TITLE Properties and immobilization of urease from leaves of Chenopodium album (C3)
AUTHOR H.M. El-Shora
Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt
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ABSTRACT In Chenopodium album, leaf excision and light both increase urease (EC. 3.5.1.5) activity. Dithiothreitol (DTT), reduced glutathione (GSH), cysteine and diazoinedicarboxylic acid bis(N,N-dimethylamide) (diamide) activated the crude enzyme. In contrast, crude urease was inhibited by phenylmethylsulphonyl fluoride (PMSF) and N-a-p -tosyl-L-lysine chloromethyl ketone HCl (TLCK), suggesting the presence of serine and histidine residues in the active site. The enzyme is Ca dependent for its activity and exogenous calmodulin (CaM) did not stimulate it. However the enzyme is strongly inhibited by CaM antagonist fluphenazine, indicating the presence of a Ca-like domain. EGTA, LaCl3 and tetraacetic acid, 3,4,5,-trimethoxybenzoic acid 8-(diethyl-amino)-octyl ester (TMB-8) inhibited urease activity in vivo, and the inhibition was restored by exogenous Ca. Urease was immobilized in gelatin by covalent cross-linking with formaldehyde as organic hardener. The results indicated enhanced resistance to thermal denaturation, increased temperature optima (from 30oC to 40oC), and a rapid rate of substrate saturation were achieved after immobilization. The free urease showed remarkable loss of activity in the presence of sodium dodecyl sulphate, sodium deoxycholate, sodium taurocholate, Triton X-100, and Tween 80. The free enzyme lost 68%, 75% and 81% of its activity in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (NBS2), p-hydroxymercuribenzoate (PHMB) and phenylmercuric acetate (PMA) as thiol reagents. However, the immobilized enzyme was not affected significantly by these compounds. By increasing the incubation time, the activity of immobilized enzyme decreased faster than that of the free one.
KEYWORD Chenopodium album; Immobilization; Properties; Purification; Urease;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 42 Number 4 October 2001, page 251-258, 8 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China