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:'::K /4.4.5. Go m p a r is o n of th e P e r o x i d a t i c A c tiv ity o f B e e f-lx v e r and Ii\m,Q:al Garb a la s e Tiibes
The p é ro x id a tic a c t i v i t i e s of im m obilised p re p a ra tio n s of bo th b e e f - liv e r and fung'al c a ta la s e w e r e compared in th e fo llo w in g manner.
Ti'jo 3 m le n g th s of a c tiv a te d }üîDA~substituted n y lo n -tu b e were prep ared as d e sc rib e d in s e c tio n 3 .2 , One such tu b e d e riv a tiv e was in cu b ated
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w ith an enzyme co u p lin g s o lu tio n com prising I .4 mg ml b e e f - liv e r —•1
enzyme and th e o th e r w ith 1.4 mg ml fu n g al enzyme. A fte r w ashing, each d e riv a tiv e was assayed f o r p e ro x id a tic a c t iv it y as d e sc rib e d in s e c tio n 3 ,6 .4 . Ho d iffe re n c e in th e r a te of ÏÏGHO p ro d u ctio n of
e i th e r d e riv a tiv e could be d e te c te d . Thus in each case th e re must be s u f f ic ie n t im m obilised a c t i v i t y to a t t a i n th e h ig h e st r a ti o of
p e ro x id a tic : c a t a l a ti c a c t i v i t y .
I t has been shovm in s e c tio n 4*3,5 th a t th e fu n g a l enzyme d isp la y s s u p e rio r s t a b i l i t y . As th e p e ro x id a tic a c t i v i t i e s of th e fu n g al and b e e f - l iv e r im m obilised d e riv a tiv e s a re id e n tic a l, th e form er may
th e re fo re be more u s e fu l as an in s o lu b le enzyme fo r a u to a n a ly tic a l work.
4,4*6. S t a n d a r d Curves fo r th e d e te rm in a tio n of
HpOg was determ ined by th e p e ro x id a tic re a c tio n of im m obilised c a ta la s e as d e sc rib e d in s e c tio n 3 .6 .4 * Three HM DA-substituted
d e riv a tiv e s were p rep ared in -ohe fo llo w in g way : 1 ) IMS a lk y la tio n of 1mm bore tube 2) TOTPB all<yla'cion of 1mm bore tu b e , 3) TOTPB a llc y la tio n o f 2 mm bore tu b e . Sach "was in s e r te d in to th e flow system d escrib e d in
P ig 86 and sta n d a rd ise d s o lu tio n s of 1i#i - 5*i'>M”~HpOp, were sam pled. The sta n d a rd curves o b tain ed f o r th e th re e c a ta la s e d e riv a tiv e s a re shov/n in P ig 2 7 . The h ig h e st s e n s i ti v i ty was o b tained w ith th e TOTii’.B
a lk y la te d 2 mm bore c a ta la s e tu b e , w h ils t th e 1 mm bore a lk y la te d c a ta la n e tube d isp la y e d th e low est s e n s i t i v i t y . This dem onstra.tes th e
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4 . 4 . 7 . S t a b i l i t y of C a ta la se Tubes to th e P e ro x id a tic A ction
The s t a b i l i t y of c a ta la s e tu b es w orking in th e c a t a l a ti c mode have p re v io u sly been d isc u sse d in s e c tio n 4«3. However, a, c a t a l a s e
tube w o r k in g in th e p e ro x id a tic mode i s exposed to m ethanol and ECHO in a d d itio n to HgOg and b u f f e r . In o rd e r to determ ine th e o p e ra tio n a l b b a b ility of c a ta la s e tu b es under th e se c o n d itio n s, th e fo llo w in g experim ent was u n d ertak en .
Two 1.5 m le n g th s o f HIvlM -substituted n y lo n -tu b e im m obilised c a ta la s e were prep ared from b e e f - liv e r and fu n g al enzyme, as d e sc rib e d in s e c tio n 3 .2 , Both d e riv a tiv e s were s u b je c te d to continuous a n a ly sis o f 5 mM-HgOp as d e sc rib e d in s e c tio n 3 .6 .4 , Each d e riv a tiv e was found to be s ta b le over a p e rio d o f 60 h over which 3600 assay s were p e r form ed su g g e stin g th a t no d ecrease in s t a b i l i t y of a c a ta la s e tube is caused by exposure to c o n d itio n s imposed by th e p e ro x id a tic conversion of m ethanol to form aldehyde.
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SmaïAEY OF 8BCTIŒ 4 . 4
The p e ro x id a tic and c a t a la t ic a c t i v i t i e s of im m obilised c a ta la s e were in v e s tig a te d . The sum o f th e combined a c t i v i t i e s in th e presence
of b o th HgOg and m ethanol was f ou n d, t o e q u a l t h e c a t a la t ic a c t i v i t y
in th e absence of m ethanol. This sug g ested th a t co m p etitio n e x is ts between th e p e ro x id a tic o.nd c a t a l a t i c a c t i v i t i e s . The p resence of 2,5 M-methanol gave t h e h ig h e st r a t i o o f p e ro x id a tic : c a t a la tic a c t i v i t y d isp la y ed by th e im m obilised d e r iv a tiv e . The e x te n t of p e ro x id a tic a c t i v it y a s s o c ia te d w ith d if f e r e n t im m obilised c a ta la s e tu b e s was in v e s tig a te d , TO TPB-alkylation was found to produce a
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- f*» / d e r iv a tiv e in which th e r a ti o of p e ro x id a tic : c a t a l a ti c a c t i v i t y was h ig h e r th a n a d e riv a tiv e p rep ared by DI^IS-alkyl a t io n . In c re a s in g th e d iam eter of th e tube from 1 - 2 mim a lso in c re a se d t h i s r a t i o . The le n g th of c a ta la s e tu b e assay ed was shovm to a f f e c t th e e x te n t of
p e ro x id a tic u t i l i s a t i o n o f th e HpOg, A lin e a r resp o n se of form aldehyde