Simple method for analyzing the purity of protease-containing samples by acid-treatment SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to analyze the purity of a protein. However, it is necessary to denature (via boiling) the samples before subjecting them to electrophoresis. In the case of protease-containing samples, autolysis of the protease can occur, affecting the accuracy of results. In this study, we investigated the methods for analyzing the purity of Dispase I, a thermolysin-like neutral protease. When we analyzed D protease, a neutral metalloprotease component of Dispase I and highly purified Dispase I using the conventional SDS-PAGE method, a large number of bands were detected in both cases. These bands (putative D protease fragments) were assumed to result from autolysis. To inactivate D protease (optimal pH 7-8), 0.05 M sulfuric acid was utilized (pH 0.7-2.5). Using a conventional sample preparation solution, acid-treated Dispase I samples (without boiling) were made, and SDS-PAGE (15% w/v gel) was carried out. Our findings show that autolysis was inhibited under strong acidic conditions, and protein denaturation was achieved by treatment with sulfuric acid and SDS without boiling. Using this modified SDS-PAGE method, the purities of Dispase I and the purified enzyme were determined to be approximately 80% and 98%, respectively. Furthermore, we demonstrated that this method can be applied for the analysis of other samples including non-acidic proteases (e.g., thermolysin, subtilisin, and trypsin) and protease-contaminated samples (a mixed solution of albumin and D protease).

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity.

α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and Agl1 was inhibited in the presence of 1 % glucose, while Foagl1 was more tolerant to glucose than the other two enzymes were. The three fungal isomaltases did not show transglycosylation when isomaltose was used as the substrate and a similar result was observed for AgdC and Agl1 when p-nitrophenyl-α-glucoside was used as the substrate.