Debaryomyces hansenii UFV-1 intracellular alpha-galactosidase characterization and comparative studies with the extracellular enzyme.
Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.
Viana
Pollyanna A
PA
BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
de Rezende
Sebastião T
ST
Passos
Flávia Maria Lopes
FM
Oliveira
Jamil S
JS
Teixeira
Kádima N
KN
Santos
Alexandre M C
AM
Bemquerer
Marcelo P
MP
Rosa
José C
JC
Santoro
Marcelo M
MM
Guimarães
Valéria M
VM
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D055165:Debaryomyces / Q000201:enzymology*; D004795:Enzyme Stability; D042541:Intracellular Space / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D041942:Soy Milk / Q000737:chemistry; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000378:metabolism*
D002241:Carbohydrates; D009844:Oligosaccharides; D000519:alpha-Galactosidase