Multiple Xylanases from the Extreme Thermophile Caldicellulosiruptor Saccharolyticus

by Te’o, Valentino Setoa

Abstract (Summary)
Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. Over the last decade a considerable amount of research has focused on developing ways to reduce adsorbable organic chlorides (AOX) produced during bleaching of the kraft pulp as these by-products have serious detrimental impacts on the environment. The use of hemicellulases (particularly xylanases) within bleaching sequences has been shown to be a feasible way of enhancing the extraction of lignin during paper manufacturing, and so can be used as an alternative to chemical agents. Due to the extreme conditions (high temperature and alkalinity) during pulp production in paper manufacture, enzymes must be alkaline-tolerant, but more importantly, be thermostable, otherwise extensive plant and energy costs are incurred by the industry. This thesis describes the isolation, characterization and expression of three xylanase genes (xynE, xynF and xynI) from the extremely thermophilic microorganism Caldicellulosiruptor saccharolyticus. The three genes (xynE, xynF and xynI) code for enzymes which may be especially suited for use as bleaching agents. Also described is the correction of a frame shift previously identified by others in order to produce a functional xylanase gene. The DNAs of the xylanase genes xynE and xynF were sequenced and both were found to be part of the xylanase gene cluster located on an 18kb BamHI C. saccharolyticus fragment. Translation and homology comparisons of xynE and xynF showed both to encode enzymes which were modular in composition. Also, based on homology comparisons, XynE was expected to have endoxylanase activity while XynF was to have arabinosidase with possibly xylanase and xylosidase activities. PCR using consensus primers was used successfully to isolate a new xylanase gene from C. saccharolyticus. This new xylanase gene designated xynl was found to be located elsewhere from the xylanase gene cluster on the 18kb BamHI fragment. The DNA of xynl was sequenced and its deduced amino acid sequence was found to be highly homologous to XynA from Caldicellulosiruptor sp. Strain Rt8B.4. The sequence showed that XynI belongs to the family F/10 xylanase. Therefore, like XynE, XynI was expected to have endoxylanase activity. In addition to isolating and expressing functional xylanase genes, the sequence of the frame shift previously identified on the orf3/4 open reading frame was analysed. Correction of the frame shift was carried out using primer extension PCR in an attempt to produce a functional xylanase gene. However, despite the successful correction of the frame shift, no active xylanase enzyme was detected. It is likely that the pseudogene was defective in ways other than the obvious frame shift mutation. All three xylanases XynE, XynF and XynI (domain 2) were overexpressed and characterized in E. coli. The temperature optima of XynE and XynF were found to be 75°C while that of XynI (domain 2) was 65°C. Also, the pH-dependent activities of the three enzymes were tested and XynE and XynF were each found to have a pH optimum of 6 while that of XynI was 6.5. A11 three enzymes had characteristic shoulders over the alkaline region. Both XynE and XynF were found to have 50% residual activity at pH 9, while XynI had 50% activity at pH 10. Activity studies of the three enzymes on xylan-based substrates indicated that XynE was an exo-xylanase. XynF was found to have arabinosidase and exo-xylanase activities. XynI was a true endoxylanase as indicated by hydrolysis products. A11 three enzymes were tested and found to release reducing sugars from the fibre bound substrate, kraft pulp. However, under laboratory conditions, only XynF released lignin from kraft pulp. These results indicated that these enzymes have characteristics suitable for potential bleaching agents in the enzyme-aided bleaching of wood pulp.
Bibliographical Information:


School:The University of Auckland / Te Whare Wananga o Tamaki Makaurau

School Location:New Zealand

Source Type:Master's Thesis



Date of Publication:01/01/1996

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