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Biocatalysis – the application of enzymes or microbes in chemistry – has developed into one of the key technologies of the 21st century. Enzymes in isolated form, as a cell extract or whole cell biocatalysts can be used to replace or supplement purely chemical process routes with the goal to make chemical synthesis more efficient, environmentally friendly, sustainable and potentially more cost-effective.

Where does industrial biocatalysis stand today? To comprehensively answer this question, an analysis of the Swiss manufacturing environment was carried out and set in the context of global developments. Interviews with Swiss companies, active in different industrial sectors, in tandem with an analysis of global research and patent trends highlight that the accelerating scientific progress in bioinformatics, protein engineering, molecular biology and high-throughput-screening fuels the industrial use of biocatalysis by making accessible new enzymatic activities. Existing hurdles to the full industrial exploitation of biocatalysis such as commercial enzyme availability, regulatory requirements, education deficits and public perception of biotechnology are discussed and contrasted with the many opportunities biocatalysis has to offer. The development of new business ideas and forms of collaborations between industry and academia will guarantee the field a prosperous future.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.

Saccharomyces cerevisiae is a much preferred host for biotechnological applications. However, the expression of entire heterologous pathways, required for some potential products, is technically challenging in yeast. A possible tool would be polycistronic gene expression. Recent studies demonstrated that short 5' untranslated regions (5'UTRs) found upstream of certain genes support cap-independent translation in vitro. In this study 5'UTRs were used as linkers between genes in polycistronic constructs. Expression levels of genes located in the first, second and third position after a promoter were studied by replacing the respective gene by a promoterless green fluorescence protein (GFP) gene. S. cerevisiae transformed with these constructs was grown on different carbon sources and GFP expression was assayed. Our results demonstrate that (i) ribosomal read-through does not suffice for polycistronic gene expression in vivo, (ii) 5'TFIID and 5'HAP4 but not 5'L-A significantly improve the expression of a reporter gene located second in a bicistron, (iii) 5'TFIID, 5'HAP4 and 5'YAP1 but not 5'L-A can drive expression of a promoterless reporter gene, and (iv) expression driven from 5'TFIID, 5'HAP4 and 5'YAP1 is induced in the presence of raffinose or galactose but not in the presence of glucose. This implies that these elements unlike typical internal ribosome entry site-like structures contain small, potentially useful promoters which support carbon source-regulated expression.

Diets enriched with bioactive food components trigger molecular changes in cells that may contribute to either health-promoting or adverse effects. Recent technological advances in high-throughput data generation allow for observing systems-wide molecular responses to cellular perturbations with nontoxic and dietary-relevant doses while considering the intrinsic differences between cancerous and noncancerous cells. In this chemical profile, we compared molecular responses of the colon cancer cell line HT29 and a noncancerous colon epithelial cell line (HCEC) to two widely encountered food components, sulforaphane and selenium. We conducted this comparison by generating new transcriptome data by microarray gene-expression profiling, analyzing them statistically on the single gene, network, and functional pathway levels, and integrating them with protein expression data. Sulforaphane and selenium, at doses that did not inhibit the growth of the tested cells, induced or repressed the transcription of a limited number of genes in a manner distinctly dependent on the chemical and the cell type. The genes that most strongly responded in cancer cells were observed after treatment with sulforaphane and were members of the aldo-keto reductase (AKR) superfamily. These genes were in high agreement in terms of fold change with their corresponding proteins (correlation coefficient r(2) = 0.98, p = 0.01). Conversely, selenium had little influence on the cancer cells. In contrast, in noncancerous cells, selenium induced numerous genes involved in apoptotic, angiogenic, or tumor proliferation pathways, whereas the influence of sulforaphane was very limited. These findings contribute to defining the significance of cell type in interpreting human cellular transcriptome-level responses to exposures to natural components of the diet.

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μ max (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of q p > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated q p occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q p(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in q p were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

The cell surface glycoprotein of Halobacterium halobium is soluble in dilute buffer at neutral pH. At low counterion concentrations, the protein is monomeric (Ms,D = 209 kDa) and exhibits the characteristics of a highly charged polyelectrolyte. Evidence obtained from intrinsic fluorescence and far-UV circular dichroism shows that the monomer at low salt loses both its native conformation and its inherent tendency to form high molecular mass assemblies. In 4M NaCl, 25 mM KCl, and in the presence of divalent ions (greater than or equal to 50mM Mg2+ or Ca2+), association to well-defined assemblies of up to approximately 4 X 10(6) Da occurs. At low Mg2+ concentration and in the presence of Ba2+, a wide size-distribution of aggregates is observed. The assembly pattern of the protein may be correlated with salt-dependent alterations in the morphology of the bacterium.