OBTAINING AND PROPERTIES OF INTRACELLULAR AMINOACYLASE ESCHERICHIA COLI - Biochemistry and Biotechnology: Research and Development - page 37

used: g/l: KH 2 PO 4 - 3; Na 2 HPO 4 - 6; NaCl - 0,5; NH 4 Cl - 1; agar - 0,2; MgSO 4 - 0,25,
tiaminechloride - 0,01; glucose - 6; Na succinate - 6; ampicillin - 0,1; pH medium 7,0 ± 1.
N-acetyl-D, L-methionine, dithiothreitol, Na succinate, ortophtalic aldehyde produced in
Serva (Germany), DEAE cellulose (Pharmacia, Sweden), reagents produced in FSU countries
were used as well.
Aminoacylase activity was determined by method [8]. Reaction mixture contained: 0,045
M N-acetyl-D, L-methionine; 0,05 M Na, K-phosphate buffer, pH 7,0; 0,2 mM CoCl 2 , 1mM
dithiothreitol and 2,5 μg protein of ferment solution. The samples were incubated at 37 ° C for
5 min. The reaction was stopped by adding trichloroacetic acid (final concentration - 6%).
The liberated L-methionine was determined by adding 1,25 ml distilled water and 1,25 ml
reagent of ortophtalic aldehyde (60 ml 0,1M borate buffer, pH 9,7; 10mg of ortophtalic
aldehyde; 0,005 ml of mercaptoethanol) to 0,05 ml of sample, followed by measurement of
optical density of obtained solutions at wavelength 340 nm. As a unit of acylase activity is
accepted ferment amount catalyzing 1 μmol L-methionine for 1h at above-mentioned
conditions. The concentration of proteins was determined by absorption at 280 nm.

Table 1. Isolation and purification of intracellular aminoacylase E. coli

№ Purification stage Protein,

mg

Total
activation,
U x10 3

Specific
activity,
U/mg

Total
yield,
%

Purification,
fold

1 Cell extract 1100 2200 2000 100 1

2 Precipitation with

streptomycine sulfate

1000 2100 2100 95 1,05

3 Fractioning with

ammonium sulfate

160 1680 10500 80 5

4 Chromatography on

DEAE cellulose, at pH
7,0

25,8 814 31500 37 16

5 Chromatography on

DEAE cellulose at pH
6,0

10,5 660 63000 30 32

Figure 1. Chromatography of intracellular aminoacylase E. coli on DEAE cellulose, at pH 6,0. Stepwise
natrium chloride gradient is shown with arrows: 1 - 0,1 M NaCl; 2 - 0.15 M NaCl; 3 - 0,2 M NaCl; 4 -
0.25 M NaCl. Active fractions of the ferment is shadowed.