Characterization of lipase from pseudomonas putida 3SK and in situ lipid hydrolysis using the whole cell in twol phase system

Abstract

Extracellular lipase was produced during the late logarithmic growth phase by solvent resistant strain, Pseudomonas putida 3SK. The enzyme was purified to 21 fold with 5.3\% recovery by ion exchange chromatography and gel filtration chromatography. Molecular weight of the purified enzyme was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lipase had an optimum pH range of 8.0 - 9.0 and was stable within the pH range of 4.0 to 10.0. The optimal temperature was $37\,^\circ\!C$ and it was stable up to $75\,^\circ\!C$. When the effects of organic solvents were tested, isooctane was the most suitable for enzymatic fat hydrolysis in the two phase system. Enzyme activity was inhibited by mercury ions whereas its activity was stimulated by calcium ion and taurocholic acid. Also, batch and continuous hydrolysis of olive oil in organicaqueous two phase system have been investigated using whole cell of Pseudomonas putida 3SK as a source of lipase. In two phase system, the strain was fully viable and grown well, and also produced extracellular lipase simultaneously. The optimum conditions for the hydrolysis of triglyceride in two phase system were determined. Isooctane was selected as the most suitable organic solvent in this system. The volumetric ratio of aqueous phase to isooctane was maximum at the ratio of 5:2(v/v). Degree of hydrolysis depending on olive oil concentration in the solvent was maximal at 13.5\% (w/v) and then decreased with the increase of the substrate concentration. At the optimized condition, more than 95\% of degree of hydrolysis was achieved within 24 h at $30\,^\circ\!C$ when the reaction was carried out in a two phase batch stirred reactor. For a long term operation, a continuous stirred reactor was designed. When the reaction was carried out in the continuous stirred reactor, degree of hydrolysis was reached to 91 \% at the dilution rate of 0.2 h$^{-1}$. Satisfactory performance of the two phase bioreact...