ISOLATION, PURIFICATION AND SOME PROPERTIES - Biochemistry and Biotechnology: Research and Development - page 13

At the beginning of the cultivation the highest LO biosynthesis occurred at 37 о C (curve 1a),
but after 6-8 days the process slowed down and no further LO biosynthesis was observed. The
highest LO yield was obtained at 29 о C: on the 11-12 day up to 125 U/g wheat bran (curve
2a). At 24 о С (curve 3a) LO accumulation proceeded slowly LO and the total time of
cultivation enhanced up to 16 days.
The influence of pH on LO biosynthesis was fulfilled at 29 о С and рО 2 70-80% of
saturation. Without pH maintaining during the first 4 days the pH of the medium shifted to
the acidic area рН 5.8 - 4.5 (figure 5), but later the pH value slow increase up to 7.8 (curve
1a) was observed which coincided with the appearance of LO activity. The maintenance of
pH values not lower than 5.8 (curves 2, 3) did not improve the results. The highest LO
biosynthesis - 170 U/g of wheat bran (10 U/ml) was obtained in the case when pH was hold
not higher than 6.0 (curves 4, a and b).
So the fermentation in the worked out optimal conditions (pH not higher than 6.0,
рО2=70-80%, temperature 29 о С) made it possible to increase the LO biosynthesis and to
obtain 170 U/g of wheat bran (10 U/ml). The analysis of literature data points that so high
level of LO enzymatic activity was not observed beforehand.
А new improved technique of LO purification based on ammonium sulfate precipitation
(25-55% of saturation), chromatography on octyl-sepharose and DEAE-Toyopearl was used.
This approach made it possible to obtain homogeneous enzyme with high specific activity 99
U/mg (25 o C) and good yield 66%.
High thermal stability of LO was shown: the enzyme retained its activity up to 50ºC.
Optical absorption spectrum of LO are analogous to those of flavoproteins with maximums at
278, 390 and 465 nm (shoulder at 490 nm) [9]. The prosthetic group of the enzyme proved to
be FAD and each subunit possesses one molecule of FAD. The pH optimum of L-lysine
oxidation by LO from Trichoderma sp. 6 was determined to be 7.8-8.0. This data coinside
with the information about the enzymes from the other sources [5,9]. LO is a stereo specific
enzyme.
Figure 5. Influence of pH on LO synthesis by Trichoderma sp . 6 in bioreactors. рО 2 -70-80%,
temperature -29 о С. a - LO activity; b - рН. 1. pH without corrtction; 2. pH 5.8; 3. pH ≥ 6.0; 4. рН ≤
6.0.