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Aims: The study of molecular mechanisms related to obesity and associated pathologies like type 2-diabetes and non-alcoholic fatty liver disease requires animal experimental models in which the type of obesogenic diet and length of the experimental period to induce obesity deeply affect the metabolic alterations. Therefore, this study aimed to test the influence of aging along a rat model of diet-induced obesity in gene expression of the hepatic transcriptome., Main Methods: A high-fat/high-fructose diet to induce obesity was used. Mid- (13 weeks) and long-term (21 weeks) periods were established. Caloric intake, bodyweight, hepatic fat, fatty acid profile, histological changes, antioxidant activity, and complete transcriptome were analyzed., Key Findings: Excess bodyweight, hepatic steatosis and altered lipid histology, modifications in liver antioxidant activity, and dysregulated expression of transcripts related to cell structure, glucose & lipid metabolism, antioxidant & detoxifying capacity were found. Modifications in obese and control rats were accounted for by the different lengths of the experimental period studied., Significance: Main mechanisms of hepatic fat accumulation were de novo lipogenesis or altered fatty acid catabolism for mid- or long-term study, respectively. Therefore, the choice of obesity-induction length is a key factor in the model of obesity used as a control for each specific experimental design., Competing Interests: Declaration of competing interest At the time of submission, the authors have no competing interests (financial, professional or personal) that are relevant to the submitted work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.

Proteolysis of two purified recombinant enzymes, namely, the Aspergillus niger phytase (r-PhyA) and the Escherichia coli pH 2.5 acid phosphatase (r-AppA), by pepsin and trypsin was investigated in this study. After r-PhyA and r-AppA were incubated with different concentrations of pepsin or trypsin, their residual phytase activities and amounts of inorganic phosphorus released from soybean meal were determined. Both enzymes retained more than 85% of their original activities at the trypsin/phytase ratios (w/w) 0.001 and 0. 005, while r-AppA and r-PhyA lost 60 and 20% of the original activity at the ratio of 0.01 or 0.025, respectively. In contrast, there was a 30% increase in phytase activity after r-AppA was incubated with pepsin at the ratios of 0.005 or 0.01. Meanwhile, r-PhyA lost 58 to 77% of its original activity under the same conditions. Trypsin and pepsin affected the hydrolysis of phytate phosphorus from soybean meal by r-AppA and r-PhyA in a similar way to their residual phytase activities. All of these in vitro proteolyses were confirmed by SDS-PAGE analysis. Our results demonstrate different sensitivities of r-AppA and r-PhyA to trypsin and pepsin, suggesting active trypsin resistant r-PhyA and pepsin resistant r-AppA polypeptides., (Copyright 1999 Academic Press.)