Effect of glycosylation on the stability of fungal xylanase exposed to proteases or rumen fluid in vitro

Abstract

The effects of glycosylation on the stability of an exogenous xylanase exposed to proteases or rumen fluid in vitro, were examined. Commercial xylanase produced by Trichoderma longibrachiatum was purified by gel filtration chromatography, followed by ammonium sulfate precipitation and dialysis. A partially purified xylanase, consisting of proteins with molecular weights of 20 and 10 kDa was identified. Glycoproteins present in this purified xylanase complex were identified with the fluorescein isothiocyanate-labeled lectin method (FITC) prior to being enzymatically deglycosylated with the endo- N-glycosidase, PNGase F. Staining with FITC-labeled concanavalin A confirmed the presence of nano-gram quantities of glycoproteins. The naturally glycosylated- or deglycosylated xylanases were incubated with a 1:3 rumen fluid: McDougall's buffer solution (Rf), Prevotella ruminicola culture supernatant (Pr), or a commercial protease from Bacillus subtilis (Bs) for 0, 3, 6, 9, and 24 h at 39 °C. The protease activities of Rf dilution, Pr, and Bs were 0.018, 0.046, and 1.009 mg azocasein degraded/(ml h), respectively. Results indicated that, in Rf, the PNGase F-deglycosylated xylanase activity was lower ( P < 0.05) than the xylanase activity of the native enzyme after incubation for 3 and 6 h, but did not differ after incubation for 9 and 24 h. In Pr, however, xylanase activity did not differ after incubation for 3 and 6 h, but was lower ( P < 0.05) at 9 and 24 h of incubation for the PNGase F-deglycosylated enzyme. Therefore, the glycosylation effect was observed earlier during the incubation period when the enzymes were incubated with the numerous proteases from Rf, in comparison with Pr, where the proteases were produced only by P. ruminicola. In Bs, where the effect of glycosylation was tested under severe proteolytic conditions, xylanase activity was not different for native and PNGase F-deglycosylated enzymes and both enzymes lost 60% of the original activity within the first 3 h of incubation, whereas only 20% of losses were observed for the same period in Rf and Pr. These results indicate that glycosylation enhances xylanase stability and therefore is an important contributing characteristic towards the stability of exogenous enzyme supplements for ruminants.