Esterase activity of an aqueous extract of the green bean was
separated into fourteen bands, while aqueous extracted pea esterases
revealed seven bands, by polyacrylamide-gel electrophoresis. The
fourteen bands of bean esterase activity formed three groups; slow,
intermediate and fast moving. α-Naphthyl acetate, propionate, and
n-butyrate and AS naphthol acetate were hydrolyzed at various rates
by the bean and pea esterases. No hydrolysis of β-naphthyl laurate
was observed, indicating the absence of a lipase in the aqueous
extracts of these vegetables. Since all the esterase bands active
toward α-naphthyl acetate were inhibited by organophosphorus compounds
(diisopropylphosphorofluoridate, diethyl p-nitrophenyl thiophosphate
and diethyl p-nitrophenyl phosphate), these esterases were
classified as carboxylesterases (carboxylic ester hydrolase, EC
3.1.1.1).
To study the carboxylesterases of the green bean in greater
detail, a protamine sulfate treated aqueous extract was separated
into three fractions (S₁, S₂ and S₃) by chromatography on Sephadex
G-100. Subsequent analysis of each fraction by polyacrylamide-gel
electrophoresis demonstrated the presence of the slow moving group
in fraction S₁, slow and intermediate moving groups in fraction S₂
and the fast moving group in fraction S₃. Hence, these studies suggest
that the three groups of esterase activity in beans were dissimilar
in molecular size and the relative molecular size was
slow > intermediate > fast moving group.
Chromatography of fraction S₁ on carboxymethyl (CM) cellulose
with sodium chloride linear-gradient elution resulted in three fractions
(CM₁, CM₂ and CM₃). Similarly, fraction S₂ yielded three
fractions (DE₁, DE₂ and DE₃), while fraction S₃ produced two
fractions (DE₄ and DE₅), by chromatography on microgranular
diethylaminoethyl (DEAE) cellulose. Polyacrylamide-gel electrophoresis
revealed the presence of the first three bands of the slow
moving group in CM₁ and only the first two bands in CM₂. DE₁
possessed mainly the first two bands and DE₂ the last two bands of
the five bands in the slow moving group. The five bands of the intermediate
moving group of esterase activity was found only in fraction
DE₃. The first two bands of the four bands of the fast moving group
were separated into fraction DE₄, while the last two bands were in
DE₅.
Nine substrates and various concentrations of three inhibitors
were used to characterize some of the fractions obtained from ion-exchange
chromatography. Although most of the fractions hydrolyzed
the substrates used in this study, each fraction differed to some
extent in substrate specificity. Inhibitor studies indicated the
presence of a sensitive and a resistant component of esterase activity
in each fraction studied. These results suggest that the esterase
fractions were composed of two enzymes. To account for the fourteen
bands of esterase activity a hypothetical model of polymers
consisting of two monomers was proposed. This hypothesized model
suggests that the slow moving group contained six pentamers, the
intermediate group five tetramers and the fast moving group four
trimers. Most characteristics of the carboxylesterases of beans
observed in these studies could be explained on the basis of the
hypothetical model. / Graduation date: 1969
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/26816 |
| Date | 09 December 1968 |
| Creators | Veerabhadrappa, Patnagere Siddaveera Sheety |
| Contributors | Montgomery, Morris W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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