Characterization of β-galactosidase from Lactobacillus acidophilus: Stability and kinetic study

Abstract

β-Galactosidase is the industrially important enzyme that catalyzes both, lactose hydrolysis and synthesis of different bioactive galactosides. In this study, optimal conditions (fermentation temperature, inoculum concentration and lactose concentration) for accomplishing high yields of β-galactosidase activity from Lactobacillus acidophilus ATCC 4356, bacteria regarded as safe for human consumption, were investigated. Using the response surface methodology (RSM), it was concluded that the highest activity and specific activity were obtained by 2-day shake-flask culture fermentation at 28°C, provided that the lactose content in the fermentation medium was 1.48%, and the inoculum concentration was 2.8%. The optimum temperature and pH for the obtained enzyme were 45°C and 6.5, respectively. More importantly, these conditions simultaneously ensure a high enzyme stability. The Km and Vmax values were 0.44 mM and 25.64 mM/h (for o-nitrophenyl-β-D-galactopyranoside), and 3.79 mM and 3.10 mM/h (for lactose), and the substrate excess inhibition was not observed. On the other hand, the enzyme was inactivated in the presence of Ca2+, Ba2+, and Cu2+.

β-Galaktozidaza je značajan industrijski enzim koji se tradicionalno koristi za katalizovanje reakcije hidroloze laktoze, ali poslednjih godina nalazi i sve značajniju primenu u reakcijama sinteze različitih bioaktivnih galaktozida. U ovoj studiji, ispitani su optimalni uslovi za postizanje visokih prinosa β-galaktozidaze iz Lactobacillus acidophilus ATCC 4356, bakterije mlečne kiseline koja se smatra bezbednom za korišćenje u prehrambenim proizvodima. Statističkim planiranjem eksperimenata i metodom odzivnih površina, zaključeno je da se najveća aktivnost i specifična aktivnost dobijaju nakon dvodnevne fermentacije na temperaturi od 28°S, kada je koncentracija laktoze 1,48%, a koncentracija inokuluma 2,80%. Temperaturni i rN optimum dobijenog enzima iznose 45°S i 6,8, redom, pri čemu ovi uslovi istovremeno omogućuju i izuzetnu stabilnost enzima. U kinetičkoj studiji određene su vrednosti kinetičkih parametara, Km i Vmax: 0,44 mM i 25,64 mM/h (za o-nitrofenil- β-D-galaktopiranozid), odnosno 3,79 mM i 3,10 mM/h (za laktozu). Takođe, nije primećena inhibicija supstratom u višku, dok se enzim inaktivira u prisustvu jona Ca2+, Ba2+, i Cu2+. PR Projekat Ministarstva nauke Republike Srbije, br. III 46010 .