Elena V. Emelyanova

Lipid and γ-linolenic acid production by Mucor inaquisporus

Abstract The production of lipid and γ-linolenic acid (GLA) was studied in Mucor inaquisporus . In batch culture on ethanol, rapid lipid accumulation occurred during logarithmic growth phase of the culture and a direct proportional relationship was demonstrated between specific growth rate of the culture and GLA content of the lipid. The highest value of GLA in the lipid (36% w/w) was reached at the highest growth rate in batch culture. It has been shown that M. inaquisporus is suitable for GLA production in continuous culture. GLA content in biomass of continuous culture (2·7% w/w) was comparable to the highest GLA amount in biomass achieved in batch culture.

γ-linolenic acid production by Cunninghamella japonica in solid state fermentation

Abstract The production of lipid and γ-linolenic acid (GLA) by Cunninghamella japonica was studied in solid state fermentations (SSF) on cereal substrates. A number of cereals were used as sources of nutrients for fungal SSF and the substrate which favoured the highest accumulation of lipid and GLA in fermented substrate was determined. The treatment of cereals, addition of nutrients to the grain and thickness of the solid substrate influenced fungal growth, lipid and GLA production. Rice and millet gave the highest yields of GLA in the biomass (7·03–7·87%, w/w). The GLA content in the lipid obtained was 20% of the total fatty acids.

Relationship between magnesium and iron uptake by the yeast Candida ethanolica

Abstract During magnesium-deficiency continuous cultivation of the yeast Candida ethanolica in a bistat at a high value of growth-limitation, the response of the culture to the pulsed addition of iron salts into the fermenter was studied. The culture responded only to an addition of trivalent iron (‘apparent deficiency’ of iron) but did not react similarly to divalent iron. Under the influence of Fe 3+ the specific growth rate of the culture and the growth yield from the carbon substrate were changed. The intensity and duration of the response of the culture to an addition of trivalent iron depended on both the amount of added Fe 3+ ions and on the value of growth-limitation by magnesium. It is assumed that different systems of di- and trivalent ions uptake are present in C. ethanolica . It is proposed that iron-binding compounds- siderophores (iron(3)-specific ligands for assimilation of Fe 3+ ), probably, possessing a very high affinity for magnesium ions, can be used by the cell for Mg 2+ uptake, but, due to the slow rate of this uptake, it can be detected only in media with high magnesium deficiency.

Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process.

ABSTRACT The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L−1. The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200–250 mg L−1) and high (4 g L−1) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.

Dioxygenases of chlorobiphenyl-degrading species Rhodococcus wratislaviensis G10 and chlorophenol-degrading species Rhodococcus opacus 1CP induced in benzoate-grown cells and genes potentially involved in these processes

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding αand β-subunits of protocatechuate 3,4-dioxygenase and to two genes of theR. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.

Morphological, physiological, and biochemical characteristics of a benzoate-degrading strain Rhodococcus opacus 1CP under stress conditions

Ability of actinobacteria Rhodococcus opacus 1CP to survive under unfavorable conditions and retain its biodegradation activity was assessed. The morphological and ultrastructural features of R. opacus 1CP cells degrading benzoate in the presence of oxidants and stress-protecting agents were investigated. The cells of R. opacus 1CP were resistant to oxidative stress caused by up to 100 mM H2O2 or up to 25 μM juglone (5-oxy-1,4-naphthoquinone). After 2 h of stress impact, changes in the fatty acid composition, increased activity of antioxidant enzymes, and changes in cell morphology and ultrastructure were observed. The strain retained its ability to degrade benzoate. Quercetin had a protective effect on benzoate-degrading cells of R. opacus 1CP. The strategy for cells survival under unfavorable conditions was formulated, which included decreased cell size/volume and formation of densely-packed cell conglomerates, in which the cells are embedded into a common matrix. Formation of conglomerates may probably be considered as a means for protecting the cells against aggressive environmental factors. The multicellular conglomerate structure and the matrix material impede the penetration of toxic substances into the conglomerates, promoting survival of the cells located inside.

Kinetics of interaction between substrates/substrate analogs and benzoate 1,2-dioxygenase from benzoate-degrading Rhodococcus opacus 1CP

Benzoate 1,2-dioxygenase (BDO) of Rhodococcus opacus 1CP, which carried out the initial attack on benzoate, was earlier shown to be the enzyme with a narrow substrate specificity. A kinetics of interaction between benzoate 1,2-dioxygenase and substituted benzoates was assessed taking into account the enlarged list of the type of inhibition and using whole cells grown on benzoate. The type of inhibition was determined and the constants of a reaction of BDO with benzoate in the presence of 2-chlorobenzoate (2CBA), 3,5-dichlorobenzoate (3,5DCBA), and 3-methylbenzoate (3MBA) were calculated. For 2CBA and 3MBA, the types of inhibition were classified as biparametrically disсoordinated inhibition and transient inhibition (from activation towards inhibition), respectively. The process of not widely recognized pseudoinhibition of a BDO reaction with benzoate by 3,5DCBA was assessed by the vector method for the representation of enzymatic reactions. Ki value was determined for 2CBA, 3MBA, and 3,5DCBA as 337.5, 870.3, and 14.7 μM, respectively.

Survival and biodegradation activity of Gordonia polyisoprenivorans 135: Basics of a biosensor receptor

The effects of stress factors (drying, low temperature, and long-term storage) on the survival of the actinobacteria Gordonia polyisoprenivorans 135, which is used as a biosensor receptor to detect sodium benzoate in water solutions, were studied, as well as the effects of these factors on the biodegradation of aromatic compounds. The cells remained viable after starvation and subsequent long-term storage in suspension. Immobilization of G. polyisoprenivorans 135 cells prevented the loss of the viability when they were dried prior to storage. The immobilized cells, which were used as a biosensor receptor element, were active in relation to benzoate for more than 9 months. Storage of the receptor at 4°C for four months led to a sensor response to 10–4 and 10–3 M benzoate at a level of 69 and 79% of the maximum, respectively, while the response was 58 and 51% (70 pA/s and 140 pAs), respectively, after 9 months of storage. These results suggest that actinobacterial cells can survive an adverse environment and can be used to create highly stable and sensitive bioreceptors.