Ludmila S. Slokoska

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.

Comparison of antioxidant enzyme biosynthesis by free and immobilized Aspergillus niger cells

Effect of immobilization on antioxidant enzyme synthesis by growing and non-growing cell culture of Aspergillus niger 26 was studied. Entrapped cells showed a greater than 1.5-fold increase in the superoxide dismutase (SOD) activity and a moderate elevation in catalase activity. The immobilization did not cause changes in the spectrum of SOD isoenzymes. The observed increase in SOD activity required de novo synthesis of this enzyme, because it was suppressed by inhibitors of the transcription and translation. The addition of various viscous substances (agar, Na-alginate and pectin) stimulated the SOD synthesis. Despite these results, it was found that the changes in SOD activity are induced in response to growth in the state of immobilization rather than to presence of alginate. Immobilized A. niger cells exhibited about a 4- to 5-fold higher level of cyanide-resistant respiration. This latter phenomenon might use as an indicator of intracellular oxy-intermediate generation in cell culture growing under stress conditions. The results are discussed relative to association between physiological stress caused by immobilization and oxidative stress.

Effect of cultural conditions on the synthesis of superoxide dismutase by Humicola lutea 110

Abstract Superoxide dismutase (SOD) was produced by the fungal strain Humicola lutea 110. Some of the factors influencing both enzyme and biomass production were studied in shake-flask cultures: glucose and nitrogen concentration, inoculum quantily, and temperature. The best results were obtained for a fermentation performed with 4% glucose, 0.152% nitrogen source, and 8% inoculum at 30°C. The time course of SOD biosynthesis showed two maxima (84.1 and 120.8 U/mg protein SOD activity respectively), which correspond to the maxima of the biomass. The dramatic increase in SOD activity during the late stationary stage indicated that the processes of O2− generation were intensified in the cells. Mn-SOD was responsible for the modulation of total SOD activity at both maxima.

Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26

The involvement of the regulatory mechanism induction in the biosynthesis of polymethylgalacturonase (PMG) by immobilized cells of Aspergillus niger 26 was studied. The immobilization altered the spectrum of pectin-degrading enzymes. The free cell cultures produced four pectinolytic enzyme activities, namely PMG, polygalacturonase (PG), pectinesterase (PE) and pectinylase (PL), while the entrapped mycelium synthesized PMG and PG only. The best inducer for PMG synthesis was highly esterified apple pectin. The effects of various pectin concentrations and time of incubation with inducer showed the induction model of PMG biosynthesis. The deinduction experiments provided evidence that the presence of the inducer in every cycle was obligatory for the PMG biosynthesis during repeated replacement cultivation.

Immobilization of acid proteinase producer Humicola lutea 120-5 with photo-crosslinkable prepolymer

Abstract Spores of fungal strain Humicola lutea 120-5 were immobilized with photo-crosslinkable prepolymer. The entrapped spores were allowed to germinate and develop in situ. The immobilized growing mycelium thus obtained was reused in batch mode for extracellular acid proteinase production. The influence of prepolymer concentration, initial inoculum gel volume, spore content, and duration of the growth cycle on the enzyme activity and mycelium growth were studied. The best yield of the acid proteinase was reached with 15 ml initial gel volume, 20% prepolymer concentration, 10 6 spores per ml gel and 56 h duration of one cycle. The optimization of these factors, which affect the stability and productivity of the immobilized system, resulted in the increase of the enzyme activity (up to 180% compared with free cells), decrease of concentration of outgrowing cells in the medium, and the realization of semicontinuous cultivation. The productive life was nearly 10–12-times longer, and the cumulative enzyme activity was 18-times higher in comparison with those of free culture. Therefore, the immobilized growing Humicola lutea 120−5 in the photo-crosslinked polymer could be used for a long-term production of an acid proteinase.

IMMOBILIZATION OF POLYMETHYLGALACTURONASE PRODUCING ASPERGILLUS NIGER ON LUFFA SPONGE MATERIAL

Abstract The vegetable sponge of Luffa cylindrica was studied as a matrix for the immobilization of Aspergillus niger 26. p roducer of polymethylgalacturonase (PMG). Entrapped spores could grow and multiply within the lattice of the sponge. The influence of loofa sponge inoculum content, initial spore inoculum content, and du ration of the growth cycle on the enzyme activity and mycelium growth was studied. The best yield of PMG was reach ed with 1 piece of loofa sponge (approx. 0.10 g dry weight), 109 spores per g carrier and 48 h duration of one cycle. Data obtained during long-term semicontinuous cultivation showed that production capacity increased significantly and the production period was extended more than 10 times compared with the free cell culture.

Production of acid proteinase by Humicola lutea 120-5 immobilized in mixed photo-cross-linked polyvinyl alcohol and calcium-alginate beads

Abstract Humicola lutea 120-5 spores were immobilized in a mixed photo-crosslinked polyvinyl alcohol and calcium-alginate gel. Maximum enzyme synthesis was established with 1:8 (v:v) gel beads: growth medium inoculum and 48 h duration of one cycle. The free cells were very unstable in replacement fermentations. The operational stability of the immobilized system indicated the possibility of the application of Humicola lutea 120-5 in a semi-continuous process for the production of acid proteinase.

Effect of Dissolved Oxygen Concentration on Superoxide Dismutase Production by Humicola lutea Cells

Cultures of the fungal strain Humicola lutea 110 were grown in a 3-l bioreactor. Effects of dissolved oxygen concentration (DO) on cell growth, intracellular protein content and antioxidant enzyme activities (SOD and catalase) were investigated. Controlling DO from 20 to 60% lead to: (I). The lethal phase of growth was reached faster; (ii) strong reduction of the intracellular protein content, and (iii) increase of antioxidant enzyme activities. The most efficient SOD biosynthesis was achieved at the 1st maximum of activity in the culture grown under DO uncontrolled conditions.

Effect of cu/zn-superoxide dismutase from the fungal strain Humicola lutea 103 on antioxidant defense of Graffi tumor-bearing hamsters

A novel Cu/Zn-containing superoxide dismutase (SOD) was isolated from the fungal strain Humicola lutea 103. Previously, a protective effect of this enzyme (HLSOD) against tumor growth and also superoxide production in Graffi tumor-bearing hamsters (TBH) were established. The aim of the present study was to investigate the effect of HLSOD on the activity of endogenous SOD and catalase in the cells from TBH during tumor progression. Our results point out that transplantation of Graffi tumor causes a significant decrease in SOD activity in the cells from liver of the hosts (from 35 to 59% compared to the control). In the tumor cells relatively low levels of SOD (about 7 U mg protein-1) were found, and Cu/ZnSOD was the main isoenzyme in total SOD activity. Tumor growth resulted in a reduction of catalase activity, which correlated with the process of tumor progression. A single dose (65 U) treatment with HLSOD caused an increase in endogenous SOD and catalase activity in healthy animals and resulted in restoration of the antioxidant ability in liver cells of the hosts at the early stage of tumor progression. The results show the possible participation of HLSOD in the host oxidant-antioxidant balance, which is probably one of the factors of its immunoprotective action established earlier.

Immunoprotective Effect of Cu/Zn Superoxide Dismutase on Myeloid Graffi Tumor-Bearing Hamsters

Abstract Investigation on the immunoprotective activity of Cu/Zn superoxide dismutase from Humicola lutea 103 AL (HLSOD) in hamsters with transplanted myeloid tumor was performed. Survivability, tumor growth and tumor transplantability were followed. The immune status of tumor-bearing animals, injected with the optimal protective HLSOD dose, was examined during 27 days after tumor transplantation by the following parameters: (i) the number, migration and phagocytic activity of peritoneal macrophages, (ii) the phagocytic activity of blood polymorphonuclear leukocytes (PMNs), (iii) the responsibility in vitro of spleen lymphocytes to T and B cell mitogens. It was established that intraperitoneal inoculation of HLSOD produced a protective effect on the development of tumors. Elongation of the latent time for tumor appearance and inhibition of the tumor growth were observed. The decreased percentage of mortality in early stage of tumor progression was established. Immunological studies on tumor-bearing hamsters (TBH) induced a tem porary immunorestoring effect on the suppressed phagocytic activities of peritoneal macrophages and blood PMNs during the first 14 days of tumor development. Moreover, HLSOD showed an expressed stimulating effect on proliferative activity in vitro of spleen B lymphocytes from healthy and TBH as well. The immunorestoring and protective effect of the enzyme was probably due to improve of the oxidant-antioxidant balance in peritoneal phagocytes. The tem porary character of the effect can be explained with the interference of immunosuppressing factors produced by tumor tissue as well as by the presence of tumor antigens, tumor cells and antigen-antibody complexes in the circulation.