Extraction of Lipase from Burkholderia cepacia by PEG/Phosphate ATPS and Its Biochemical Characterization

摘要

This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS) and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred atpH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gum-arabic in 0.1M sodium phosphate buffer at pH 8.0 and 37 deg C were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG 1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108- and 209-folds. Lipase presented optimal activity conditions at 37°C and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37°C. Metal ions such as Mn~(2+), Co~(2+), T and Ca~(2+) sustained enzymatic activities; however, it was inhibited by the presence of Fe~(2+), Hg~(2+) and Al3+. K_m and V_(max) values were 0.258 Ulmg and 43.90 gIL, respectively. A molecular weight of 33 kDa and an isoelectric point at pH 5.0 were determined by SDS-PAGE and IFS electrophoresis, respectively.