Document Type Master's Dissertation Author Van de Vyver, Wilhelmus Francois Joubert URN etd-08102005-164108 Document Title The effect of glycosylation on the stability of exogenous xylanase under in vitro proteolytic conditions similar to the rumen Degree MSc (Agric) Department Animal and Wildlife Sciences Supervisor
Advisor Name Title Prof N H Casey Committee Chair Keywords
- ruminants feeding and feeds
- enzymes in animal nutrition
- proteolytic enzymes
Date 2003-04-01 Availability unrestricted AbstractThe aim of this study was to evaluate the effect of glycosylation on exogenous xylanase stability when incubated under proteolytic conditions.
Xylanase produced by Trichoderma longibrachiatum, was purified using gel filtration chromatography, ammonium sulfate salt precipitation and dialysis. A partially purified xylanase with Mr of 20- and 10 kDa was identified and contained >65% of the original xylanase activity. Glycoproteins present in the xylanase were identified by thymol sulfuric acid staining or by the FITC-Iabeled lectin method, specific for glycoproteins. This naturally glycosylated xylanase was enzymatically deglycosylated with one of two endo-N-glycosidases: PNGase F or Endo H. Efficiency of deglycosylation was determined with electrophoresis by observing protein mobility shifts or by staining with FITC-Iabeled lectin. The effect of glycosylation on the stability of the exogenous xylanase was tested by incubating the glycosylated or deglycosylated xylanase with rumen fluid (Rf), Prevotella ruminicola culture supernatant (Pr) or a commercial protease from Bacillus subtilis (Bs) for 0, 3, 6, 9 and 24h at 37°C. Results indicated that glycosylated xylanase was significantly more stable (P<0.05) against proteolytic inactivation under the relatively low protease conditions of Rf and Pr (0.018 and 0.046 mg azocasein degraded/ml/h, respectively), but not under high proteolytic conditions of Bs (1.009 mg azocasein/mllh). Also, the glycosylation effect was observed earlier when incubated with the numerous proteases of Rf (3h), than with Pr (9h). These results indicate that glycosylation enhances xylanase stability and therefore is an important characteristic for exogenous enzyme supplements for ruminants.
© 2002 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.
Please cite as follows:
Van de Vyver, WFJ 2002, The effect of glycosylation on the stability of exogenous xylanase under in vitro proteolytic conditions similar to the rumen, MSc(Agric) dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08102005-164108/ >
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access dissertation.pdf 939.99 Kb 00:04:21 00:02:14 00:01:57 00:00:58 00:00:05