L-Arginine 4-nitroanilide dihydrochloride

The glycopeptidase GCP and its homologue proteins are conserved and essential for survival of bacteria. The ygjD gene (Glycopeptidase homologue) was cloned from Vibrio harveyi strain SF-1. The gene consisted of 1,017 bp, which encodes a 338 amino acid polypeptide. The nucleotide sequence similarity of the ygjD gene with that of V. harveyi FDAARGOS 107 was 95%. The ygjD gene also showed similarities of 68%, 67% and 50% with those of Salmonella enterica, Escherichia coli and Bacillus cereus. The ygjD gene was expressed in E. coli BL21 (DE3) and the recombinant YgjD was purified by Ni2+ affinity chromatography column. The purified YgjD showed a specific 37 kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and exhibited protease activities of 59,000 units/mg, 53,700 units/mg and 8,100 units/mg, respectively, on N-Acetyl-L-tyrosine ethyl ester monohydrate (ATEE), N-Benzoyl-L-tyrosine ethyl ester (BTEE) and N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) substrates. When the conserved amino acids of His111, Glu113 and His115 in the YgjD were replaced with alanine, respectively, the protease activities of the mutants were partly decreased. The two conserved His111 and His115 of YgjD were mutated and the protein lost the protease activity, which implied that the two amino acid played very important roles in maintaining its protease activity. The addition of the purified YgjD to the culture medium of V. harveyi strain SF-1 can effectively promote the bacteria growth. These results indicated that the protease activities may be involved in the survival of bacteria.

The aims of this study were to, first, determine the intracellular aminopeptidase activity (APEi) and second, purify and biochemically characterize one intracellular aminopeptidase enzyme from the phytopathogen fungus Sporisorium reilianum (psrAPEi), the causal agent of head smut in corn. The fungus produced APEi activity in all media cultures evaluated. The psrAPEi was purified by a procedure that involved ammonium sulfate fractionation and four chromatographic steps using an FPLC system (Fast Protein Liquid Chromatography). Results showed an estimated molecular mass of 52.2 kDa. Enzymatic activity was optimal at pH 7.0 and 35 °C and was inhibited by EDTA-Na2, 1,10-phenanthroline, bestatin, and PMSF. This aminopeptidase showed a preference for leucine, arginine, and lysine at the N-position. The Km and Vmax values were 3.72 μM and 188.0 μmol/min, respectively, for L-lysyl-4-nitroanilide. This is the first study to report on intracellular aminopeptidase activity in S. reilianum and the purification and characterization of an intracellular metallo-serine-aminopeptidase (psrAPEi).

Trypsin (EC 3.4.21.4) was successfully immobilized on the surface of Fe3O4 magnetic nanoparticles that had been pre-treated with gallic acid (GA). Measurements of protein load by using Bradford assay and the trypsin-catalyzed hydrolysis of Nα-Benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) were made for the immobilized enzyme. By using magnetic nanoparticles, which provides easy separation and decent support material for enzyme immobilization with high surface area to volume ratio, and by employing biocompatible material gallic acid, immobilized enzyme system was synthesized along with improving trypsin activity and stability. Immobilized trypsin (TR) was more stable than the free one and demonstrated higher enzymatic activity at elevated temperatures (45-55°C) and in the alkaline pH region (6-10.5). Fe3O4 NPs-GA-TR retained 92% of its initial activity after 120days of storage at 4°C in sodium phosphate buffer (0.1M, pH 7.5), whereas the free trypsin maintained about 64% of its initial activity during the same storage period. In addition, activity of the immobilized trypsin was preserved 54.5% of its initial activity after eight times successive reuse. The Michaelis-Menten kinetic constant (Km) and maximum reaction velocity (Vmax) for free trypsin were 5.1mM and 23mM/min, respectively, whereas Km and Vmax values of immobilized trypsin were 7.88mM and 18.3mM/min, respectively. The performance of the immobilized trypsin was demonstrated by carrying out the hydrolysis of bovine serum albumin (BSA) within 1h, and the assay was performed by using liquid chromatography-mass spectrometry (LC-MS/MS) technique. The hydrolysis of bovine milk as a real food was investigated by immobilized trypsin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).