Evaluating the effect of glycation on lipase activity using boronate affinity chromatography and mass spectrometry.

Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from .

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in . EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality.

Targeting proline in (phospho)proteomics.

Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome.

Aspergillus niger Prolyl Endoprotease for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies.

To monitor the structural integrity of therapeutic proteins, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5.

Metabolic Engineering toward Sustainable Production of Nylon-6.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid.

Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine.

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway.

Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA.

Background: The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis.

Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate.

Sample preparation and biostatistics for integrated genomics approaches.

Genomics is based on the ability to determine the transcriptome, proteome, and metabolome of a cell. These technologies only have added value when they are integrated and based on robust and reproducible workflows. This chapter describes the experimental design, sampling, sample pretreatment, data evaluation, integration, and interpretation.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery.

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity.

Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-L-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger.

The carbon starvation response of Aspergillus niger during submerged cultivation: insights from the transcriptome and secretome.

Background: Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from endogenous resources becomes mobilized to fuel maintenance and self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution.

Is proteomics a reliable tool to probe the oxidative folding of bacterial membrane proteins?

The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity.

Peroxicretion: a novel secretion pathway in the eukaryotic cell.

Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations.