الفهرس 
Materials and MethodsOnly 14 pages are availabe for public view
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Abstract
Plant pcroxidases are hemoproteins which catalyze the oxidation of unusually broad structural
spectrum of hydrogen donors by H20z. Although conventional and nonconventional starting materials
for peroxidase production are available in developing
countries, including Egypt, no real enzyme production technology is existing.
The main objective of this work is the production of peroxidase from locally available source and
from plant tissue culture for analytical purposes especially preparation of clinical diagnostic
kits. To achieve this aim, establishing of simple reproducible procedure(s) for purification of
peroxidase from turnip roots and establishing procedure for peroxidase production by plant cell
culture were carried ouL
Soluble (S) and ionically bound (IB) peroxidase isoenzymes were extracted from turnip roots by
juicing and by 2M NaCI in 0.1 M ammonium phosphate buffer, pH 6.8 respectively. Sand IB peroxidase
isoenzymes were precipitated using 75% ammonium sulfate saturation with 75.5% recovery.
The isoenzyme patterns of Sand IB fractions were examined by polyacrylamide gel electrophoresis
(PAGE) using three types of running buffers (pH 8.3 , 7.0 and 4.0). PAGE of the crude and ammonium
sulfate preparations of the Sand IB fractions at pH 8.3 and pH 7.0 showed the presence of seven
isoenzymes, three anionic (A 1,
A 2, and A 3) migrate toward anode and four cationic isoenzymes (C 1, C2 , C3 and C4)
migrate toward cathode.
Two simple reproducible procedures for purification of the main peroxidase isoenzymes from turnip
root juice (soluble form) were established. The first procedure included cation exchange
chromatography on cellulose phosphate column, rechromatography on cellulose phosphate,
chromatography on Sephadex G-1 00 column and cation exchange chromatography on carboxymethyl
cellulose column. The
second procedure was hydrophobic interaction chromatography on phenyl Sepharose CL-48 column.
A simple one step purification was established for the purification of the ionically bound
peroxidase isoenzymes (18). The purification procedure included cation exchange chromatography on
cellulose phosphate column and hydrophobic chromatography on phenyl Sepharose CL-48 column.
The purified SP2 was found to be homogenous as judged by PAGE. The homogenous preparation was found
to be:
a- slightly cationic on the basis of its EM and its chromatographic behavior on cellulose phosphate
column,
b-its molecular weight was estimated to be 41,007 Da,
c- it exhibited a broad pH optimum with maximum activity at pH 5.5,
d- the Km values for phenol and H202 were estimated to be 0.5 mM and 0.2 mM respectively.
The ionically bound isoenzyme IBP2 was found to be similar to SP2 in its EM and chromatographic
behavior on cellulose phosphate column. The pH optimum was
around 6 and the Km values for phenol and H202 were found to be 1 mM and 0.2 mM respectively.
Also the main purified IBP4 was found to be homogenous as judged by PAGE. The homogenous
preparation was found to be:
a- cationic isoenzyme on the basis of its EM and its chromatographic behavior on cellulose
phosphate column,
b-its molecular weight was estimated to be 39,810 Da,
c-it exhibited a broad pH optimum with maximum at pH 6.0,
d- the Km values for phenol and H202 were found to be 8.3 mM and 0.025 mM respectively.A water soluble heat stable preparation of naturally occurring compound (NOC) was separated from
turnip root. The addition of NOC protected the enzyme from
inhibition by high concentration of H20z. The extracted fractions of NOC were found
to be free from peroxidase activity.
On economic basis two grades of the soluble peroxidase namely ammonium sulfate fraction (A) and
cellulose phosphate preparation, SP2 (B) were prepared in solid
salt-free form by lyophilization of the dialyzed fractions. The enzymatic diagnostic kits for
determination of glucose and cholesterol prepared using either turnip peroxidase A orB were found
to be efficient in determination of glucose and cholesterol in 8 and 7 serum samples compared to
the diagnostic kits prepared from HRP (product of BDH) and international diagnostic kits. Beside
that the blank reagents (containing all the reagents except glucose or cholesterol) were clear and
colorless, and the rate of color appearance due to cholesterol or glucose at 500 nm was equal to
that of the reference
kits.
Calli of B. rapa, were initiated from stem grown on solidified MS-media
containing 1 mg each of 2,4-D and kinetin and subcultured every 2 weeks, were used for preparation
of the cell suspension culture. Excretion of peroxidase by suspension culture is known to be under
the control of the ealcium concentration in the culture filtrate and the relatively high levels of
calcium supplied may increase excretion and synthesis of cationic peroxidase. The effect of Ca2+
ion concentration on excretion of peroxidase into the culture filtrate and its relation to sucrose
and phosphorus depletion was studied. Cells of calli were grown on liquid MS-media containing 1.0
mg/l each of 2,4-D and kinetin and 1, 3, 5, and 10 mM Ca2+ for 5 weeks.
The increase in cell weight started after 3 and 2 weeks of culturing at 1 and 3 mM
CaCl 2 respectively, reaching almost the same weight after 5 weeks. The cell weight decreased with
increasing the Ca2+ concentration. Carbohydrate and Pi were utilized completely by the cells after
5 weeks of growth and the increase in cell fresh weight
coincides with their decrease
The decrease in Ca2+ concentration from the normal value of the MS medium (3 mM) did not affect the
growth ( 17 and 16 mg/ 100 ml media at I and 3 mM Ca2 + respectively after 5 weeks of culturing) or
excretion of protein and the increase in growth coincide with excretion of proteins. Also the
increased protein excretion coincide with the decrease in the cell extracted proteins (mg/g fresh
weight). The highest value for peroxidase activity was observed after 3 weeks of culture ( 1774.9
and
3037.2 units/g tissue). Peroxidase activity in the cell free filtrate exhibited maximum value after
2 weeks although the secreted proteins increased by increasing of growth.
Increasing Ca2+ concentration to 5 mM affected both the growth and the
peroxidase activity. A sharp DROP in the peroxidase activity in cell extract was observed after 3
and 4 weeks of culturing associated with an increase in the excreted peroxidase. The increase in
the excreted peroxidase after 3 weeks of culturing of calli increased with increasing the Ca2+ ion
concentration (3 1.7, 40.2, 232.6 and 235.4 units/100 ml cell
filtrate at 1, 3, 5, and 10 mM Ca2+ respectively). At 10 mM Ca2+ ion concentration the highest
amount of excreted peroxidase was observed after 2 weeks of culturing (729 units/100 ml cell free
filtrate).