Identification and characterization of cellobiose 2-epimerases from various aerobes.

Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of β-1,4-linked oligosaccharides to D-mannose residues. In this study, we characterized CE-like proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and β-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates.

Immobilization of a thermostable cellobiose 2-epimerase from Rhodothermus marinus JCM9785 and continuous production of epilactose.

Cellobiose 2-epimerase (CE) efficiently forms epilactose which has several beneficial biological functions. A thermostable CE from Rhodothermus marinus was immobilized on Duolite A568 and packed into a column. Lactose (100 g/L) was supplied to the reactor, kept at 50 °C at a space velocity of 8 h(-1).

α-Glucosylated 6-gingerol: chemoenzymatic synthesis using α-glucosidase from Halomonas sp. H11, and its physical properties.

6-Gingerol [(S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one] is a biologically active compound and is abundant in the rhizomes of ginger (Zingiber officinale). It has some beneficial functions in healthcare, but its use is limited because of its insolubility in water and its heat-instability. To improve these physical properties, the glucosylation of 6-gingerol was investigated using α-glucosidases (EC.

Characterization of Halomonas sp. strain H11 α-glucosidase activated by monovalent cations and its application for efficient synthesis of α-D-glucosylglycerol.

An α-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R.

Emulsion culture: a miniaturized library screening system based on micro-droplets in an emulsified medium.

A typical library screen in directed evolution primarily requires physical separation of the clones on agar plates followed by detection of clones with improved properties; using this method only limited numbers of clones relative to the number of potential variations can be assessed. In particular, screening for a secretory enzyme is difficult to perform at high clone density, because of diffusion of the signal or unfavorable utilization of the reaction product by neighboring clones. In this study, we have developed a novel method of enrichment culture: "Emulsion Culture", i.

Quantitative Y2H screening: cloning and signal peptide engineering of a fungal secretory LacA gene and its application to yeast two-hybrid system as a quantitative reporter.

A quantitative protein/peptide screening system amenable to high-throughput screening has been developed by furnishing conventional yeast two-hybrid (Y2H) system with an engineered fungal secretory beta-galactosidase gene (designated LacA3). We describe the molecular cloning and signal peptide-optimization of the original fungal LacA gene of which extracellular expression was initially toxic to the host cell. The engineered LacA, LacA3, showed less toxicity, resulting in improved cultural properties of the host.