Purification And Characterization Of Vigna unguiculata Cultiver Asparaginase

Abstract

Asparaginase (EC 3.5.1.1) activity was determined in non germinating
seeds and germinating seeds of five Egyptian cowpea (Vigna
unguiculata) cultivars (Kareem 7, Dokki 331, Kafer El-Sheikh 1,
Kaha 1 and Fodder). The specific activities of germinating seeds
asparaginase in different cultivars were higher than the specific
activities of non germinated seeds of these cultivars. Asparaginase
was purified from Fodder cultivar germinating seeds ( the highest
specific activity) and resolved into three peaks with asparaginase
activities by DEAE sepharose, designated by asp I, asp II and asp III.
The molecular weight of asp II was 70 kDa for native enzyme using
gel filtration. By using SDS-PAGE electrophoresis, asp II had
molecular weight about 35 kDa suggesting that a dimeric structure for
asp II. AspII had a Km value 1.25 mM for asparagine and a pH
optimum at 8.0. Asp II had a temperature optimum and heat stability
at 40 ^oC. The fodder cultivar asp II activity was specific for Lasparagine
and did not hydrolyze D-asparagine. It is not specific for
L-glutamine. Ni²⁺ and Co²⁺ had activator effects on asp II but other
metals ions had inhibitory effect.

Key words: Cowpea , Isoenzymes, Asparagine, Glutamine, Antineoplastic effect.