Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purifie d to homogeneity using immobilized metal affinity chromatography. The enzym e was characterized as a metalloprotease with an approximate relative molec ular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (5 0 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-p henanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzy me could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gl y-D-Phe did not inhibit Chryseobacretium indologenes Ix9a protease. Heat in activation did not follow first order kinetics, but showed biphasic inactiv ation curves. The protease has a K-m of 0.813 mu g . ml(-1) for casein as s ubstrate. Amino acid analysis showed that the protease contains a high amou nt of small amino acids like glycine, alanine, and serine, but a low concen tration of methionine and no cysteine at all. Electrospray mass spectrometr y of proteolysis fragments formed when insulin B chain was hydrolyzed showe d cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction. (C) 1999 Academic Press.