RESUMO
We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.
Assuntos
Reatores Biológicos , Enzimas Imobilizadas/química , Histidina/química , Animais , Enzimas Imobilizadas/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Estrutura Molecular , Propriedades de SuperfícieRESUMO
To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.
Assuntos
Aminoquinolinas/química , Carbamatos/química , Cromatografia em Camada Fina/métodos , Fragmentos de Peptídeos/metabolismo , Tripsina/análise , Tripsina/metabolismo , Animais , Bovinos , Fluorescência , Hemoglobinas/química , Hidrólise , Lycoris/enzimologia , Fragmentos de Peptídeos/química , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Especificidade por SubstratoRESUMO
A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.
