. Biotechnol. 23, 257–270. Bartolomé B., Faulds C.B., Tuohy M., Hazlewood G.P., Gilbert H.J. and Willianson G. (1995). Biotechnol. Appl. Bioc. 22, 65–73.

Bataillon M., Mathali P., Cardinali A.P. and Duchiron F. (1998). Ind. Crops Prod. 8, 37–43. Beldman G., Osuga D. and Whitaker J.R. (1996). J. Cereal Sci. 23, 169–180. Bradford M.M. (1976). Anal. Biochem. 72, 248–254. Dimitrov P.L., Kambourova M.S. Mandeva R.D. and Emanuilova E.I. (1997). FEMS Microbiol. Lett., 157, 27–30. Faulds C.B. and Williamson G. (1994). Microbiology. 140, 779–787. Gilead S. and Shoham Y. (1995). Appl. Environ. Microb. 61, 170–174. Herr D., Baumer F. and Dellweg H. (1978). Appl. Microbiol. Biot. 5, 29–36. López C., Blanco A. and Pastor J.F.I. (1998). Biotechnol. Lett., 20, 243–246. Miller G. L. (1959). Anal. Chem., 31, 426–428. Morales P., Madarro A., Flors A., Sendra J.M. and Pérez-González J.A. (1995). Enzyme Microb. Tech. 17, 424–429. Naudin O. and De Baynast R. (1990). Procédé de préparation de son de blé désamylacé et produit obtenu. European Patent 90 401 461.0. Subramaniyan S., Prema P. and Ramakrishna V. (1997). J. Basic Microb. 37, 431–437. Sunna A., Prowe S.G., Stoffregen T. and Antranikian G. (1997). FEMS Microbiol. Lett. 148, 209–216. Voragen A.G.J., Bergmans M.E.F, Oosterveld A., Schols H.A. and Beldman G. (1997). Carbohydr. Org. Raw Mater. III, Workshop. Van Bekkum H., Roeper H. and Voragen A.G.J. eds., p 1–16. Weinstein L. and Albershein P. (1979). Plant. Physiol. 63, 425–432. Williams R.A.D. and Da Costa M.S. (1992). The genus Thermus and related microorganisms. In: The Prokaryotes, New-York: Springer-Verlag, p. 3745–3753.

Received: 13 August Revisions requested: 10 September Revisions received: 7 October Accepted: 7 October


1998 1998 1998 1998

1071