Biomedical Engineering Reference
In-Depth Information
To define better the associated minor proteins a more sensitive method - silver staining
was used.
Molecular weight determination by gel filtration was fulfilled on the colomn with
Toyopearl HW-55 (1,5 х 95 сm). The elution was carried out by Tris-HCl buffer (25 mМ, рН
7.8). Alcohol dehydrogenase (150 кDа), bovine serum albumen (67 кDа),
а
-chymotrypsin (25
кDа) and cytochrome
с
(13 кDа) were used for column calibration. V
0
of the column was
measured with the help of Blue Dextran 2000.
Cofactor analysis. Aqueous solution containing LO (100 μg/ml) and trichloracetic acid
(10%)
kept for 5 min at 100
o
C. The denaturized proteins were withdrawn by centrifugation
(10000 g, for 10 min). The supernatant was neutralized by КОН and used for the
measurement of optical density at 460 nm (ʵ=11300 х mol
-1
х cm
-1
) (Shimadzu). Qualitative
analysis of the cofactor was carried out with HPLC, colomn Separon C
18,
4х150, 5 μm (Czech
Republic).
R
ESULTS AND
D
ISCUSSION
Genus
Trichodermа
fungi were tested for their ability to produce LO using
solid-phase
cultivation method. All strains were actively growing on wheat bran (medium 1), but only 4
strains appeared to produce LO, the highest LO level being shown by
Trichoderma
sp. 6
(Table 1). The addition of different nitrogen sources enhanced the LO biosynthesis 5-10
times.
Figure 1. The influence of nitrogen sources on LO synthesis by
Trichodеrma
sp. 6. 1 - NH
4
NO
3
; 2 -
NH
4
Cl; 3 - NaNO3; 4 - (NH
4
)
2
SO
4.
The bulk cultivation of four mentioned fungi strains using the media and conditions
described in “MATERIAL AND METHODS showed that all strains were growing
intensively in the used media (visual observation), including the Capek's medium with
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