Purification, Characterization, and cDNA Cloning of a Novel α-Galactosidase from Mortievella vinacea

Abstract

A novel α-galactosidase, designated α-galactosidase II, was isolated from the culture filtrate of Mortievella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51–62 kDa, estimated by SDS–PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial α-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain α-galactosyl residue from 63-mono-α-d-galactopyranosyl-β-1,4-d-mannotetraose but also terminal α-galactosyl residue from 63-mono-α-d-galactopyranosyl-β-l,4-d-mannotriose. In addition, the enzyme acted on galactomannans effectively. α-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41,334 Da. The derived amino acid sequence of the enzyme showed 31–49% sequence similarity with those of α-galactosidases from other origins. © 1997, Taylor & Francis Group, LLC. All rights reserved.