Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen.
The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.
Liu
You-Yan
YY
College of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, PR China.
Xu
Jian-He
JH
Wu
Hui-Yuan
HY
Shen
Duan
D
D000327:Adsorption; D019149:Bioreactors; D002175:Candida / Q000201:enzymology*; D005591:Chemical Fractionation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007660:Ketoprofen / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011136:Polysorbates; D013237:Stereoisomerism
D004800:Enzymes, Immobilized; D007527:Isoenzymes; D011136:Polysorbates; D007660:Ketoprofen; D008049:Lipase