Multiple endoxylanases ofButyrivibrio sp

Folia Microbiol. 46 (I), 94-96 (2001)

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Multiple Endoxylanases of Butyrivibrio sp. M. ZOREC, T. CEPELJNIK, F . V . NEKREP, R. MARIN~EK LOGAR

Zootechnical Department, Biotechnical Faculty, University of Ljubljana, 1230 Dome_ale, Slovenia Received 20 December 2000 ABSTRACT. Butyrivibrio sp. Mz 5 with a high xylanolytic activity was isolated Four major xylanases were detected in the cell-associated fraction using the zymogram technique. The xylanolytic activity was inducible with the oat spelts xylan; two endoxylanases (51 and 145 kDa) were formed constitutively. The bulk of the xylanolytic activity was cell-bound and growthphase dependent; the maximum activity in the cell-associated

fraction was achieved after 16 h of incubation. The highest xylanolytic activity was determined in a medium with 0.5 % oat spelts xylan. Under optimum conditions (the highest xylanolytic activity produced), the two cell-bound xylanases (51 and 58 kDa) were isolated by anion exchange chromatography on CIM DEAE 8 tubes attached to a MPLC system, and gel filtration.

Following cellulose, hemicellulose is the most abundant polysaccharide in forages; xylan is the predominant hemicellulose. Because of its structural heterogeneity, bacteria and fungi evolved xylanolytic systems consisting of a number of different enzymes with different specificities. For some endoxylanases, their action is often prevented by the presence of various substituents on the xylan backbone. On the other hand, these substituents promote the binding of other xylanases and thus enhance their action. Five major and six minor xylanases were detected in the principal rumen bacterial xylanolytic species Butyrivibriofibrisolvens H17c by zymogram techniques (Lin and Thompson 1991). Electrophoretically distinct xylanases could be formed by post-translational modifications (glycosylation or/and proteolysis) and/or could be products of different genes or different alleles of the same gene (Wong et al. 1988). In addition, many cellulolytic enzymes have xylanolytic activity as a secondary function. The genus Butyrivibrio is composed of obligately anaerobic, curved rod-shaped bacteria that ferment saccharides under the production of large amounts of butyric acid. All ruminal isolates have been routinely classified as B. fibrisolvens, although they actually comprise a number of distinct species or even genera (Mannarelli t988). Strains of B. fibrisolvens are almost uniformly highly xylanolytic (Hespetl et al. 1987) and are the most important ruminal bacteria involved in the digestion of xylans. The knowledge of native xylan-degrading systems in butyrivibria strains would help in the selection of target strains and genes for the most effective construction of recombinant xylanolytic butyrivibria to improve the fiber degradation in rumen. Butyrivibrio sp. Mz 5 was previously isolated from the rumen of a black-and-white Friesian cow and its xylanolytic activity was shown to be 1.65 times higher than the activities of all known xylandegrading rumen bacterial species (Marin~ek Logar et al. 2000). After determining optimum conditions for the expression of the xylanolytic activity, we partially purified the enzymes for further characterization and possible biotechnological applications.

MATERIALS AND M E T H O D S

Growth conditions. Butyrivibrio sp. Mz 5 was grown in a medium without tureen fluid (Deutsche Sammlung yon Mikroorganismen, DSM; medium no. 330) or modified medium containing 0.5 % oat spelts xylan as the sole saccharide source (330-xylan); concentrations of 0, 0.1, 0.3, 0.5, 0.7, 1, 2 and 5 % were used for testing the influence of substrate concentration on xylanase activity expression. Cultures were incubated at 37 ~ in CO2 (100 %) atmosphere in Hungate tubes. Enzyme assays. Cells were harvested after l-d growth by centrifugation at 5 Hz. The supernatant was collected and frozen, pellets were washed in 50 mmol/L sodium phosphate buffer (pH 6.5) and frozen in distilled water. Endoxylanase and carboxymethyl cellulase (CMCase) activities were determined spectrophotometrically using the reducing-sugar method (Lever 1977) and protein was determined according to Lowry. Activities are expressed in gmol of reducing sugar per s per g of protein (gkat/g). Influence of growth phase on xylanolytic activity. Hungate tubes containing 330-xylan medium were inoculated with a well-grown culture of Mz 5. Supernatants and ceils were collected after 0, 4, 8, 12, 16, 18, 20, 24, 48 and 66 h of incubation. SDS-PAGE xylanograms. Proteins were separated by SDS-PAGE (Laemmli 1970). The separating gels contained 0.2 % oat spelts xylan or CMC. The cells and supernatant were denaturated at 80 ~ for

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10 min in loading buffer. After electrophoresis, proteins were stained with Coomassie Brilliant Blue R-250 according to the rapid Fairbanks method (Wong et al. 2000). For enzyme detection, the gels were renaturated, incubated, and stained with Congo red. After destaining, clearing zones were detected and molar mass determined according to the calibration curve with protein markers. Partial purification of cell-bound xylanases. The cell extract was prepared from 1.6 L of cell culture by osmotic shock (double freezing and additional sonication) and subsequent centrifugation. The supernatant was used for isolation of endoxylanases by anion exchange chromatography on CIM | DEAE 8 mL monolithic column (BIA) (Strancar et al. 1997); 13 mL of nonconcentrated sample was applied. Proteins were separated using a linear gradient of elution buffer (1 mol/L NaCI in 20 mmol/L Tris-HCl; pH = 7.2), 0-100 % in 25 min on Gradifrac MPLC system (Pharmacia-LKB); flow rate was 8 mL per min. Xylanolyticaly active fractions were pooled and concentrated by ultrafiltration. The proteins were further separated using gel filtration on Sephacryl HR-200 column (eluted with the same buffer) and the flow rate of 1 mL/min.

RESULTS AND DISCUSSION The xylanolytic activity was mostly cell-associated (33.8 lakat/g) and only a third of this value being determined in culture supernatant (10.5 p.kat/g). The majority of bacterial xylanases was extracellular or bound to the surface of the cell (the substrate might be too large to enter the cell; with the Mz 5 strain, the high cell-associated xylanase activity could be explained by the production of large amounts of extracellular polysaccharides which then hinder the diffusion of extracellular enzymes into the growth medium; cf. Ha et al. 1991). The surface colonies were mucoid with emanating gas bubbles. A similar morphology was also found in the highest extracellular polysaccharide-producing strains (e.g., CF strains of B. fibrisolvens; Ha et al. 1991). The SDS-PAGE xylanograms revealed 14 cell-associated xylanases with molar mass ranging from 26.7 to 145 kDa. Only the 99.8- and 77.4-kDa cell-associated endoxylanases showed weak CMCase activity (0.43 ~tkat/g) on CMCgrams. All the other enzymes were therefore considered as true xylanases. The first xylanolytically active bands in the cell-associated fraction were detected after 12 h of incubation (51- and 99.8-kDa endoxylanase). After 14 h all 4 major xylanases (51, 77.4, 99.8 and 145 kDa) were detected and were active untill the late stationary phase. The cell-associated activity reached its maximum after 16 h (59.8 ktkat/g) when the activity in the supernatant was still very low (4.2 ~tkat/g) and undetectable on the xylanogram. The maximum activity in the supernatant fraction was reached after 48 h (18.1 ~tkat/g) when cell-bound activity already slowly decreased (14.2 ~tkat/g; increasing activity in the supernatant could be possibly due to cell lysis followed by the release of xylanases into the medium). The decreased pH and efficient proteolysis affected the overall xylanolytic activity of the culture at the same time. In contrast, the 26.7-kDa xylanase appeared after 24 h in both fractions and was more active in the later growth phases (48-66 h). It seems to be more pH- and proteinase-resistant and/or it has a lower pH optimum. The best expression of xylanolytic enzyme activity in a 24-h-old culture was achieved at the concentration of 0.5 % oat spelts xylan in the growth medium (cell-associated activity 41.0 ~tkat/g, activity in the supernatant 12.5 ~tkat/g). Concentrations of xylan over 1% repressed xylanolytic enzymes with molar mass >99.8 kDa and also repressed the overall specific xylanolytic activity. At higher xylan concentration (2 and 5 %) only 4 major xylanases were detected (efficient hydrolysis need not proceed). Lower xylan concentration (0.1~).3 %) promote the release of xylanases into the growth medium perhaps by hindering extracellular polysaccharide synthesis (cf. Ha et al. 1991). The majority of the endoxylanases were inducible by oat spelts xylan. The cell-associated activity was at least 130-times lower than the activity of the xylan-grown cultures. Only the 51- and 145-kDa endoxylanases were detected as weak bands when the cells were grown without xylan (gLucose, cellobiose, maltose and soluble starch as saccharide sources; see Materials and Methods) and were considered to be constitutive. The xylanase formation in B. fibrisolvens strain NCFB 2249 was also induced by different xylans and xylooligosaccharides (Williams and Withers 1992a) and was repressed by glucose and other hemicellulosic monosaccharides (Williams and Withers 1992b). Cultures grown on a medium with 0.5 % oat spelts xylan were used for enzyme isolation. The cells were harvested after 16 h when the highest cell-associated xylanolytic activity was determined. In the first step of the purification procedure, two peaks of the enzyme activity were selected for further separation of xylanolytically active proteins. On xylanograms, the 51-, 58- and 77.4-kDa xylanases were found in the first peak and 51-, 99.8- and 145-kDa xylanases in the second. The 51- and 58-kDa xylanases showed to be the

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most resistant to all purification conditions and were finally separated and partially isolated by gel filtration in quantities sufficient for further characterization and study o f practical application. REFERENCES

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