New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 studied to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by $$\sim$$ ∼ 30% and 48%, respectively. Moreover, it produced neokestose, which represented $$\sim$$ ∼ 27% of total FOS, and yielded 31.8 g l−1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.

Enzymatic synthesis of novel fructosylated compounds by Ffase from Schwanniomyces occidentalis in green solvents

Glycerol, as a good acceptor for Ffase, produces novel fructosylated derivatives with biotechnological potential.

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 revised to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by `30% and 48%, respectively. Moreover, it showed a shift towards neokestose, which represented ~27% of total FOS, and yielded 31.8 g l-1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.

Análisis del genoma de Pycnoporus cinnabarinus mediante electroforesis de campo pulsado

En el presente trabajo se describe la separación óptima de moléculas de DNA cromosómico intacto del hongo filamentoso Pycnoporus cinnabarinus, mediante eléctroforesis de campo pulsado usando un sistema de contorno de campo eléctrico homogéneo (CHEF), con un electrodo hexagonal.Se estudió una serie de condiciones experimentales en las cuales se hizo variar la duración del intervalo de tiempo en cada dirección del campo y el tiempo total de la corrida.De esta manera fue posible separar el DNA cromosómico de P. cinnabarinus en cuatro bandas que, dependiendo e las condiciones de electroforesis,pueden ser resueltas por lo menos en siete bandas. Utilizando los patrones de migración del DNA cromosómico de Saccharomyces cerevisiae y Schwanniomyces occidentalis comoestándar, estimamos que los tamaños moleculares del DNA cromosómico de P. cinnabarinus oscilan entre 1.9 y 2.5 megabases (Mb).