Specific detection of L-glutamate in food using flow-injection analysis and enzymatic recycling of substrate

A flow injection analysis (FIA) system for specific determination of L-glutamate in food samples based on a bi-enzymatic amplification system has been developed. The content of L-glutamate in the sample was amplified by cycling between L-glutamate dehydrogenase (GIDH) and a novel enzyme, D-phenylglycine aminotransferase (D-PhgAT). In this system, GIDH converts L-glutamate to 2-oxoglutarate with concomitant reduction of NAD(+) to NADH. D-PhgAT transfers an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate, thus recycling L-glutamate. Accumulation of NADH in the course of the enzymatic recycling was monitored both by fluorescence and UV absorbance and used for quantification of L-glutamate. The assay was characterized by high long-term stability (at least 70 days) and good reproducibility (within-day and between-day RSDs were 4.3-7.3% and 8.9%). The fluorimetric assay was slightly more sensitive with a L-glutamate detection limit of 0.4 muM and linear range of 2.5-50 muM. The assay was specific for L-glutamate, with recoveries between 95-103% in the presence of 17 different amino acids tested one by one. The method was applied to analysis of real food samples and results were correlated with a commercial Boehringer Mannheim assay kit. (C) 2004 Elsevier B.V. All rights reserved

Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement

Supplementary Material for: Effective Improvement of <i>D</i>-Phenylglycine Aminotransferase Solubility by Protein Crystal Contact Engineering

Structure-guided genetic engineering of <i>D</i>-phenylglycine aminotransferase (<i>D</i>-PhgAT) aimed at increasing protein solubility was attempted. In silico analyses predicted the Asn439 and Gln444 as highly solvent-exposed β-turn residues involved with protein crystal contact (CC) potential candidates for solubility-improving mutations. They were replaced with Asp and Glu creating the N439D and Q444E single mutants, and N439D/Q444E double mutant with 2.5-, 3.3- and 5.9-fold increases in solubility, respectively. The protein CC prevention effect rather than the net charge effect accounted for the dramatically improved solubility since the N439D, Q444E and N439D/Q444E mutations altered the isoelectric point of <i>D</i>-PhgAT by only 0.1, 0.1 and 0.3 units, respectively. Examination of the <i>D</i>-PhgAT structural model revealed that the N439D mutation weakened the CC attraction force and the Q444E mutation created electrostatic repulsion at the CC point. Analysis of circular dichroism spectra, melting temperature, and <i>D</i>-PhgAT-specific activity showed that the mutations posed no unfavorable effect on the conformational stability and catalytic performance of the enzyme. The protein solubility-improving strategy employed on <i>D</i>-PhgAT in this study was successful with minimal protein structure modification required. It should be applicable with a high chance of success for other proteins, especially those with 3-D structural models available