Partitioning and resolution of mixture of two lipases from Bacillus stearothermophilus SB-1 in aqueous two-phase system

The partitioning and resolution of two lipases (acidic and neutral) was ach ieved in an aqueous two-phase system from crude culture supernatant of Baci llus stearothermophilus SB-1. The effects of changes in PEG molecular weigh t, varying PEG/phosphate system, pH and sodium chloride concentration were examined on the partition coefficient, K in PEG/phosphate system. The best system was PEG 6000 (70% w/v)/phosphate (40% w/v) and NaCl (3% w/v) at pH 7 .0 where the total lipase partitioned to the top PEG phase. The two lipases were then selectively eluted by addition of fresh salt phase in a pH depen dent manner with acidic lipase moving to the bottom phase at pH 4.0 with 5. 27-fold purification while neutral lipase shifted to the bottom phase at pH 6.0 with 15.25-fold purity. The two lipases were easily recovered from the salt phase by immobilisation on accurel. Thus the mixture of two lipases w as resolved and purified into acidic and neutral within 2 h with complete r ecycling of both the phase components. It has also been found that addition of PEG 6000 leads to an increase in the enzyme activity of the lipase mixt ure. (C) 1999 Elsevier Science Ltd. All rights reserved.