A novel extracellular subtilisin-like protease from the hyperthermophile Aeropyrum pernix K1: biochemical properties, cloning, and expression.
A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degrees C) with maximal activity at 90 degrees C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl(2) the activity, as a function of the temperature, reached a maximum at 90 degrees C but at 120 degrees C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione- S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.
Catara
G
G
Institute of Protein Biochemistry, CNR, Via Marconi 10, 80125 Naples, Italy.
Ruggiero
G
G
La Cara
F
F
Digilio
F A
FA
Capasso
A
A
Rossi
M
M
D041522:Aeropyrum / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013381:Subtilisins