ISOLATION, PURIFICATION AND SOME PROPERTIES - Biochemistry and Biotechnology: Research and Development - page 9

LO activity was assayed at 25 oC by measuring the velocity of hydrogen peroxide
formation in 20 mM Tris-HCl buffer (pH 8.0) in the presence of o-dianisidine (0.2 mM),
peroxidase (5 μg/ml) and L-lysine (2.0 mM) on “Shimadzu” spectrophotometer (E436 = 11,0
mM-1·sm-1) [5].
Substrate specifity of LO was determined under the same conditions. 0.5 мМ L-lysine, L-
leucine, L-phenylalanine, L-citrulline, L-alanine, L-asparagine, L-histidine, L-ornithine, L-
glutamine, L-threonine, L-tyrosine, L-isoleucine, L-valine, glycine and D-lysine were used as
the substrates, LO was added at the concentration equal to 2.0 g/ml .
pH-dependence of the velocity of L-lysine enzymatic oxidation was measured under the
described above conditions using 50 mM buffer solutions: formiate-acetate (pH 4.0-6.0), Tris-
acetate (рН 6.0-7.5); Tris-HCl (рН 7.0-9.0).
The LO thermostability was studied in 2.5 mM Tris-HCl buffer by incubation of 2.0
g/ml enzyme solution during 10 min at 20, 28, 37, 50, 60, 70 80 о C before enzymatic activity
determination.
LO stability at 37 о C was studied by incubation of 5 mg/ml enzyme solution during 5
days. The aliquots were taken periodically for enzymatic activity determination.
The effectiveness of biosynthesis was estimated as the amount of International Units of
LO activity per 1 ml of fermentation mixture (U/ml) or per 1 g of dry wheat bran used for
fermentation (U/g). (It should be noted that fungal mycelia as usual grow penetrating through
the wheat bran, so it was not possible to withdraw both the biomass and all the proteins
including LO from the swallen solid substrate and to determine the ratio of U/per g of
protein).
LO isolation and purification. Stage 1. After the filtration and centrifugation of cultural
liquid the ballast proteins were removed during one day at 4 о С by ammonium sulfate
addition up to 25% of saturation and subsequent centrifugation (6000 g, 40 min). Ammonium
sulfate was again added to the supernatant up to 55% of saturation and the solution was
incubated at 4 о С for 12 hours.
Stage 2. The insoluble proteins, including LO, were separated by centrifugation (6000 g,
30 min) and dissolved in tris-HCl buffer (50 мМ, рН 8.0), containing ammonium sulfate
(20% of saturation). The obtained solution was centrifuged to withdraw the insoluble proteins
and applied to the column (2 х 30 сm) with Octyl-Sepharose СL-4B, equilibrated with tris-
HCl buffer (25 мМ, рН 8.0), containing ammonium sulfate (20% of saturation). The column
was washed with the same buffer. The enzyme was eluted by stepwise lowering the degree of
ammonium sulfate saturation: 20, 15 and 10%.
Stage 3. The fractions with LO activity were collected, united, dialyzed during 12 hours
against tris-HCl buffer (50 мМ, рН 8.0) and applied to the the column (2 х 40 сm) with
DEAE-Toyopearl, equilibrated and washed by tris-HCl buffer (25 mМ, рН 8.0). The enzyme
was eluted by the fractional enhancement of NaCl concentrations: 0.15; 0.2; и 0.3 М.
Molecular weight and purity of the enzyme were determined by native electrophoresis [6]
using PROTEAN II xi system BIO-RAD (USA). 50 μl analyzed samples containing 50 μg of
protein were added into 50 μl of SDS sample buffer (62.5 mM Tris-HCl buffer, pH 6.8,
containing 1.0% ß-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue). 20-25 µl of
protein samples were applied on the gel for analysis.
After electrophoresis the gel was stained by Coomassie Brilliant Blue R250.