Synthesis of b(1 ? 3) and b(1 ? 6) galactooligosaccharides from lactose and whey using a recombinant b-galactosidase from Pantoea anthophila

BACKGROUND: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by bgalactosidases. RESULTS: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with b(1 ? 6) and b (1 ? 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. CONCLUSIONS: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues / Bacillus subtilis con actividades de endocelulasa y exocelulasa aislado en la fase termofílica del compostaje con residuos de café

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57 -#9702;C and pH 5.5 and the other, a temperature of 61 -#9702;C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24 h until 144 h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

El objetivo de este estudio fue aislar, seleccionary caracterizar bacterias con actividad celulolítica a partir de 2 diferentes pilas de compostaje de residuos de café, una con temperatura interna de 57°C y pH 5,5; la otra con temperatura interna de 61 °C y pH 9,3. Se utilizaron medios de cultivo con carboximetilcelulosa y celulosa cristalina como únicas fuentes de carbono. La actividad enzimàtica fue evaluada por formación de halos de hidrólisis, producción de azúcares reductores y zimogramas. De 20 cepas aisladas, 3 presentaron mayor actividad enzimàtica y fueron identificadas como Bacillus subtilis sobre la base de sus características morfológicas, fisiológicas y bioquímicas y del análisis de las secuencias de la región 16S del ADNr. Los extractos enzimáticos de las 3 cepas seleccionadas presentaron actividad de exocelulasa y de endocelulasa, con máximos de 0,254 y 0,519 U/ml, respectivamente; la actividad de estas enzimas se mantuvo incluso a pH ácido (4,8) o básico (9,3) y a temperaturas de hasta 60 °C. Las actividades enzimáticas halladas en este estudio se ubican dentro de las más altas reportadas para celulasas producidas por bacterias del género Bacillus. En los zimogramas se demostró actividad de endocelulasa desde las 24h hasta las 144h de incubación. Asimismo, se reporta por primera vez el efecto del pH sobre la actividad de endocelulasa observado por zimogramas. Los resultados de este estudio abren la posibilidad de hacer uso de estas enzimas en la obtención de sustratos fermentables para la producción de energía a partir de los residuos generados en grandes cantidades por la agroindustria del café.