3 ALPHA-GALACTOSIDASE GENES OF TRICHODERMA-REESEI CLONED BY EXPRESSION IN YEAST

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated fr om a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on pla tes containing the substrate romo-4-chloro-3-indolyl-alpha-D-galactopy ranoside. The genes agl1, agl2 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 alpha-ga lactosidases of plant, animal, yeast and filamentous fungal origin cla ssified into family 27 of glycosyl hydrolases whereas the deduced amin o acid sequence of AGLII showed similarity with the bacterial alpha-ga lactosidases 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-al pha-D-galactopyranoside and the small galactose-containing oligosaccha rides, melibiose and raffinose. They liberated galactose from polymeri c galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo -1,4-beta-mannanase of T, reesei. AGLII and AGLIII showed synergy in g alacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated mol ecular mass and the hydrolytic properties of AGLI indicate that it cor responds to the alpha-galactosidase previously purified from T. reesei .