Debaryomyces hansenii UFV-1 Intracellular α-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Abstract

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.

Purification and Characterization of Aspergillus terreus α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides

“…α-Galactosidases from T. reesei [51], and B. stearothermophilus NCIM-5146 [50], were competitively inhibited by galactose. On the other hand, extracellular and intracellular α-galactosidases from D. hansenii UFV-1 were noncompetitively and competitively inhibited by galactose, respectively [9,27]. The potential A. terreus α-galactosidases to hydrolyze the oligosaccharides present in soybean aqueous extract (soy milk) was evaluated and the results are shown in Table 5.…”

“…Fungal α-galactosidases are the most suitable for technological applications mainly due to their acidic optima pH and broad stability profiles [7][8][9]. The most important industrial application of α-galactosidases is presently in the sugar-making industry [10].…”

Spectroscopic and thermodynamic properties of Debaryomyces hansenii UFV-1 α-galactosidases

“…The t 1/2 value at 65 • C for the intracellular enzyme was 38 min, and this enzyme lost completely its activity at 70 • C [16]. These observations indicate that although the secondary structure of the intracellular ␣-galactosidase has been resistant at elevated temperatures (thermodynamic process), probably the loss of enzymatic activity was due to small kinetically controlled tertiary structure alterations.…”

“…Purified extracellular and intracellular ␣-galactosidases (0.2 mg/mL) from D. hansenii UFV-1 [11,16], in 5 mmol/L sodium acetate buffer, pH 5.5, were analyzed through far-UV CD spectra in the 190-260 nm range, at different temperatures (20-80 • C). CD measurements were also obtained for the extracellular enzyme (0.2 mg/mL) in 5 mmol/L Mclvaine buffer (citric acid/sodium phosphate) [17], at different pH values (3-7) and temperatures (30-80 • C).…”

“…This enzyme is very important in the processing of beet sugar, where it is used to remove raffinose which inhibits normal crystallization of sucrose [15]. The extracellular and intracellular D. hansenii UFV-1 ␣-galactosidases presented pI values of 5.15 and 4.15, and carbohydrate contents of 40 and 34%, respectively; they have the same N-terminal sequences and the extracellular enzyme presented higher kcat value, 7.16 s −1 compared to the value for the intracellular enzyme, 3.29 s −1 [16].…”