The filamentous fungus Trichoderma reesei is one of the most potent microorganisms degrading cellulosic and hemicellulosic materials. In order to completely degrade substituted hemicelluloses, the fungus produces enzymes hydrolysing the backbone of hemicelluloses and enzymes removing substituents. The aim of the present investigation was to isolate and characterize genes of T. reesei encoding side-chain releasing enzymes. The gene coding for alfa-glucuronidase (glr1), which releases glucuronic acid attached to xylose units of xylan, was isolated from an expression library of T. reesei RutC-30. The glr1 gene is the first alfa-glucuronidase gene hitherto cloned and characterised and the deduced amino acid sequence shows no similarity with any protein sequence available in the data bases. The axe1 gene encoding acetyl xylan esterase, which removes acetic acid bound to xylan was also isolated from the expression library. The catalytic domain of AXEI has an active site serine and exibits amino acid similarity with fungal cutinases, which are serine esterases. AXEI has a modular structure carrying a cellulose binding domain of fungal type at its C-terminus, separated from the catalytic domain by a linker region. Two genes, abf1 and bxl1, encoding alfa- arabinofuranosidase activity and three genes, agl1, agl2 and agl3, encoding alfa-galactosidase activity were cloned from a cDNA expression library of T. reesei RutC-30 constructed in the yeast S. cerevisiae. ABFI and BXLI correspond to a previously purified alfa-L-arabinofuranosidase and a alfa-xylosidase from T. reesei, respectively. The deduced amino acid sequence of ABFI displays high similarity with one of the alfa-L-arabinofuranosidases, ABF B, of Aspergillus niger. The deduced amino acid sequence of BXLI shows no similarity with any of the known beta-xylosidases, but is significantly similar to the beta-glucosidases grouped into the family 3 of glycosyl hydrolases. The deduced amino acid sequences of AGLI and AGLIII show similarity with the alfa-galactosidases of plant, animal, yeast and filamentous fungal origin. On the other hand, the deduced amino acid sequence of AGLII shows similarity with the bacterial alfa-galactosidases of family 36 of glycosyl hydrolases, and is thus the first reported eukaryotic alfa-galactosidase to show similarity with the corresponding prokaryotic enzymes. ABFI, BXLI, AGLI, AGLII and AGLIII were produced in yeast in order to test their action against different small synthetic and natural polymeric substrates. Both ABFI and BXLI showed alfa-L-arabinofuranosidase and beta- xylosidase activities against p-nitrophenyl-alfa-L-arabinofuranoside and p-nitrophenyl-beta-D-xylopyranoside, respectively. However, ABFI only released L-arabinose from arabinoxylans. BXLI hydrolyzed xylobiose and slowly released xylose from polymeric xylan, but did not hydrolyse L-arabinose from arabinoxylan. Both ABFI and BXLI produced in yeast displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei. AGLI, AGLII and AGLIII were able to hydrolyze the synthetic substrate p-nitrophenyl-alfa-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They showed different efficiencies when acting on polymeric galacto(gluco)mannan. AGLI was the most active enzyme towards polymeric substrates and its action was enhanced by the presence of the endomannanase of T. reesei. The calculated molecular mass and the hydrolytic properties of AGLI indicated that it might correspond to the alfa-galactosidase previously purified from T. reesei. AGLII and AGLIII were less active on the intact polymer and showed synergy in galacto(gluco)mannan hydrolysis with the mannanase of T. reesei and a beta-mannosidase of Aspergillus niger.
|Award date||14 Jun 1996|
|Place of Publication||Espoo|
|Publication status||Published - 1996|
|MoE publication type||G5 Doctoral dissertation (article)|
- genetic engineering
Margolles-Clark, E. (1996). Molecular characterization of side-chain cleaving hemicellulases of Trichoderma reesei: Dissertation. VTT Technical Research Centre of Finland. http://www.vtt.fi/inf/pdf/publications/1996/p276.pdf