Purification and characterization of ß-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis.
Bioorg Chem
; 77: 176-189, 2018 04.
Article
en En
| MEDLINE
| ID: mdl-29421696
ABSTRACT
ß-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of ß-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07â¯kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58â¯kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8-7.0 with more than 97% activity. Purified ß-galactosidase was optimally active at 50⯰C. Kinetic parameters Km and Vmax for purified enzyme were 400⯵M and 1.22â¯×â¯10-1 U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca2+, Mg2+ and Mn2+ slightly activated the enzyme whereas NH4+, Co2+ and Fe3+ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that ß-galactosidase is less stable at higher temperature (60⯰C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047â¯min-1 and t1/2 of 14.74â¯min. This is better than other studied ß-galactosidases. Both sonicated Pediococcus acidilactici cells and purified ß-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5⯰C. These findings indicate the use of ß-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca2+ shows that it is suitable for milk processing.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Oligosacáridos
/
Beta-Galactosidasa
/
Leche
/
Pediococcus acidilactici
/
Galactosa
/
Lactosa
Límite:
Animals
Idioma:
En
Revista:
Bioorg Chem
Año:
2018
Tipo del documento:
Article