Digital screening methodology for the directed evolution of transglycosidases.

Engineering of glycosidases with efficient transglycosidases activity is an alternative to glycosyltransferases or glycosynthases for the synthesis of oligosaccharides and glycoconjugates. However, the engineering of transglycosidases by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of digital imaging-based high-throughput screening methodology for the directed evolution of glycosidases into transgalactosidases.

Identification and characterization of a gamma-glutamyl transpeptidase from a thermo-alcalophile strain of Bacillus pumilus.

A gamma-glutamyltranspeptidase (GGT, E.C. 2.

Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family.

A 1242 base pair DNA fragment from Bacillus halodurans H4 isolated from alkaline sediments of Lake Bogoria (Kenya) coding for a potential protease was cloned and sequenced. The hexa-histidine-tagged enzyme was overexpressed in Escherichia coli and was purified in one step by immobilized-metal affinity chromatography (IMAC) on Ni-NTA resin. The protease (ppBH4) presents an inverted zincin motif, HXXEH, which defines the inverzincin family.

Physico-chemical and immunological properties and partial amino acid sequencing of a new metalloprotease: endoprotease Thr-N.

Previous studies have described the isolation of a new metalloprotease with a strict specificity for the amide bonds of peptide substrates having a threonine residue at the P1' position [Biochem. Biophys. Res.

Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa.

The profile of sedimentation on a 4-20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA.