Identification,Cloning,and Characterization of a Sporobolomyces singularis beta-Galactosidase-like Enzyme Involved in Galacto-Oligosaccharide Production

摘要

Sporobolomyces singularis can be used as a biocatalyst in galacto-oligosaccharide production.We isolated 2-deoxy-D-glucose-resistant mutants of S.singularis ATCC 24193 and recovered a mutant that showed 10-fold higher beta-galactosidase-like activity than the parent strain and which was insensitive to catabolite repression.Thereafter,the beta-galactosidase-like enzyme was purified from the mutant and revealed to be a glycoprotein with both beta-glucosidase- and beta-galactosidase-like activity,the Michaelis-Menten constants of which for o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-glucopyranoside were 5.40 and 1.96 mM,respectively,and the maximum velocities were 3.07 and 2.30 mumol/min per mg of protein,respectively.Its molecular mass was estimated to be 73.9 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 146 kDa by gel filtration,suggesting that it has a homodimeric structure.We sequenced the N-terminus and internal peptides of this protein and isolated both a cDNA and a gene with degenerate primers.The gene,named bg1A,has 18 introns and 19 exons and encodes a polypeptide of 594 amino acids.The BglA protein was approximately 35% identical and 50% similar to plant beta-glucosi-dases belonging to family 1 glycosyl hydrolases,but with a unique 110-amino-acid sequence at the N-terminus.The beta-galactosidase-like enzyme (i.e.,BglA protein) in S.singularis is a beta-glucosidase with high transgalactosidase activity.