Purification and properties of a termostable a-galactosidase from Lenzites elegans

SAMPIETRO, D. A.; QUIROGA, E.N.; SGARIGLIA, M.A.; SOBERÓN, J.R.; VATTUONE, M.A.

Tucumán

Congreso; VII Congreso Argentino de Microbiología General; 2011

Sociedad Argentina de Microbiología General

White rot fungi are important in th e forest ecosystems because of their unique capacity to degrade wood components (i .e ., cellulose ,hemicelluloses and lignin). The major constituents of hemicelluloses are the hetero- l ,4- b-D-xylans, hetero-l,4-b-D-mannans, galacto-glucomannans and glucomannans.The biotechnological importance in the hydro lysis of hemicelluloses for paper and feedstock industries has revived interest in the enzymology of hemicellulose degradation. Galacto-glucomannans enzymatic hydrolysis requires, among other enzymes, the action of a-galactosidases. These enzymes have other industri al uses like hydrolysis of raffinose in beet sugar molasses and the raffinose family of oligosaccharides in soybean milk. a-Galactosidases (a-D-galactoside galactohydrolase, EC 3:2.1.22) catalyze the hydrolysis of a-D-galactopyranosyl linkages from alkyl, aryl or glucosyl (mono or oligo) residues or groups. An a-galactosidase was isolated from a culture filtrate of Lenzites elegans grown on pectin as carbon source.It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and ion-exchange chromatography.The molecular mass of the native purified enzyme was 158 kDa by gel filtration .lt is a homodimer with subunits of 61 kDa.The optimal temperature for enzyme activity was 80°C This a-galactosidase showed a high thermostability, retaining 94% of its activity after preincubation at 60°C for 1 h.The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 at O°Cand from 3.9 to 6.5 at 50°Cl t was active against several a-galactosides like p-nitrophenyl-a-D-galactopy ranoside, a-D-melibiose, rafinose and stachyose.Thea-galactosidase isa glycoprotein with 26 % of structural sugars. Galactose is a non-competitive inhibitor with a Ki 0= 22 mM vs. p-nitrophenyl-a-D-galactoside and 12 mM vs. a-D-melibiose as substrates.Cations as Hg2 +, Ag 1+ and p-chloromercuribenzoate were also inhibitors of this activity,suggesting the presence of – SH groups in the active site of the enzyme. The sequence of th e N-terminal end of the agalactosidase from L. elegans was SPDTIVLDGTNFALNN. This sequence data was aligned and compared using Basic Local Al igment Researc h Tool (BLAS T) and CLUSTALW Programmes.No similarities were found in the protein sequence using protein-protein BLAST from the National Center for Biotechnology Information USA(http ://www.ncbi.nlm.n ih.gov/blast/Blast.cgi) .The alignments of sequences with enzymes belonging to GH 36 were carried out with CAZy database.Consequently, the studied a-galactosidase isolated from Lenzites elegans was classified as member of glycosyl hydrolase family 36(GH 36).Given the high optimum temperature and heat stability of L. elegans a-galactosidase, th is fungus may become an alternative source of a-galactosidase production for industrial use.