Effects of vanadate on enzymatic and non-enzymatic components of the antioxidant defense system in the filamentous fungus Phycomyces blakesleeanus (Burgeff) [NRRL 1555(−)]

[J. Lukičić]

94 str.

Biološke nauke - Biofizika / Biology - Biophysics

Zadatak ove disertacije je bio ispitivanje dejstva vanadijuma (V), prelaznog metala sa raznovrsnim fiziološkim efektima, na metabolizam končaste gljive Phycomyces blakesleeanus. Od ranije je poznato da ova gljiva ima sposobnost usvajanja, redukcije i unutarćelijskog heliranja V u obliku vanadata [V(V)] i vanadila [V(IV)]. Vanadijum je redoks aktivan metal, te je ispitan uticaj inkubacije micelije P. blakesleeanus u V(V) na produkciju reaktivnih kiseoničnih vrsta (ROS) i promene u aktivnosti enzimskih, odnosno koncentraciji neenzimskih komponenti antioksidativnog sistema. Micelije u tri karakteristične faze rasta – eksponencijalnoj (E), kasnoj eksponencijalnoj (KE) i stacionarnoj (S) - su tretirane 1, 5 ili 10 mM V(V) tokom 1, 5 ili 24 h. U E i KE fazi rasta je udeo V(V) u micelijama nakon 24 h tretmana dostigao 0,8 i 0,7%, što kandiduje ovu gljivu za bioakumulatora V. V(V) je već nakon 30 min. doveo do značajnog povećanja unutarćelijskih ROS kod micelije u E fazi rasta, dok je kod druge dve starosne grupe ovaj porast bio manji. Duža (24 h) izlaganja su dovela do većeg porasta ROS kod KE i S micelija, što ukazuje na najveće antioksidativne kapacitete micelije u E fazi, koji je očuvan zahvaljujući visokim koncentracijama neenzimskih antioksidanata poput GSH i fenola. U S fazi, osnovnu antioksidativnu ulogu nose GSH i peroksidaze, i to POD i GPx. KE faza rasta se pokazala kao najosetljivija na efekte V. Redukujući kapacitet micelije je ovde ostao umanjen i nakon 5 h tretmana i to je jedina faza na čiju vijabilnost je V(V) imao inhibitorni efekat. Izlaganje mikroorganizama stresu dovodi do sinteze lipida ili ugljenih hidrata, pa je sa tim u skladu FTIR metodom određen relativni sadržaj gradivnih biomolekula u miceliji P. blakesleeanus. Kao glavni faktor u povećanju udela lipida u biomasi istaklo se starenje micelije, ali i tretman V(V) je imao jasan uticaj. Analiza lipidnog sastava može ukazati na potencijalnu upotrebnu vrednost P. blakesleeanus u proizvodnji biodizela ili nutritivnih ulja.

The aim of this dissertation was to investigate the effect of vanadium (V), a transition metal with various physiological effects, on the metabolism of the filamentous fungus Phycomyces blakesleeanus. It is known that this fungus can take up, reduce and intracellularly chelate V in the form of vanadate [V(V)] and vanadyl [V(IV)]. Since vanadium is a redox-active metal, the influence of incubation of P. blakesleeanus mycelia in V(V) on the production of reactive oxygen species (ROS) and the changes in the activity of enzymatic and non-enzymatic components of the antioxidant system was investigated. Mycelia in three characteristic growth phases - exponential (E), late exponential (LE) and stationary (S) - were treated with 1, 5 or 10 mM V(V) for 1, 5 or 24 hours. In growth phases E and LE, the proportion of V(V) in the mycelia reached 0.8 and 0.7% after 24 h of treatment, making this fungus a candidate for V bioaccumulation. V(V) led to a significant increase of intracellular ROS in mycelia in the E-growth phase after 30 min, whereas this increase was lower in the other two age groups. Longer exposures (24 h) led to a greater increase in ROS in LE and S mycelia, indicating the highest antioxidant capacities of mycelia in the E phase, which was maintained due to high concentrations of non-enzymatic antioxidants such as GSH and phenolic compounds. In the S phase, GSH and peroxidases, namely POD and GPx, played the most important antioxidant role. The LE growth phase proved to be the most sensitive to the effects of V. The reduction capacity of the mycelium remained reduced even after 5 hours of treatment, and it is the only phase on whose viability V(V) had an inhibitory effect. Exposure of microorganisms to stress leads to synthesis of lipids or carbohydrates, therefore, the relative content of biomacromolecules in the mycelia of P. blakesleeanus was determined using the FTIR method. Ageing of the mycelium was the main factor for the increase in lipid content in the biomass, but V(V) treatment also had a clear impact. The analysis of the lipid composition could give an indication of the potential usefulness of P. blakesleeanus for biodiesel or food/feed oils production.

vanadat, oksidativni stres, antioksidativna zaštita, Phycomyces blakesleeanus, reaktivne kiseonične vrste, biosorpcija, lipidi, kriva rasta

vanadate, oxidative stress, antioxidant defense, Phycomyces blakesleeanus, reactive oxygen species, biosorption, lipids, growth curve

577.3:582.28(043.3)

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