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.

The popularity of non-alcoholic beers has been increasing over the past few years. Maltose-negative strains of different genera are frequently used to obtain beers of low alcohol content. S. cerevisiae hybrids with other Saccharomyces species offer interesting inherited flavour characteristics; however, their use in non-alcoholic beer production is rare. In this work, we constructed six hybrids of maltose-negative S. cerevisiae parental strains (modified to produce higher amounts of organic acids) and S. mikatae (wild-type). Growth behaviour, osmotolerance and fermentation features of the offspring were compared with parental strains. One hybrid with mitochondrial DNA inherited from both parents was used to produce non-alcoholic beer in which organic metabolites were evaluated by HPLC and HS-SPME-GC-MS. This hybrid produced non-alcoholic beer (≤0.05% (v/v)) with an increased organic acid content, just as its parent S. cerevisiae, but without producing increased amounts of acetic acid. The beer had a neutral aromatic profile with no negative off-flavours, similar to the beer produced by the parent S. mikatae, which was used for the first time to produce non-alcoholic beer. Overall, both parents and hybrid yeast produced non-alcoholic beers with increased amounts of higher alcohols compared with esters.

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (K_m) and 435.7 U/mg (V_max). These results could be applied to the expression of other plant enzymes.

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.