Biomedical Engineering Reference
In-Depth Information
The purpose of this work is obtaining (isolation and purification) of intracellular
aminoacylase
Escherichia coli
and investigation of it's properties. It was found that in
logarithmically growing culture
E. Coli
, possessing aminoacylase activity, intracellular
aminoacylase manifests essential fermentative activity in stationar phase of cell growth.
Based on this, to isolate the investigating ferment,
E. Coli
cells, having aminoacylase activity
were used in stationary phase. Fermentation period was 20 h at 37
°
C. Data concerning
isolation and purification of intracellular aminoacylase are shown in table 1. Cell extract was
obtained by degradation of the cells under ultrasonic desintegrator, followed by centrifugation
at 13000 rpm, 20 min, + 4
°
C. Precipitation of fractions by nuclear acids was performed by
ammonium sulfate at 55-80 % saturation, followed by chromatography on DEAE cellulose at
pH 7,0 with a gradient of phosphate buffer concentration from 0,1 M to 0,7 M in the presence
of 1mM CoCl
2
and 1mM dithiothreitol - managed to escape from most part of ballast
proteins. At secondary chromatography active fractions of investigating ferment on DEAE-
cellulose at pH 6,0 intracellular aminoacylase
E. coli
is eluted if used sodium chloride
gradient concentration from 0,1 to 0,4 M in initial buffer. Active ferment eluted under
influence of 0,25 M NaCl on the same buffer (Fig. 1). Based on developed scheme it was
possible to obtain intracellular aminoacylase
E. coli
with 30% yield activity and 32-fold
purified.
Intracellular aminoacylase
E. coli
, obtained with above-mentioned method was used to
study it's physico-chemical properties. The isolated ferment is stable upon storage in 0.1 M
phosphate buffer at pH 7,0 in the presence of 1mM CoCl
2
and 1mM dithiothreitol at 4
°
C for
several months. pH of the ferment optimal activity by hydrolysis of N-acetyl-D, L-methionine
makes up 7,0. The optimum value for the ferment stability is the pH interval 6,0-7,5. In the
presence of Co
2+
ions, the intracellular aminoacylase
E. coli
is rather thermostable, it doesn't
lose its activity at 50
°
C for 60 min. However, at as high temperature as 60
°
C, it's total
inactivation occurs within 30 min. The temperature optimum of the ferment in the presence of
1mM CoCl
2
is observed at 37
°
C.
The total inactivation of intracellular aminoacylase
E. coli
with p-chloromercuri-benzoate
indicates availability of free SH-groups that are essential for activity. The isolated ferment is
inhibited with ethylenediaminotetraacetic acid that proves metal-dependence of intracellular
aminoacylase
E. coli
. Studies of the effect of metal ions on the ferment have shown that the
ions of Co
2+
provide maximum activating action, activating intracellular aminoacylase
E. coli
for 100%, ions of Ca
2+
, Mn
2+
- is two times less, and the action of other investigated ions of
metals (Cd
2+
, Pb
2+
, Sn
2+
) on the ferment activity is ineffective.
Thereby it can be concluded that isolation and purification method of intracellular
aminoacylase
E. coli
is developed, which allows to obtain the ferment with 30% yield activity
and 32-fold purified. Physico-chemical properties of investigated ferment are studied.
M
ATERIALS AND
M
ETHODS
Strain of
Escherichia coli LGE 36
, containing recombination plasmid with
arg E
gene,
coding aminoacylase synthesis was used in this work (All-Russian collection of industrial
microorganisms, “Institute of Genetics and Selection of Industrial Microorganisms”,
Moscow). To maintain and reproducе the strain, medium with following composition was
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