The purification of lipase produced by Candida parapsilosis : preliminary investigation on the enantioselectivity of the enzyme towards hydrolysis of an ester of a racemic arylpropionic acid

Date

4-1998

Degree

Bachelor of Science in Agricultural Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Ma. Jamela R. Revilleza

Co-adviser

Milagros M. Peralta

Abstract

The study aimed to purify a lipase produced by Candida parapsilosis and use this in the resolution of a racemic aryl propionic methyl ester. Two chromatographic protocols were used to effect enzyme purification from the culture medium which served as crude extract. These were hydrophobic interaction chromatography (HIC) using phenyl sepharose column and affinity chromatography (AF) using a heparin column. Eluate obtained by phenyl sepharose chromatography using ethylene glycol — 50 mM phosphate buffer showed specificity for the hydrolysis of the S-enantiomer of an aryl propionic acid methyl ester . This S-isoenzyme was isolated in relatively pure form upon elution using 100% ethylene glycol. It was identified to have a molecular weight of 34.3 KD by SDS-PAGE. Affinity chromatography using a derivatized heparin column yielded fractions that had lower activities towards the substrate relative to the crude enzyme preparation. However, there was a substantial increase in enantioselectivity , with the 0.3M NaCI fraction giving 100% S-(+)-acid and the 0.2M NaCI fraction giving rise to the S(+)-acid in 24% ee. This compares favorably with the enantioselectivity of only 10% ee for the crude enzyme preparation. The lower than expected activities of the purified fractions relative to the crude enzyme seems to indicate partial denaturation of the enzyme by the high salt concentration and the use of 100% ethylene glycol.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 1998 A13 M38

Document Type

Thesis

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