ISOLATION, PURIFICATION AND SOME PROPERTIES - Biochemistry and Biotechnology: Research and Development - page 14

Figure 6. The optical absorbtion stectra of L-lysine alpha-oxidase (0.5 mg/ml LО in 25 mM Tris-НСl
buffer рН 8.0). 1.- without L-lysine ; 2. - in the presence of 0.5 mМ L-lysine.
It does not affect D-lysine. L-lysine is the main substrate of LO (100% activity). Among
the tested amino acids (see MATERIAL AND METHODS) only structural analogs of L-
lysine (L-ornithine and L-arginine) are oxidized, but much slower (less then 8%). Substrate
specificity of LO from the new strain Trichoderma sp. 6 resembles the specificity amino acid
oxidases from the other strains of genus Trichoderma ( T. viride Y-244-2 [9] and T.
harzianum Rifai [5] ). Molecular weight of enzyme determined by native electrophoreses and
gel filtration was equal to 115-116 кDа. According to SDS electrophoresis data LO is a
dimeric molecule with identical subunits of 57-58 kDa. High termal stability of LO was
shown: the enzyme retained its activity up to 50 º C. The enzyme remained active at 37 ºC at
least 5 days.
The spectra of optical absorption are presented in Figure 6. LO isolated demonstrated the
spectra typical for flavin-containing proteins with maxima at 278, 390 и 465 nm. In the
presence of L-lysine the maxima at 390 and 465 nm disappeared (figure 6, curve b). After
exhaustion of lysine (5-6 min) spectra were recovered in the presence of oxygen. These data
are indicating that flavin-containing coenzyme participates as the prosthetic group of LO.
Further qualitative and quantitative analysis of the cofactor ( see MATERIAL AND
METHODS ) showed that prosthetic group of the enzyme proved to be FAD and each LO
subunit possessed one molecule of FAD. The biological properties of LO are mainly based on
its catalytic activity: during the reaction L-lysine level is decreased and hydrogen peroxide
formation occurs. L-lysine is an important molecule for cell growth and hydrogen peroxide
presents one of the reactive oxygen species, which takes part in the oxidative stress
development. LO antibacterial effect towards different strains of Bacillus subtilis,
Staphylococcus, Pseudomonas and Aspergillus and anti fungal effect towards Clostridium
sporogenes was shown. The strains of tumor cells are sensitive to the lack of L-lysine and to
the action of oxidative stress caused by hydrogen peroxide.
The main difference between LO and L-asparaginase (the only enzyme used in tumor
therapy) is that LO does not effect Fisher lymphadenosis L5178Y. Eight strains of tumor cells
were shown to be sensitive to LO treatment. Currently further pre-clinical studies of LO anti
tumor activity are carried out in Blokhin's Cancer Research Center of Russian Academy of
Medical Sciences.