PREPARATION AND CHARACTERIZATION OF PSEUDOMONAS AERUGINOSA LIPASE IMMOBILIZED ON CALCIUM ALGINATE MICROSPHERES

Abstract

Lipases are promising industrial biocatalysts because they are stable and active enzymes obtained from numerous
sources. Lipase from Pseudomonas aeruginosa was immobilized on calcium alginate and characterized. Sodium
alginate was mixed with purified lipase and the enzyme/alginate mixture was pumped drop wise in 0.4M CaCl2. The
molecular mass of the lipase was found to be 32 kDa as determined by sodium dodecyl sulphate polyacrylamide gel
electrophoresis. The immobilized lipase had pH and temperature optima in the range of 7.5 to 8.0 and 45 to 55°C
respectively and was stable at pH (7.5 to 8.5) and the temperature range of 45 to 60°C as compared to the free lipase.
The KM and Vmax values were 0.13mmole higher than that of free lipase and 50.0μmole/minutes respectively as
compared to free lipase which had KM and Vmax values of 0,07mmole and 55.5μmole/minutes. The enzyme activity
was enhanced in the presence of Mg2+and Ca2 with the residual activities of 97.1% and 111.4% as compared to free
lipase that had residual activity of 97.2^ and 110%. Immobilization improved the residual activity from 61.9% and
26.1% in the presence of Fe2+ and Ni2+ in free lipase to 64.5% and 50.0%in immobilized lipase. Slight inactivation of
immobilized lipase occurred in the presence of Cu2 with the residual activity of 62.8% as compared to free lipase
which had 64.1%. In the presence of methanol and n-hexane, the enzyme had residual activities of 113.1% and
113.1%, respectively. The immobilized lipase was recycled up to 5 cycles and the result showed that calcium
alginate\immobilized lipase could be used as an operative immobilized enzyme system.