[Show abstract][Hide abstract] ABSTRACT: Using RT-PCR and rapid amplication of cDNA ends (RACE) techniques, a 1345bp full-length cDNA fragment was obtained from Aspergillus sulphureus. The gene, designated MANN, codes for a 383-residue with a calculated mass of 41,389Da. MANN displayed amino acid sequence similarity to the beta-mannanase of Aspergillus aculeatus and Trichoderma reesei, members of the glycoside hydrolase family 5. The recombinant beta-mannanase gene was successfully expressed in a fully active form in Pichia pastoris. Southern blot analysis showed that the recombinant beta-mannanase gene had successfully integrated into the P. pastoris X-33 genome. SDS-PAGE and Western blot assays demonstrated that the recombinant beta-mannanase, a 48kDa glycosylated protein, was secreted into the culture medium. The enzyme had high specific activity toward locust bean gum and an extremely broad pH range of 2.2-8.0. It showed highest activity at pH 2.4 and 50 degrees C and was stable at temperature below 40 degrees C. The K(m) and V(max) values for locust bean gum at 50 degrees C and pH 2.4 are 0.93mgmL(-1) and 344.83Umg(-1), respectively. The isoelectric point of the recombinant protein is 4.89. The enzymatic activity of recombinant A. sulphureus beta-mannanase was not significantly affected by a range of ions or EDTA.
No preview · Article · Mar 2007 · Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Two experiments were conducted to evaluate the effects of dietary energy level and source of oil on leptin mRNA and long form leptin receptor (Ob-Rl) mRNA expression in dorsal, abdominal and visceral adipose tissues in young growing pigs. In experiment one, 15 barrows (initial body weight 15.0 kg) were used to examine the effects of dietary energy levels on leptin mRNA and Ob-Rl mRNA expression. The pigs were randomly allotted to one of three dietary treatments (n=5 per treatment) containing 13.4, 15.1 or 16.7 MJ DE/kg diet for 28 days. Based on the results of experiment one, experiment two was designed to examine the effects of oil sources including soybean oil (rich in n-6 polyunsaturated fatty acids) or fish oil (rich in n-3 polyunsaturated fatty acids) on leptin mRNA and Ob-Rl mRNA expression in the same adipose tissues examined in experiment one. The energy content of these diets was 15.1 MJ/kg. Fourteen barrows (initial weight 20.5 kg) were allocated to either of the two dietary treatments (n=7 per treatment), which was supplemented with either soybean or fish oil (both 5.73% of the diet) and fed to the pigs for 21 days. At the end of both experiments, blood samples were collected to determine plasma leptin and insulin concentrations. Adipose tissues were sampled to determine leptin and Ob-Rl mRNA expression using real-time fluorescence quantification PCR. In experiment one, plasma leptin concentrations were enhanced (P=0.02), and insulin concentrations were decreased (P<0.01) in pigs fed the high-energy diet (16.7 MJ DE/kg). Dorsal adipose tissue leptin mRNA expression was increased by feeding the diet containing 15.1 MJ/kg DE compared with the diets containing 13.4 and 16.7 MJ/kg DE. There was no difference in leptin mRNA expression in abdominal and visceral adipose tissue. In experiment two, there were no differences in plasma leptin and insulin concentrations between pigs fed with either fish oil or soybean oil diets. Nevertheless, fish oil decreased both leptin mRNA and Ob-Rl mRNA expression in dorsal adipose tissues compared with soybean oil (P<0.01). These experiments indicate that the source of oil plays a more potent role in regulation of leptin mRNA expression relative to dietary energy levels by an insulin-independent mechanism. Plasma leptin concentrations may also be regulated by a post-transcriptional mechanism.
No preview · Article · Oct 2006 · Domestic Animal Endocrinology