The molecular basis of Sanfilippo syndrome type B.

The Sanfilippo syndrome type B is a lysosomal storage disorder caused by deficiency of alpha-N-acetylglucosaminidase; it is characterized by profound mental deterioration in childhood and death in the second decade. For understanding the molecular genetics of the disease and for future development of DNA-based therapy, we have cloned the cDNA and gene encoding alpha-N-acetylglucosaminidase. Cloning started with purification of the bovine enzyme and use of a conserved oligonucleotide sequence to probe a human cDNA library. The cDNA sequence was found to encode a protein of 743 amino acids, with a 20- to 23-aa signal peptide immediately preceding the amino terminus of the tissue enzyme and with six potential N-glycosylation sites. The 8.5-kb gene (NAGLU), interrupted by 5 introns, was localized to the 5'-flanking sequence of a known gene, EDH17B, on chromosome 17q21. Five mutations were identified in cells of patients with Sanfilippo syndrome type B: 503del10, R297X, R626X, R643H, and R674H. The occurrence of a frameshift and a nonsense mutation in homozygous form confirms the identity of the NAGLU gene.

Atividade β-D-N-Acetilglucosaminidásica de enzimas imobilizadas extraídas da Artemia franciscana e possíveis aplicações biotecnológicas

A β-D-N-acetilglucosaminidase extracted and partially isolated from crustacean Artemia franciscana by ammonium sulfate precipitation and filtration gel chromatography Bio Gel A 1.5m. the enzyme was immobilized on ferromagnetic Dacron yielding a insoluble active derivative with 5.0 units/mg protein and 10.35% of the soluble enzyme activity. β-D-N-acetilglucosaminidase-ferromagnetic Dacron was easily removed from the reaction mixture by a magnetic field, it was reused for ten times without loss in its activity. The ferromagnetic Dacron was better activated at pH 5.0. The particles visualized at scanning electron microscope (SEM) had presented different sizes, varying between 721nm and 100µm. Infra red confirmed immobilization on support, as showed by primary amino peaks at 1640 and 1560 cm-1 . The immobilize enzyme presented Km of 2.32 ± 0.48 mM and optimum temperature of 50°C. Bought presented the same thermal stable of the soluble enzyme and larger enzymatic activity at pH 5.5. β-D-N-acetilglucosaminidase-Dacron ferromagnético showed sensible for some íons as the silver (AgNO3), with loss of activity. The β-D-N acetilglucosaminidase activity for mercury chloride (HgCl2), whom is one of the most toxic substance joined in nature, it was presented activity already diminished at 0,01mM and lost total activity at 4mM, indicating sensitivity for this type of metal. β-D-N-acetilglucosaminidase-ferromagnetic Dacron showed degradative capacity on heparan sulfate, the enzyme still demonstrated degradative capacity on heparan sulphate, suggesting a possible application to produce fractions of this glycosaminoglycan

Microbial community composition shapes enzyme patterns in topsoil and subsoil horizons along a latitudinal transect in Western Siberia

Soil horizons below 30 cm depth contain about 60% of the organic carbon stored in soils. Although insight into the physical and chemical stabilization of soil organic matter (SUM) and into microbial community composition in these horizons is being gained, information on microbial functions of subsoil microbial communities and on associated microbially-mediated processes remains sparse. To identify possible controls on enzyme patterns, we correlated enzyme patterns with biotic and abiotic soil parameters, as well as with microbial community composition, estimated using phospholipid fatty acid profiles. Enzyme patterns (i.e. distance-matrixes calculated from these enzyme activities) were calculated from the activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase), which had been measured in soil samples from organic topsoil horizons, mineral topsoil horizons, and mineral subsoil horizons from seven ecosystems along a 1500 km latitudinal transect in Western Siberia. We found that hydrolytic enzyme activities decreased rapidly with depth, whereas oxidative enzyme activities in mineral horizons were as high as, or higher than in organic topsoil horizons. Enzyme patterns varied more strongly between ecosystems in mineral subsoil horizons than in organic topsoils. The enzyme patterns in topsoil horizons were correlated with SUM content (i.e., C and N content) and microbial community composition. In contrast, the enzyme patterns in mineral subsoil horizons were related to water content, soil pH and microbial community composition. The lack of correlation between enzyme patterns and SUM quantity in the mineral subsoils suggests that SOM chemistry, spatial separation or physical stabilization of SUM rather than SUM content might determine substrate availability for enzymatic breakdown. The correlation of microbial community composition and enzyme patterns in all horizons, suggests that microbial community composition shapes enzyme patterns and might act as a modifier for the usual dependency of decomposition rates on SUM content or C/N ratios. (C) 2015 The Authors. Published by Elsevier Ltd.