The Effect of Accessibility of Insoluble Substrate on the Overall Kinetics of Enzymatic Degradation.

The enzymatic reaction kinetics on cellulose and other solid substrates is limited by the access of the enzyme to the reactive substrate sites. We introduce a general model in which the reaction rate is determined by the active surface area, and the resulting kinetics consequently reflects the evolving relationship between the exposed substrate surface and the remaining substrate volume. Two factors influencing the overall surface-to-volume ratio are considered: the shape of the substrate particles, characterized by a single numerical parameter related to its dimensionality, and the distribution of the particle sizes.

Optimizing bioprocessing efficiency with OptFed: Dynamic nonlinear modeling improves product-to-biomass yield.

Biotechnological production of recombinant molecules relies heavily on fed-batch processes. However, as the cells' growth, substrate uptake, and production kinetics are often unclear, the fed-batches are frequently operated under sub-optimal conditions. Process design is based on simple feed profiles (e.

Site-Specific Immobilization Boosts the Performance of a Galectin-1 Biosensor.

The analysis of protein-bound glycans has gained significant attention due to their pivotal roles in physiological and pathological processes like cell-cell recognition, immune response, and disease progression. Routine methods for glycan analysis are challenged by the very similar physicochemical properties of their carbohydrate components. As an alternative, lectins, which are proteins that specifically bind to glycans, have been integrated into biosensors for glycan detection.

An industrial-grade Nicotiana benthamiana line for the production of glycoproteins carrying fucose-free galactosylated N-glycans.

β1,4-galactosylation is a typical human N-glycan formation with functional impact on proteins, particularly known for IgGs. Therefore, the expression of recombinant proteins with controlled galactosylation is an important quality parameter in the biotech industry. Here we describe the establishment of a plant-based expression platform for the manufacturing of recombinant proteins carrying β1,4-galactosylated N-glycans.

Keeping the Distance: Activity Control in Solid-Supported Sucrose Phosphorylase by a Rigid α-Helical Linker of Tunable Spacer Length.

Enzyme immobilization into carrier materials has broad importance in biotechnology, yet understanding the catalysis of enzymes bound to solid surfaces remains challenging. Here, we explore surface effects on the catalysis of sucrose phosphorylase through a fusion protein approach. We immobilize the enzyme via a structurally rigid α-helical linker [EAK] of tunable spacer length due to the variable number of pentapeptide repeats used ( = 6, 14, 19).

Biocatalytic sulfation of aromatic and aliphatic alcohols catalyzed by arylsulfate sulfotransferases.

Many relevant metabolites, as well as chemical commodities, contain at least one sulfate ester group. Consequently, biocatalytic strategies to attach sulfate to a molecule under mild conditions are of high interest. In order to expand the enzymatic toolbox available, five new arylsulfate sulfotransferases (ASSTs) were identified in this study.

Predictive dynamic control accurately maps the design space for 2,3-butanediol production.

2,3-Butanediol is a valuable raw material for many industries. Compared to its classical production from petroleum, novel fermentation-based manufacturing is an ecologically superior alternative. To be also economically feasible, the production bioprocesses need to be well optimized.

Expanding the high-pH range of the sucrose synthase reaction by enzyme immobilization.

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.

Development of a whole-cell SELEX process to select species-specific aptamers against Aspergillus niger.

Background: Spores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations.

Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein.

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli.

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production.

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce.

Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements.

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples.

Electrochemically Induced Nanoscale Stirring Boosts Functional Immobilization of Flavocytochrome P450 BM3 on Nanoporous Gold Electrodes.

Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex.

From natural to synthetic: Promoter engineering in yeast expression systems.

Synthetic promoters are particularly relevant for application not only in yeast expression systems designed for high-level heterologous protein production but also in other applications such as metabolic engineering, cell biological research, and stage-specific gene expression control. By designing synthetic promoters, researcher can create customized expression systems tailored to specific needs, whether it is maximizing protein production or precisely controlling gene expression at different stages of a process. While recognizing the limitations of endogenous promoters, they also provide important information needed to design synthetic promoters.

Urban air quality affects the apple microbiome assembly.

Exposure to air pollution affects health of all organisms on earth but the impact on the plant microbiome is less understood. Here, we link the Air Quality Index with the dust and apple epiphytic and endophytic microbiome across the city of Graz (Austria). The microbiome of the apple episphere, peel endosphere and pulp endosphere, and surrounding dust was analyzed.

Enhancing NA immunogenicity through novel VLP designs.

Current influenza virus vaccines poorly display key neuraminidase (NA) epitopes and do not robustly induce NA-reactive antibodies; instead, they focus on the induction of hemagglutinin (HA)-reactive antibodies. Next-generation influenza vaccines should be optimized in order to activate NA-reactive B cells and to induce a broadly cross-reactive and protective antibody response. We aimed at enhancing the immunogenicity of the NA on vaccines by two strategies: (i) modifying the HA:NA ratio of the vaccine preparation and (ii) exposing epitopes on the lateral surface or beneath the head of the NA by extending the NA stalk.

Into the metabolic wild: Unveiling hidden pathways of microbial metabolism.

Microbial metabolism has been deeply studied over decades and it is considered to be understood to a great extent. Annotated genome sequences of many microbial species have contributed a lot to generating biochemical knowledge on metabolism. However, researchers still discover novel pathways, unforeseen reactions or unexpected metabolites which contradict to the expected canon of biochemical reactions in living organisms.

NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger.

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence.

Quantification of human intravenous immunoglobulin from plasma and in process samples by affinity chromatography.

Advances in affinity chromatography now make it possible to analyze immunoglobulin G from plasma and its fractions with a simple chromatographic method. Ligands derived from camelid antibodies have been developed which have affinity to all 4 subclasses of human IgG without a cross reactivity to other immunoglobulins. The commercially available Capture Select FcXL is the basis for a simple method for direct quantification of immunoglobulin G from plasma or from fractions from cold ethanol precipitation.

Enzyme Machinery for Bacterial Glucoside Metabolism through a Conserved Non-hydrolytic Pathway.

The flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions.

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii.

Background: Amidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.