ISOLATION, PURIFICATION AND SOME PROPERTIES - Biochemistry and Biotechnology: Research and Development - page 8

immunological reactivity. This aim could be achieved by using different sources of
enzyme.
The aim of the present work was to find new strains-producers of LO, capable of
intensive enzyme biosynthesis in the bioreactors, and to investigate LO from the new
strain with the purpose of its practical application in oncology.

M ATERIAL AND M ETHODS

Microorganisms. Genus Trichodermа fungi were tested for their ability to synthesize LO.
12 strains under study were from the All-Russian Culture Collection of the Institute of
Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences: T.
harzianum Rifai VKM F-1959, T. longibrachiatum Rifai VKM F-2025, T. virens (J.H. Mill.,
Giddens and A.A. Foster) Arx VKM F-1117, T. aureoviride Rifai VKM F-2026, F-2027, T.
viride Pers. VKM F-2721, F-1130, F-1132, F-1133, F-1134, F-1135, F-2430. 8 strains were
isolated from soil (fields near Pushchino, Moscow region).
The isolation of the strains from soil samples. Enriched cultures were obtained on
dry sterile wheat bran (a few passages). Soil samples were suspended in sterile distilled water
and added to sterile dry wheat bran (10g). After 5 days of cultivation at 28 o C 100 ml of
sterile water were added into each flask. After shaking for 1 h aliquots were inoculated to an
agar medium, containing the crushed wheat bran (10%) and NaNO 3 (5%). The grown up
colonies were treated by the solution containing L-lysine (2.0 мМ), o-dianizidine (0.1 мМ)
and peroxidase (5 μg/ml). Fungi from the colored (brownish) colonies were tested

again for their ability to synthesize LO under solid-phase fermentation.
The solid-phase cultivation of the fungi. Fungi from colored colonies as well as
museum strains were grown at 28 0 С under periodical shaking in the 750-ml Erlenmeyer
flasks with 10 g of sterilized bran and 10 ml of 4% solution of different nitrogen salts (or
distilled water) (media N 1). The cultivation was carried out for 15 days. After fermentation
100 ml of Tris-HCl buffer (25 мМ, рН 8.0) were added to each flask and the mycelium
with wheat bran were shaked for 1 hour (220 rpm). The insoluble residue was removed
by centrifugation at 10000 g for 10 min and supernatant was used for enzyme isolation
and purification.
The bulk cultivation of the fungi. Fungi were grown at 28 0 С on a shaker (200 rpm) in
750-ml Erlenmeyer flasks with 100 ml of a Capek's medium, containing (g/l): NaN0 3 - 4.0;
KH 2 PO 4 -1.0; MgSО 4 -0.5; КСl-0.5 FeSO 4 -0.01, and supplemented with Burkholder trace
elements solution and the sole source of carbon and energy such as glucose (5.0%) (medium
2a), or starch (5.0%) (medium 2b), or sucrose (5.0%) (medium 2c), or glucose (1.0%) and
corn extract (1.0 %) simultaneously (medium 2d). The pH of the media was 7.2.
The fungi were grown also on the liquid media, containing 10 g of wheat bran per 100 ml
of distilled water and supplied with different nitrogen sources (media 3). After fermentation
the cultural liquid was centrifuged (10000 g for10 min). The insoluble residue was removed
and supernatant was used for enzyme isolation and purification.
LO biosynthesis monitoring. The concentration of LO in the reaction media was tested
every day. To determine LO activity the aliquots of growing media were taken, filtrated and
centrifuged at 10000g for 10 min.