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 purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed 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.
Venter
H
H
Department of Food Science, University of the Orange Free State, Bloemfontein, South Africa.
Osthoff
G
G
Litthauer
D
D
D000595:Amino Acid Sequence; D000596:Amino Acids; D001051:Apoenzymes / Q000378:metabolism; D002118:Calcium / Q000032:analysis; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005417:Flavobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018629:Spectrometry, Mass, Secondary Ion; D013379:Substrate Specificity; D013816:Thermodynamics; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D001051:Apoenzymes; D002413:Cations, Divalent; D010446:Peptide Fragments; D011480:Protease Inhibitors; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium