GLUCOSE OXIDAZE PREPARATIONS DERIVED FROM  
PENICILLIUM FUNGI FOR BIOCATALYTIC SYSTEMS 
Mikhailova R.V., Semashko T.V., Demeshko O.D. 
Institute of Microbiology, National Academy of Science, Belarus 
Glucose oxidase (EC 1.1.3.4) (GOX) is widely used in food, chemical industries and 
medicine. Significant GOX application trend is generation of derived biosensors and biofuel cells. 
Earlier highly active producers of extracellular GOX Penicillium adametzii and P. funiculosum 
were isolated at laboratory of enzymes, Institute of Microbiology, National Academy of Science, 
Belarus and methods for producing GOX with different purity grade were elaborated. 
Aim of this study is to obtain and characterize purified glucose oxidase by Penicillium fungi 
to be further used in biocatalytic systems. 
Purification of GOX from P. adametzii and P. funiculosum was carried out by the schemes 
comprising combination of concentration by ultrafiltration of fungal cultural filtrate (membranes 
with nominal minimal mass retention limit 10 kDA), gel-filtration (Sephacryl S-300) and ion-
exchange chromatography (DEAE ceramic). Derived enzymes were homogeneus, characterized by 
high specific activity 224-238 U/mg protein, GOX activity constituted 1289.8-1317.2 U/ml. 
Spectrophotometric and spectrofluorometric analyses have shown that GOX spectra are 
typical for flavoproteins. The absorption spectrum peaks were recorded at 380 and 455 nm, an 
excitation spectrum – 373 and 447 nm, and an emission spectrum – 530 and 562 nm. 
Secondary structure of GOX produced by P. adametzii and P. funiculosum was deciphered. 
It was shown that both enzymes were characterized by presence of structural fragments with diverse 
conformation (α-helix, β-sheet, irregular sequences) and minimum of molar ellipticity at 212 and 
216 nm. 
Despite the fact that the studied enzymes show 99 % similarity in amino acid composition, 
they differ considerably in physical-chemical properties. 
Optimal conditions for enzyme action were found in temperature range 55 -70 °C 
(P. adametzii), 55 - 60 °C (P. funiculosum) and pH range 4.5-7.5 and 5.0-8.0, respectively. 
Distinctions in thermal and pH stability of fungal GOX were revealed. Incubation of P. adametzii 
enzyme at 50°C during 1 hour resulted in 18% loss of activity, in contrast to 45 % reduction for 
P. funiculosum GOX. The enzymes of P. adametzii and P. funiculosum retained stability at pH 5.0 
and pH 4.9-9.0, respectively. GOX of P. funiculosum was distinguished by higher stability in 
alkaline pH zone. After 24 h exposure to pH 11.0 and 12.0 P. funiculosum enzyme preserved 62 % 
and 57 % of initial activity. 
The buffer system for expression of maximal enzyme activity was defined (0.01 M 
phosphate buffer, pH 7.0). 
Parameters of GOX interaction with ferrocene and its derivatives (ferrocenecarboxylic acid, 
ferrocenecarboxylaldehyde, methylferrocene-methanol) were investigated. It was found that in 
comparison with ferrocene-modified enzyme, affinity of GOX modified with its derivatives to β-D-
glucose increased by 1.5-2.1 times (Km=9.4-13.3 mM). The best GOX catalytic characteristics 
resulted from methylferrocene-methanol modification. The obtained findings were corroborated by 
fluorometric FAD/ FADH2 studies. 
Currently research is under way at the Centre of Nanotechnology and Materials Science - 
NanoTechnas, Faculty of Chemistry, Vilnius University to generate biofuel cell with improved 
characteristics incorporating above - described GOX. 
This work was financially supported by Foundation of Basic Studues, Belarus republic 
(project B11LIT-012).