Letno poročilo 2005

Laboratorij za biotehnologijo
Laboratory of Biotechnology
Pripravili smo rekombinantni senzor za
spremljanje kalcija, pH in proteazne aktivnosti,
ki omogo~ajo in vivo spremljanje koncentracije
kalcija, vrednosti pH in aktivnost citosolnih
proteaz gliv. Osnovni gradniki senzorjev so
zeleni fluorescirajo~i proteini. Senzor za kalcij
smo v povezavi z University of Edinburgh
analizirali z metodo FLIM-FRET (Förster resonance
energy transfer in fluorescence lifetime
imaging microscopy), ki je potrdila mo`nost
uporabe omenjenega senzorja v glivah. Z
rekombinatnim senzorjem za pH smo dolo~ili
znotraj celi~no pH vrednost pri glivah in potrdili,
da je uravnavanje znotrajceli~ne vrednosti pH
povezano z uravnavanjem znotrajceli~ne
koncentracije kalcija, saj pri glivah z okvarjenimi
kalcijevimi ~rpalkami pride do dviga znotrajceli~ne
vrednosti pH. V na{em laboratoriju smo
pridobili keratinazo s fermentacijo s submeznim
gojenjem glive v bioeraktorju in jo testirali pri
na{ih sodelavcih v Angliji. Rezultati so pokazali,
da je z encimom mo`no izbolj{ati propustnost
modelne keratinske membrane. V letu 2005
smo preizkusili okrog 100 sevov gliv na
izbranem modelnem substratu za Bayer-
Villigerjevo reakcijo, s katerim je mo`no pridobiti
po dve stereo in dve opti~ni izomeri. [tevilne
glive so bile sposobne transformirati substrat,
vendar so bili produkti razli~ni. Glede na profil
in opti~no ~istost dobljenih produktov smo glive
lahko razvrstili v {tiri skupine.
SODELOVANJE Z INDUSTRIJSKIMI
IN DRUGIMI PARTNERJI
- Lek, d.d., Ljubljana: raziskave na podro~ju
biotehnologije ter razvoju novih zdravil
- BIA Separations d.o.o, Ljubljana: dva skupna
projekta
MEDNARODNO SODELOVANJE
mednarodni projekti:
- Dva evropska projekta v okviru 5. okvirnega
programa: ANTICO in ANEPID
- Dva evropska projekta v okviru 6. okvirnega
programa: Eurofungbase in TSEUR
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activation- and inhibition in presence of different
metabolites.
A recombinant sensor for calcium, pH and proteolytic
activity detection was prepared which
enable in vivo detection of calcium concentration,
pH value and activity of cytosolic proteases
in fungi. Essential building blocks of the sensors
are green fluorescent proteins. The calcium
sensor was analyzed, in cooperation with partners
from the University of Edinburgh with the
FLIM-FRET (Förster resonance energy transfer
in fluorescence lifetime imaging microscopy)
method, confirming the possibility of application
of the sensor in fungi. With the recombinant
sensor for the pH we determined the
intracellular pH value in fungi and we confirmed
that the intracellular control was connected
with the control of the intracellular calcium concentration,
since in fungi with damaged calcium
pumps an increase of intracellular pH occurred.
In our Laboratory, a fungal keratinase
was produced in a bioreactor by submerged
fermentation and our British partners tested the
enzyme. Results showed that by using the enzyme
it was possible to increase permeability
of a keratinous membrane. In 2005 we tested
around 100 fungal strains on a selected model
substrate, for Baeyer-Villiger oxidation, which
enables the production of two stereo- and two
optical isomers. Several fungi were able to transform
the substrate. However, the obtained
products were different. According to the profile
and optical purity of the products the fungi
could be classified into four different groups.
COLLABORATION WITH INDUSTRIAL AND
OTHER PARTNERS
- Lek, d.d., Ljubljana, Menge{ unit, Slovenia:
research of fungal metabolism and molecular
biology of Streptomycetes
- BIA Separations d.o.o, Ljubljana, Slovenia:
two joint projects
INTERNATIONAL COLLABORATION
international projects:
- Two projects within the 5 th Framework Programme:
ANTICO in ANEPID