Three cx-galactosidase genes, agll, ag12 and ag13, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-a-D-gnlactopyranoside. The genes ugll, ag12 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the a-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial a-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-a-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(g1uco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-/?-mannanase of T reesei. AGLII and AGLIII showed synergy in galacto(g1uco)mannan hydrolysis with the endo-l,4-/?-mannanase of 7: reesei and a B-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the a-galactosidase previously purified from 7: reesei.

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The action of ~xylosidase from Trichoderma reesei against different substituted xylo-oligosaccharides was studied. The enzyme cleaved off all unsubstituted xylose units from the non-reducing end of 1,2-1inked uronic acid substituted xylooligosaccharides. Surprisingly, an L-arabinofuranosyl group linked ¢z-1,3 to the xylopyranosyl ring was found to protect the [$-l,4-xylosidic linkage before the substituted xylose unit from being cleaved by the ~-xylosidase. Most probably the 1,3-1inked substituent sterically hinders the hydrolysis. According to the results of the present work, []-xylosidase of T. reesei is not able to remove all unsubstituted xylose units from the non-reducing end of substituted xylo-oligosaccharides, as had been believed previously.l~ ,. y words: Trichoderma reesei [3-xylosidase; Hydrolysis; Sttbstituted xylo-oligosaccharide; High-performance aaion-exchange chromatography; NMR been cloned and sequenced [13]. 13-Xylosidases are key enzymes in the production of xylose from different xylooligosaccharides. [3-Xylosidase of T. reesei has also been reported to act slowly on polymeric xylan [13,14]. Substituents, such as an acetyl group attached to the terminal xylose, inhibit the action of this enzyme [15]. In addition to the effÉcient hydrolysis of linear xylo-oligomers, 13-xylosidases are also able to liberate xylose from the non-reducing end of substituted xylo-oligosaccharides. Therefore, I]-xylosidases, in combination with side group cleaving enzymes, can be used in the structural determination and controlled production of different substituted xylo-oligomers. In the present work, several substituted xylo-oligosaccharides were treated with 13-xylosidase from 71 reesei in order to investigate its action against different substituted oligosaccharides.