299
1077 (1991) 299-307 1991 ElseVIer Science Publishers B.V. 0167·4838/91/$03.50
RlOch,mll.'a et BlOphy.'m'll A('1(I.
ADONIS 0167483891001638
BBAPRO 33881
Purification and characterization of a catalase-peroxidase and a typical catalase from the bacterium Klebsiella Pneumoniae Ayala Hochman and Iris Goldberg The Department of Bwchemlslrv, GeorKe S. WISe Facull)' 0/ Life SCIences. Tel-AvIV Uml'crsIlY. TeJ-Avw (Israel) (Ret:Clved 17 October 1990)
Key won:h: Catalase·peroxldase; Typical catalase; Catalytic activity: (KlehslelJa pneuffwmae)
The bacterium Kleh.-
~
~ ~
20
20
"..0
60
80
T I ME
o L-J'--'---''- '--'--'--'--'-- -'---'---'---' ---' o 40 80 120 160 200 240 TIME
(min)
Fig. 8. Effect of 3-amino-l.2 ,4-triazole on the catalatic .activity of
KpCP and KpT. The enzyme was incubated at 30°C with 20 mM 3-amino-1,2,4-triazole and 4 mM sodium ascorbate in SO mM potassium phosphate (pH 6.8). Samples were removed at the indicated lime points and assayed for catalase activity. KpT (_); KpCP (e).
0
20
40
60
80
(mIn)
Fig. 9. Effect of incubation with H 0 on the catalatic activity of 2 2 KpCP (A) and KpT (B). 1 ml of enzyme preparation s. diluted to similar concentrati ons of 11-13 units/ml were dialyl.ed agalllsl the indicated I.:oncentrati om (in mM) of Hill' in 33 mM phosphate buffer (pH 6.8) contain"mg 5 mM EDTA, at 30°('. Samples were removed at the indicated time points and were assayed for catalase a'tivity as descrihed under Materials and Method~. Ea'h preparation was also dialyzed against buffer without H 202' as a control.
305
~lool===~;:::;·::·::.:.: ..;'·~...; ------l
,
~
100
C 80
~.o
,..
....
;; 40 ~
u
20
'" ~ 20 '"
40
60
80
100
0
20
40
60
BO
100
TIM E (mlo)
'" ~
~ 0O'------J,O---::':--:'---':--~-:':--::::-"~.~O"" Fig. 10. Effect of incubation at vanous temperatures on the activity of the calalases. The enzyme!. KpCP (e) and KpT (.) were incubated at the mdicated (emperature~. and samples were removed after 30 min and assayed Immediately. at 30°C, for catalase activity.
Fig. 12. Errect of incubation with urea on the activity of the catalases. KpCP (Al and KpT (8) WOfO incubatod with 4 M (e) Of 8 M (.). samples were removed al the indicated time points and assayed for catalase activity after IOO-fold dilution in the reactIOn mixture.
activity, while KpCP was completely inactivated after 12 min. Discussion
the pH range of 6.2-8.1. Incubation of the enzyme for 30 min at pH 5.2 and 10.8 caused 50% inhibition. KpT retained full activity when incubated for 30 min at pH values between 5.0 and 11.0 (Fig. 11), and lost 50% of its activity at pH 4.4 and 11.7. Effect of urea. KpT was found to be more stable than KpCP in the presence of urea (Fig. 12). 60 min incubation with 4 M urea caused 6% inhibition of the activity of KpT and 72% inhibition of KpCP. When incubated in the presence of 8 M urea KpT lost 70% of the
~IOO
....1 \I
o
I
1077 (1991) 299-307 1991 ElseVIer Science Publishers B.V. 0167·4838/91/$03.50
RlOch,mll.'a et BlOphy.'m'll A('1(I.
ADONIS 0167483891001638
BBAPRO 33881
Purification and characterization of a catalase-peroxidase and a typical catalase from the bacterium Klebsiella Pneumoniae Ayala Hochman and Iris Goldberg The Department of Bwchemlslrv, GeorKe S. WISe Facull)' 0/ Life SCIences. Tel-AvIV Uml'crsIlY. TeJ-Avw (Israel) (Ret:Clved 17 October 1990)
Key won:h: Catalase·peroxldase; Typical catalase; Catalytic activity: (KlehslelJa pneuffwmae)
The bacterium Kleh.-
~
~ ~
20
20
"..0
60
80
T I ME
o L-J'--'---''- '--'--'--'--'-- -'---'---'---' ---' o 40 80 120 160 200 240 TIME
(min)
Fig. 8. Effect of 3-amino-l.2 ,4-triazole on the catalatic .activity of
KpCP and KpT. The enzyme was incubated at 30°C with 20 mM 3-amino-1,2,4-triazole and 4 mM sodium ascorbate in SO mM potassium phosphate (pH 6.8). Samples were removed at the indicated lime points and assayed for catalase activity. KpT (_); KpCP (e).
0
20
40
60
80
(mIn)
Fig. 9. Effect of incubation with H 0 on the catalatic activity of 2 2 KpCP (A) and KpT (B). 1 ml of enzyme preparation s. diluted to similar concentrati ons of 11-13 units/ml were dialyl.ed agalllsl the indicated I.:oncentrati om (in mM) of Hill' in 33 mM phosphate buffer (pH 6.8) contain"mg 5 mM EDTA, at 30°('. Samples were removed at the indicated time points and were assayed for catalase a'tivity as descrihed under Materials and Method~. Ea'h preparation was also dialyzed against buffer without H 202' as a control.
305
~lool===~;:::;·::·::.:.: ..;'·~...; ------l
,
~
100
C 80
~.o
,..
....
;; 40 ~
u
20
'" ~ 20 '"
40
60
80
100
0
20
40
60
BO
100
TIM E (mlo)
'" ~
~ 0O'------J,O---::':--:'---':--~-:':--::::-"~.~O"" Fig. 10. Effect of incubation at vanous temperatures on the activity of the calalases. The enzyme!. KpCP (e) and KpT (.) were incubated at the mdicated (emperature~. and samples were removed after 30 min and assayed Immediately. at 30°C, for catalase activity.
Fig. 12. Errect of incubation with urea on the activity of the catalases. KpCP (Al and KpT (8) WOfO incubatod with 4 M (e) Of 8 M (.). samples were removed al the indicated time points and assayed for catalase activity after IOO-fold dilution in the reactIOn mixture.
activity, while KpCP was completely inactivated after 12 min. Discussion
the pH range of 6.2-8.1. Incubation of the enzyme for 30 min at pH 5.2 and 10.8 caused 50% inhibition. KpT retained full activity when incubated for 30 min at pH values between 5.0 and 11.0 (Fig. 11), and lost 50% of its activity at pH 4.4 and 11.7. Effect of urea. KpT was found to be more stable than KpCP in the presence of urea (Fig. 12). 60 min incubation with 4 M urea caused 6% inhibition of the activity of KpT and 72% inhibition of KpCP. When incubated in the presence of 8 M urea KpT lost 70% of the
~IOO
....1 \I
o
I
