Stephan Hann

A β-galactosidase (Bbreβgal-III) derived from the human isolate Bifidobacterium breve DSM 20213, which belongs to the glycoside hydrolase (GH) family 42, was successfully overproduced in Escherichia coli, purified to apparent homogeneity, and studied with respect to its biochemical and molecular properties as well as its transgalactosylation activity for the synthesis of galacto-oligosaccharides (GOS) in comparison to the previously reported two GH2 β-galactosidases, Bbreβgal-I and Bbreβgal-II, originating from this strain. Bbreβgal-III is a homotrimer with a molecular mass of approximately 240 kDa and shows distinct characteristics in terms of catalytic efficiency, pH dependence of activity and stability over a broad pH range, substrate specificities, and preference for the formation of specific GOS structures when compared with the other two GH2 β-galactosidases. These results suggest that multiple β-galactosidases in B. breve play divergent roles in substrate utilization under various conditions, especially in the degradation of various human milk oligosaccharide structures containing one or more β-linked galactose moieties as selective substrates for the growth and well-being of breast-fed infants. Bbreβgal-III displays transgalactosylation activity with a total GOS yield of 17% of total sugars with the preference to form 6′-galactosyllactose, which is also a predominant product during transgalactosylation of lactose. This finding paves the way for further work on this enzyme using engineering strategies to enhance transgalactosylation activity and hence the yield of the transgalactosylation products or for the biosynthesis of the GOS mixtures with specific structures, which could result in functionally enhanced prebiotic GOS.

As part of established biomanufacturing development, screening and early phase bioprocess development occurs at bench scale (microplates and shake flasks) whereby conventional offline sampling can only provide limited feedback on fermentation bioprocess parameters including strain productivity. To address these limitations, a new sensitive and selective online analytical platform consisting entirely of commercially available components with a small footprint (valves, 2DLC hardware, LC separation, and online tandem mass spectrometry) was developed for online monitoring of chip-based microbioreactors. Fermentations of microbial cell factories (Saccharomyces cerevisiae) were cultivated in 20 μL bioreactors, requiring perfusion of cell culture media at low μL/min rates delivered by syringe pump modules, operated in a multiplexed configuration with a flow-through stream selection valve, and monitored with a 2DLC-MS/MS system adapted for microscale operation. This allows uninterrupted multiplexed microperfusions to be monitored with online measurements of metabolites from parallel fermentations without the occurrence of blockages or cross-contamination between independent fermentations. Fermentations of lactic-acid-producing strains ofS. cerevisiaewere continuously monitored over 5-24 h, demonstrating the suitability of the platform for online monitoring of product quantity and key metabolites for fermentation biotechnology. Offering minimal consumption of biological material and using <1.5 mL of cell culture media over 24 h per experiment, this new platform can be used for monitoring a broad range of biomolecules, rapid strain selection, and screening of microenvironmental factors and is adaptable for targeting other key biotechnology products.

The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. Pleckstrin homology domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly found that PHLPP2 has no catalytic activity in vitro. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1,000 Mya, but that lost activity at the base of the metazoan lineage. Surface conservation indicates that while PHLPP2 has lost catalytic activity, it may have retained substrate binding. Finally, using phylogenomics, we identify coevolving genes consistent with a scaffolding role for PHLPP2 on membranes. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a focus on the noncatalytic roles of PHLPP1 and PHLPP2.

Fungal enzyme systems for the degradation of plant cell wall lignin, consisting of, among others, laccases and lignin-active peroxidases, are well characterized. Additionally, fungi and bacteria contain dye-decolorizing peroxidases (DyP), which are also capable of oxidizing and modifying lignin constituents. Studying DyP activity on lignocellulose poses challenges due to the heterogeneity of the substrate and the lack of continuous kinetic methods. In this study, we report the kinetic parameters of bacterial DyP from Amycolatopsis 75iv2 and fungal DyP from Auricularia auricula-judae on insoluble plant materials and kraft lignin by monitoring the depletion of the cosubstrate of the peroxidases with a H₂O₂ sensor. In the reactions with spruce, both enzymes showed similar kinetics. On kraft lignin, the catalytic rate of bacterial DyP reached 30 ± 2 s^-1, whereas fungal DyP was nearly 3 times more active (81 ± 7 s^-1). Importantly, the real-time measurement of H₂O₂ allowed the assessment of continuous activity for both enzymes, revealing a previously unreported exceptionally high stability under turnover conditions. Bacterial DyP performed 24,000 turnovers of H₂O₂, whereas the fungal DyP achieved 94,000 H₂O₂ turnovers in 1 h with a remaining activity of 40 and 80%, respectively. Using mass spectrometry, the depletion of the cosubstrate H₂O₂ was shown to correlate with product formation, validating the amperometric method.

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15× and over 83% of compounds quantified within 10× error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

Root exudates are composed of primary and secondary organic compounds that function as signalling molecules and play important roles in plant-environment interactions. The quantity and composition of root exudates vary depending on the species, genotype, and environmental conditions, including the presence and identity of neighbouring plants. Although cover crops are commonly used in agricultural practices for their ecosystem services, such as weed suppression, pathogen control, and soil structure improvement, studies on their root exudates are limited. Our study provides the first characterization of the root exudates of black oat interacting with weed neighbours, redroot pigweed and blackgrass, as well as with neighbours of the same species, black oat. We investigated how these interactions influence the black oat root exudation patterns. Furthermore, we investigated the impact of black oat presence on neighbours and how exposure to black oat root exudate treatments affects the root traits of weeds. The upregulated compounds detected in root exudates in response to neighbouring plants primarily belonged to the organic oxygen compounds superclass, with most of which identified as amino acids and carbohydrates. In the presence of redroot pigweed, a general increase in root exudation was observed, with amino acids and sugar sulphates being upregulated. The presence of black oat had varying effects among the neighbouring plants. While significant decreases observed and black oat in redroot pigweed root traits, an increase was observed in blackgrass. Similarly, more pronounced effects were observed in redroot pigweed compared to blackgrass upon root exudate application. This study provides a characterization and insights into the dynamic nature of root exudates and their influence on root traits and plant interactions.