A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g.

Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer.

Background: Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.

Surface charge engineering of a Bacillus gibsonii subtilisin protease.

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general.

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol-xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum.

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol-xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.

Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products.

Two long-standing questions about glucansucrases (EC 2.4.1.

Identification of new acceptor specificities of glycosyltransferase R with the aid of substrate microarrays.

Finding opportunities to construct sugar motifs and to transfer them to targets of biological relevance and rapid identification of glycosylation events are important goals for glycobiology and a field of increasing interest. Here we have applied an enzyme microarray screening system for the identification of new acceptor specificities of the glycosyltransferase R (GTFR) from Streptococcus oralis (E.C.

A two-photon fluorescence-correlation study of lectins interacting with carbohydrated 20 nm beads.

We present results of a two-photon fluorescence-correlation study carried out with glycosylated and untreated 20 nm fluorescing spheres that interacted with the carbohydrate-binding proteins soybean agglutinin (SBA) and concanavalin A (Con A). The assay principle allows protein-carbohydrate binding interactions to be determined without protein labeling. This assay might serve as a simple model system for studying physical and chemical interactions between proteins and carbohydrates, for example, at cell or virus surfaces.