Efficient and rapid purification of lentil -galactosidase by affinity precipitation with alginate.

-Galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40oC and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65oC and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl--D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of -galactosidase.

Culture conditions for the production of a-galactosidase by Aspergillus parasiticus MTCC-2796: a novel source / Condiciones del cultivo para la producción de a-galactosidasa por Aspergillus parasiticus MTCC-2796: una nueva fuente

Aspergillus parasiticus microbial type culture collection (MTCC)-2796, a new source of a-galactosidase is an efficient producer of enzyme in basic medium under submerged fermentation conditions. Maximum a-galactosidase production (156.25 Uml-1) was obtained when the basic medium is supplemented with galactose (0.5 percent w/v) and raffinose (0.5 percent w/v) as carbon source and yeast extract as nitrogen source. Enzyme production was also enhanced considerably in the presence of wheat bran (1.0 percent w/v). Enzyme secretion was strongly inhibited by the presence of Hg2+, Cu2+, and Co2+ in the medium and to some extent by Zn2+ and Ni2+, while marginal increase in the enzyme production was observed when Mg2+ and Mn2+ were added in the medium. Among amino acids checked (aparagine, cysteine, glutamine, leucine and proline), glutamine (1 mM) was found to be an enhancer for the enzyme production. The temperature and pH range for the production of enzyme were 25ºC to 35ºC and 6.5 to 7.5, respectively with maximum activity (50 Uml-1) at 30ºC and pH 6.5 under static fermentation condition.

Fabry disease: A treatable lysosomal storage disorder.

Fabry disease is a lysosomal storage disease with an X-linked inheritance pattern, which presents in childhood as acroparaesthesias. Its non-specific symptoms often lead to delays in the diagnosis. We report the case of a 13-year-old boy who presented with typical acroparaesthesia of Fabry disease, his younger brother had gastrointestinal manifestations of the disease and their mother’s symptoms suggested that she is a carrier. Enzyme replacement therapy helped in ameliorating the patient’s symptoms and preventing complications such as renal failure, stroke and cardiovascular disorders.

Reduction of non-digestible oligosaccharides in soymilk: application of engineered lactic acid bacteria that produce alpha-galactosidase

Human consumption of soy-derived products has been limited by the presence of non-digestible oligosaccharides (NDO), such as the alpha-galactooligosaccharides raffinose and stachyose. Most mammals, including man, lack pancreatic alpha-galactosidase (alpha-Gal), which is necessary for the hydrolysis of these sugars. However, such NDO can be fermented by gas-producing microorganisms present in the cecum and large intestine, which in turn can induce flatulence and other gastrointestinal disorders in sensitive individuals.The use of microorganisms expressing alpha-Gal is a promising solution to the elimination of NDO before they reach the large intestine. In the present study, lactic acid bacteria engineered to degrade NDO have been constructed and are being used as a tool to evaluate this solution. The alpha-Gal structural genes from Lactobacillus plantarum ATCC8014 (previously characterized in our laboratory) and from guar have been cloned and expressed in Lactococcus lactis. The gene products were directed to different bacterial compartments to optimize their possible applications. The alpha-Gal-producing strains are being evaluated for their efficiency in degrading raffinose and stachyose: i) in soymilk fermentation when used as starters and ii) in situ in the upper gastrointestinal tract when administered to animals orally, as probiotic preparations. The expected outcomes and possible complications of this project are discussed.