Kouichi Miyata

Serratia Protease: Part I. Purification and General Properties of the Enzyme†

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease pro...

Serratia Protease:Part III. Characteristics of the Enzyme as Metalloenzyme

Protease from a strain of Serratia contained one gram atom of zinc ion per mole and the zinc ion was essential for the activity. Also zinc-free apoenzyme was isolated as a crystalline form from the native-enzyme. Several metalloenzymes were prepared by the addition of corresponding metal ions to the apoenzyme. Studies on activities toward the hydrolysis of casein showed that relative activities of native- (zinc), cobalt- and manganese-enzyme were 1.0, 1.2 and 0.8, respectively. Toward the hydrolysis of hippurylleucinamide, however, specific activity of cobalt-enzyme was about 10 times that of the native- (zinc-) enzyme. Spectroscopic studies did not reveal any significant differences in conformations among native-enzyme, apoenzyme and the other metalloenzymes.

Determination of Serratia protease by radioimmunoassay

Abstract A specific, highly sensitive radioimmunoassay has been developed for the determination of Serratia protease. The radioimmunoassay (RIA) was based upon competition of the protease with 125 I-labeled protease for antiprotease, followed by a second antibody to separate bound enzyme from free enzyme. The RIA provided a range of 1 to 10 ng for determining the enzymes under conditions in which the enzymatic activity could not be measured. The assay was completely inhibited in the presence of human plasma. The inhibition resulted from a complex formation of the enzyme with plasma α 2 macroglobulin. By treatment of the complex with acetone, however, the RIA could be achieved.

Serratia Protease:Part II. Substrate Specificity of the Enzyme

The substrate specificity of Serratia protease was determined using various synthetic substrates. The enzyme did not participate in the hydrolysis of di- and tri-peptides except benzoylglycylleucinamide which was split at a limited rate into hippuric acid and leucinamide. The enzyme action on larger peptides was also studied. The enzyme cleaved the gly-leu bond in eledoisin related peptide and the gly-phe bond in bradykinin. The enzyme split oxidized insulin B-chain at twelve different peptide bonds.