Spatial requirement for PAMO for transformation of non-native linear substrates.

Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, and therefore it is an ideal candidate for the synthesis of industrially relevant ester or lactone compounds. However, its limited substrate scope has largely limited its industrial applications. Linear substrates are interesting from an industrial point of view, it is thus necessary to identify the essential spatial requirement for achieving high conversions for non-native linear substrates.

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations.

Production of Galacto-oligosaccharides by the β-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme.

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K.

Screening β-fructofuranosidases mutant libraries to enhance the transglycosylation rates of β-(2→6) fructooligosaccharides.

β-Fructofuranosidases can divert their hydrolytic activity towards transglycosylation for the synthesis of high value-added products, including prebiotic fructooligosaccharides (FOS). A directed evolution strategy has been employed to enhance the transferase rate of the β-fructofuranosidase (SoINV) from the Schwanniomyces occidentalis yeast for the production of β-(2→6)-linked FOS. To screen for transferase activity of the SoINV functionally expressed in Saccharomyces cerevisiae, a high-throughput screening protocol based on two colorimetric assays was validated (with coefficient of variance below 11%).

New insights into the fructosyltransferase activity of Schwanniomyces occidentalis ß-fructofuranosidase, emerging from nonconventional codon usage and directed mutation.

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k(cat)/K(m)) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis.

Characterization of a beta-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose.

beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)).