Hydrolytic enzymes from germinating mustard (Brassica juncea) seeds : partial purification, characterization and preliminary investigation of its use as a catalyst for the kinetic resolution of an a- arylpropionic acid

Date

4-2000

Degree

Bachelor of Science in Agricultural Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Milagros M. Peralta

Co-adviser

Ma. Jamela R Revilleza

Abstract

ABSTRACT

NADRES, ENRICO TAPIRE. University of the Philippines Los Banos, April, 2000. "HYDROLYTIC ENZYMES FROM GERMINATING MUSTARD (Brassie,: juncea) SEEDS: PARTIAL PURIFICATION, CIIARACTERIZATION AND PRELIMINARY INVESTIGATION OF ITS USE AS A CATALYST FOR THE KINETIC RESOLUTION OF AN a-ARYLPROPIONIC ACID".

Adviser: Dr. Milagros M. Peralta

Co-adviser: Dr. Ma. Jamela R. Revilleza

The presence of proteases and lipases was detected from different brands of germinating mustard seeds extracted with 0.1 N Tris buffer pH 7.5, with 1 mM dithiothreitol (DTT). When the crude extract (7.29 U/mg) of Good Earth, chosen as a model system for the study, was subjected to ammonium sulfate fractionation, the hydrolases were concentrated in the supernatant at 100% saturation (100S fraction). This fraction was then passed through a hydrophobic interaction column, yielding negative adsorption, an indication of the enzyme's predominant hydrophilic surface. Partitioning of the 100S fraction using 15% polyethylene glycol yielded a fraction with a lipase specific activity of 1335 U/mg, a purification factor of 185. On the other hand, gel filtration of the 100S fraction resulted in the localization of a fraction with a specific activity of 7600 U/mg, a 1370 fold increase in purification. Analysis of the active fractions via SDS-PAGE identified a protein band with an approximate molecular size of 41.2 kD, approaching the value obtained when a lipase active fraction was identified after gel filtration chromatography using Sephacryl S-100.

The purification of the lipase from the local brand, Philippine Kaneko was patterned after the optimized protocol for Good Earth. Although initial lipase activity of the crude extract was higher than that of Good Earth, the partially purified fraction obtained towards the end of the scheme produced a lower activity. The decline was explained by the higher activity of proteases that could have digested the lipase.

The crude extract, 100S and gel filtration lipase active fractions of both Chinese and local brands were evaluated as potential catalysts in the kinetic resolution of a.- arylpropionic acid esters. All three fractions from the former were S (+) enantioselective where product has 100% ee. Evaluation of the enantioselectivity of the samples obtained from the local brands revealed that the crude extract showed low selectivity (18.33% ee). As purification progressed, enantioselectivity was enhanced especially for the gel filtration fraction producing the S (+) acid at 100 % ee. The results signified the presence of other hydrolases in the crude extract with different stereochemical preference, which were removed during purification. This facilitated the identification of an S(+) enantioselective lipase comparable to that of the Chinese brand.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2000 A13 N33

Document Type

Thesis

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