The presence of Ca²⁺ (up to 0.1 mol/L) in the cultivation media was found to induce the formation of conidia in submerged mycelia of Trichoderma viride in a concentration-dependent manner. Ca²⁺ dramatically stimulated conidiation after 70 h of cultivation. The effect was present in the dark, and illumination stimulated it only marginally. Low (less than 100 μmol/L) Ca²⁺ concentrations induced the formation of chlamydospores. Sr²⁺ could substitute Ca²⁺ in conidiogenesis with lower efficiency (almost 2 orders of magnitude), while the efficiency of Mg²⁺, Mn²⁺, or Ba²⁺ was lower by almost 3 orders of magnitude. Our results demonstrate that mycelial Ca²⁺ homeostasis has powerful effects on the conidiation and mycelial morphogenesis in T. viride, and they suggest that there is an additional mechanism of conidiation in addition to those induced by light and starvation.

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron—sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc₁ complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron—sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus—human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron—sulphur flavoprotein.

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (Mut^S) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/L_medium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

Sulforaphane and other natural isothiocyanates released from the respective plant glucosinolates by the plant enzyme myrosinase (β-thioglucoside glucohydrolase) show extensive anticancer and antimicrobial effects. In this study, myrosinase from garden cress (Lepidium sativum) seeds was purified to electrophoretic homogeneity by a fast and easy strategy consisting of fractionation by isoelectric precipitation with ammonium sulphate (AS) and affinity chromatography using sulforaphane (SFN) attached to cellulose resin. The overall purification of enzyme with respect to crude extract was 169-fold and recovery of 37%. Under non-reducing conditions, two protein bands exhibiting myrosinase activity with masses of about 114 and 122 kDa, respectively, and a 58 kDa protein band with no activity were detected by SDS-PAGE and zymography on polyacrylamide gel. MALDI-Tof/Tof of tryptic fragments obtained from the respective protein bands detected sequence motifs homologous to the regions responsible for glycoside-substrate binding and similarities to members of the enzyme subfamilies β-glucosidases and myrosinases GH. The enzyme hydrolyzed both the natural (sinigrin, sinalbin, glucoraphanin) and the synthetic (p-nitrophenol-β-D-glucopyranoside (pNPG)) substrates. The highest catalytic activity of purified enzyme was achieved against sinigrin. The K_M and V_max values of the enzyme for sinigrin were found to be 0.57 mM, and 1.3 mM/s, respectively. The enzyme was strongly activated by 30 μM ascorbic acid. The optimum temperature and pH for enzyme was 50 °C and pH 6.0, respectively. The purified enzyme could be stored at 4 °C and slightly acidic pH for at least 45 days without a significant decrease in specific activity.

Lepidium draba (hoary cress) is a perennial plant belonging to the Brassicaceae family that produces two dominant glucosinolates (GLSs): glucoraphanin (GRN) and sinalbin (SBN). They represent the stored form, which is converted upon the myrosinase (Myr) hydrolysis activity to active compounds, mainly isothiocyanates (ITCs) such as sulforaphane (SFN) or p-hydroxybenzyl isothiocyanate (pHBITC). Research on ITCs that have proven anticancer, antimicrobial, and chemoprotective properties is usually conducted with pure commercially available compounds. However, these are chemically reactive, making it difficult to use them directly for preventive purposes in dietary supplements. Efforts are currently being made to prepare dietary supplements enriched with GLS and/or Myr. In this study, we report a simple but efficient chromatographic procedure for the isolation and purification of GLSs from MeOH extract from hoary cress based on a combination of ion exchange and gel permeation chromatography on DEAE-Sephadex A-25 and Sephadex LH-20. To obtain the Myr required for efficient hydrolysis of GLSs into antibacterial ITCs, we developed a rapid method for its extraction from the seeds of Lepidium sativum (garden cress). The yields of GLSs were 22.9 ± 1.2 mg GRN (purity 96%) and 10.4 ± 1.1 mg SBN (purity 92%) from 1 g of dry plant material. Both purified GLSs were used as substrates for the Myr. Analysis of the composition of hydrolysis products (HPs) revealed differences in their hydrolysis rates and in the degree of conversion from GLSs to individual ITCs catalyzed by Myr. When GRNs were cleaved, SFNs were formed in an equimolar ratio, but the formation of pHBITCs was only half that of cleaved SBNs. The decrease in pHBITC content is due to its instability compared to SFN. While SFN is stable in aqueous media during the measurement, pHBITC undergoes non-enzymatic hydrolysis to p-hydroxybenzyl alcohol and thiocyanate ions. Testing of the antimicrobial effects of the HPs formed from GRN by Myr under premix or in situ conditions showed inhibition of the growth of model prokaryotic and eukaryotic microorganisms. This observation could serve as the jumping-off point for the design of a two-component mixture, based on purified GLSs and Myr that is, usable in food or the pharmaceutical industry in the future.