Isolation of a Pseudomonas lipase produced in pure hydrocarbon substrate and its application in the synthesis of isoamyl acetate using membrane-immobilised lipase

摘要

Pseudomonas pseudomallei 12Sm produced an extracellular lipase growth on n-hexadecane as the sole carbon source. Sephadex G-150 filtration and native PAGE analyses of the mmonium sulphate (60%, w/v) precipitated protein of the cell-free culture broth showed the molecular mass (M_r) of the lipase protein to be about 14 3 kDa. The lipase was stable at high alkaline pH and the optimum activity was at pH 10. Using a simple entrapment technique, the lipase was immobilised in a polyvinyl alcohol (PVA) membrane (PAM) and was applied in the synthesis of isoamyl acetate by transesterification reaction. The stability of the immobilised lipase in organic solvens ws much igher than the native lipse, particularly in the polarity (log P) range of 0.23-0.85. The relative actiivty of the PAM-immobilised lipase was sigificantly higher than the native or celite-immoiblised lipase in the temperature range of 35-50 deg C. The retention of activity of the PAM-immobilised lipase at room temperature for 3 months was 62 and 70% higher than the free and celite-immobiised lipase, respectively. The apparent Miochaelis-Menten constant (K_m) for immobilised and free lipase were 5.55 and 178muM, respectively. The catalytic efficiency of the lipase (K_cat) did not significantly altered upon immobilisation in the PAM. Water at a concentration of 0.2% in the reaction mixture increased the activity of the immogibilised upton 4.3-fold and this was sustained upton the fifty reaction cycle.