Purification and characterization of a unique alkaline elastase from Micrococcus luteus.
Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.
Clark
D J
DJ
Departments of Biological and Biomedical Sciences, Quinnipiac College, Hamden, Connecticut 06518, USA. clark@quinnipiac.edu
Hawrylik
S J
SJ
Kavanagh
E
E
Opheim
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D016982:Micrococcus luteus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012867:Skin / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature
D015842:Serine Proteinase Inhibitors; D010196:Pancreatic Elastase