The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.
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Production and Characterization of Trichoderma Reesei and Thermomyces Lanuginosus Xylanases

Hairong Xiong

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology for public examination and debate in Auditorium KE 2 (Komppa Auditorium) at Helsinki University of Technology (Espoo, Finland) on the 30th of October, 2004, at 12 o'clock noon.

Overview in PDF format (ISBN 951-22-7318-7)   [617 KB]
Dissertation is also available in print (ISBN 951-22-7317-9)

Abstract

This thesis describes the production and properties of xylanases from Trichoderma reesei Rut C-30 and Thermomyces lanuginosus DSM 10635. The thermostability of several T. reesei xylanase II mutants was also studied. T. reesei Rut C-30 responds to the pH of the growth environment by modifying its enzyme production patterns. The production of the xylanases I, II and III by T. reesei at different pH conditions correlates with the pH-dependent activity pattern of the enzymes. The xylanase was preferentially produced when it is most active in that particular pH environment. The highest total xylanase production with T. reesei Rut C-30 was achieved at pH 6 on a lactose-based medium. Among the pentoses and hexoses tested, L-arabinose was the most effective inducer of the xylanases. Furthermore, in co-metabolism of L-arabinose and D-glucose, the addition of the former relieved the repression of D-glucose on xylanase production. The replacement of part lactose with L-arabinose resulted in significant improvement in xylanase production. Small amounts of bulky L-arabinose-rich plant materials, such as sugar beet pulp and oat husk hydrolysates, stimulated the xylanase production. While xylanase production was improved by these approaches, the cellulase production was not enhanced. In addition, the stability and activity of T. reesei xylanase II mutants containing different combinations of disulphide bridges were studied. The most stable combination mutant showed about 5000-fold half-life at 65 °C compared to the wild type xylanase II. The molecular system controlling xylanase induction was fundamentally different in T. reesei and T. lanuginosus. Unlike T. reesei Rut C-30, T. lanuginosus DSM 10635 xylanase was not induced by L-arabinose or lactose. The amino acid sequence of DSM 10635 xylanase was most likely the same as that of T. lanuginosus DSM 5826 xylanase. The temperature-dependent inactivation curve of the DSM 10635 xylanase decreased slowly at neutral or slightly alkaline pH, whereas at low pH, the inactivation was fast. The thermostabilizing effect of the substrate, birchwood xylan, on DSM 10635 xylanase was observed to be significant only under acidic conditions.

This thesis consists of an overview and of the following 5 publications:

  1. Xiong H., von Weymarn N., Leisola M. and Turunen O., 2004. Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30. Process Biochemistry 39, number 6, pages 729-733.
  2. Xiong H., Turunen O., Pastinen O., Leisola M. and von Weymarn N., 2004. Improved xylanase production by Trichoderma reesei grown on L-arabinose and lactose or D-glucose mixtures. Applied Microbiology and Biotechnology 64, number 3, pages 353-358.
  3. Xiong H., von Weymarn N., Turunen O., Leisola M. and Pastinen O., Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates. Bioresource Technology, in press.
  4. Xiong H., Fenel F., Leisola M. and Turunen O., 2004. Engineering the thermostability of Trichoderma reesei endo-1,4-β-xylanase II by combination of disulphide bridges. Extremophiles 8, number 5, pages 393-400.
  5. Xiong H., Nyyssölä A., Jänis J., Pastinen O., von Weymarn N., Leisola M. and Turunen O., 2004. Characterization of the xylanase produced by submerged cultivation of Thermomyces lanuginosus DSM 10635. Enzyme and Microbial Technology 35, number 1, pages 93-99.

Keywords: xylanase, cellulase, Trichoderma reesei, Thermomyces lanuginosus, pH, L-arabinose, oligosaccharides, characterization, cultivation, induction, purification, thermostability

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© 2004 Helsinki University of Technology

Last update 2011-05-26