Properties and Immobilization of Urease from Leaves of Chenopodium Album (C3)

藜葉片尿素酶之特性及固定化作用

H.M.El-Shora

《Botanical Bulletin of Academia Sinica》 42卷4期 (2001/10) Pp. 251-258

https://doi.org/10.7016/BBAS.200110.0251

摘要

當藜葉片被切除或光照時其尿素酶的活性會增加，Dithiothreitol（一種氫硫基化合物）、還原態的無價值麩胱肽、半胱氨酸、以及醯二氨可活性化此粗抽的酵素。相反的，此粗抽的酵素活性受PMSF及TLCK之抑制，此結果建議於其活性部位含有絲氨酸及級織氨酸。此酵素活性需鈣離子，外加鈣調素(CaM)並不激化其酵素活性，然而鈣調素拮抗物fluphenazine強烈的抑制其活性，此結果建議存在一個Ca-Like domain.EGTA、LaCl3及TMB-8於活體內抑制尿素酶的活性且外加的鈣可以恢複此受抑制的活性。利用甲荃作爲有機硬化劑可將尿素酶經共價結合至gelatin上，固定化作用結果導致該酵素增加抗熱解離作用、增加溫度的最適值(30℃to40℃)、及快速的基質飽合作用。於sodium deoxycholate、sodium taurocholate、Triton X-100及Tween80下此自由態的酵素活性顯蓍減少，於含thiol藥劑如NBS2、PHMB及PMA，其活性亦分別減少68%、75%及81%，然而固定化的酵素活性並沒有顯蓍受這些化合物的影響。增加反應時間，固定化的酵素活性減少速率比自由態的酵素活性減少速率爲快。

關鍵字

藜 ； 固定化作用 ； 特性 ； 純化 ； 尿素酶

並列摘要

In Chenopodium album, leaf excision and light both increase urease (EC. 3.5.1.5) activity. Dithiothreitol (DTT), reduced glutathione (GSH), cystcine and diazoinedicarboxylic acid bis(N, N-dimcthylamide) (diamide) activated the crude enzyme. In contrast, crude urease was inhibited by phenylmethylsuiphonyl fluoride (PMSF) and N- a-p -tosyl-L-lysine chloromethyl ketone HC1 (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, LaC13 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 30CC to 40CC), 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,Y-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.

並列關鍵字

Chenopodium album ； Immobilization ； Properties ； Purification ； Urease