ENZYMES OF Erwinia carotovora TRANSAMINATING PHENYLPYRUVIC ACID
99
organisms of SPC Armbiotechnology, NAS RA, containing 50 mM Lꢀphenylalanine, 25 mM 2ꢀketogluꢀ
INMIA No. 8724) was used in this work. tarate, 0.05 mM PLP, 0.1 M HepesꢀNaOH, pH 7.2, and
The bacterium was grown on a medium with enzyme preparation of required quantity. The amount of
pH 7.0 containing (%): aspartic acid, 2; yeast extract, 0.1; phenylpyruvate generated was determined by
pyridoxine, 0.01; KH2PO4, 0.5; FeSO4, 0.001; MgSO4, absorption in the presence of 1 M NaOH (ε320
0.05. The cells were grown on circular shakers 17,500 M–1·cm–1) [5]. In determination of the pH optiꢀ
(200 rpm) at 30°C. The cells were harvested by centrifuꢀ mum of enzyme activity the reaction mixture contained
gation at 5000g for 40 min at 4°C (Kꢀ26 centrifuge; 100 mM concentrations of Tris, phosphate, carbonate,
Germany), washed in solution A (20 mM HepesꢀNaOH, and borate. The pH was adjusted by adding concentrated
pH 7.2, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluꢀ HCl or NaOH. One enzyme unit is defined as the
oride (PMSF), 5 mM mercaptoethanol (ME), 0.05 mM amount of enzyme that catalyzes the synthesis of 1 µmol
pyridoxal phosphate (PLP)), and after centrifugation of product per minute under the mentioned conditions.
were kept at –18°C.
The concentration of protein was measured by the
The cells were disrupted by ultrasonic treatment for method of Groves and Davis by the absorption in the
20 min at 20 kHz frequency and 300 W power (Labsonic ultraviolet region [26].
2000; B. Braun, Germany) in solution A. The cell debris
was removed by centrifugation for 20 min at 20,000g.
The molecular weights of the enzymes were deterꢀ
mined by gel filtration in a column packed with Toyopearl
The enzymes were purified at 4°C according to the 50F (1.5 × 54 cm). The column was calibrated using the
sixꢀstage scheme presented below. In the second stage the following protein standards with known molecular
crude enzyme extract obtained in the previous stage was weight: bovine serum albumin (67 kDa), egg albumin
subjected to anionꢀexchange chromatography on DEAEꢀ (45 kDa), chymotrypsinogen (25 kDa), and horse myoꢀ
cellulose equilibrated with solution A. The extract was globin (17.8 kDa).
applied to the column (2.5 × 20 cm) and was washed
The molecular weights of the enzyme subunits were
with two column volumes of solution A. Proteins were determined by disc electrophoresis in 12.6% polyacrylꢀ
eluted with a linear gradient of sodium chloride concenꢀ amide gel in the presence of SDS by the method proꢀ
tration (0ꢀ0.5 M) prepared in the same solution (500 ml). posed by Pharmacia (Sweden) [27]. The mixture of
The active fractions were combined.
Pharmacia low molecular weight proteins (rabbit phosꢀ
The proteins were further purified on a hydroxyapꢀ phorylase B, 94 kDa; bovine serum albumin (BSA),
atite column (2.5 × 7 cm) prepared according to Mazin et 67 kDa; egg albumin, 43 kDa; bovine erythrocyte carbonꢀ
al. [25]. The combined active fractions were applied to ic anhydrase, 30 kDa; soybean trypsin inhibitor,
the column, and adsorbed proteins were eluted with a linꢀ 20.1 kDa; bovine αꢀlactalbumin, 14.4 kDa) was used
ear gradient of concentration of phosphate buffer (0ꢀ as molecular weight standards.
0.2 M) prepared in solution B (1 mM EDTA, 0.1 mM
PMSF, 5 mM ME, 0.05 mM PLP, pH 7.2).
The isoelectric points of enzymes were determined
by isoelectric focusing in 5% polyacrylamide gel plates
In the fourth stage again the anionꢀexchange chroꢀ using the Pharmacia protocol [27]. The 1.6% solution of
matography on DEAEꢀToyopearl was used for effective LKB ampholyte (Sweden) was used (75% with pH 4.0ꢀ
separation of PAT3 from PAT1 and PAT2. The extract 6.0 and 25% with pH 3.5ꢀ9.0). Filter papers saturated
was applied to the column (1.5 × 25 cm), which was then with 0.5 M NaOH and 0.25 M H2SO4 were used as elecꢀ
washed with two column volumes of solution A. The trode buffers. The following wide pI spectrum
adsorbed proteins were eluted with a linear gradient of Pharmacia isoelectric point markers were used:
sodium chloride concentration (0ꢀ0.25 M) in buffer A trypsinogen (pI 9.30), lentil lectin basic group (pI 8.65),
(200 ml).
lentil lectin neutral group (pI 8.45), lentil lectin acidic
For separation of PAT1 from PAT2 another chroꢀ group (pI 8.15), myoglobin basic group (pI 7.35), myoꢀ
matography on hydroxyapatite (1.5 × 10 cm) was used. globin acidic group (pI 6.85), human anhydrase (pI
The proteins were applied to the column, which was then 6.55), bovine anhydrase (pI 5.85), βꢀlactoglobulin A (pI
washed with two column volumes of solution A, and the 5.20), soybean trypsin inhibitor (pI 4.55), amyloglucosiꢀ
adsorbed proteins were eluted with a linear concentration dase (pI 3.50).
gradient of phosphate buffer (0ꢀ0.2 M) prepared in soluꢀ
The substrate specificity of the enzymes was deterꢀ
tion B (200 ml). At this stage the chromatography mined at concentration of amino group donors 50 mM
of PAT3 on hydroxyapatite was performed separately (Lꢀtyrosine, 10 mM), and 25 mM 2ꢀketoglutarate was
according to the method described above.
used as an amino group acceptor. The concentration of
For the final stage gel filtration on Toyopearl 50 F Lꢀglutamic acid was determined qualitatively by thin
(1.0 × 65 cm) was used. The aminotransferases were elutꢀ layer chromatography on silica gel plates (Silufol,
ed by solution A containing 0.1 M NaCl.
Czechia) following Kirchner [28] and quantitatively by
The enzyme activity, if not mentioned specifically, means of glutamate dehydrogenase by NAD reduction in
was measured in reaction medium of 200 µl final volume Trisꢀhydrazine buffer.
BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012