Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp.
An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C). The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++. An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.
Prescott
M
M
Thermophile Research Unit, University of Waikato, Hamilton, New Zealand.
Peek
K
K
Daniel
R M
RM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D004795:Enzyme Stability; D004852:Epoxy Compounds / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000494:pharmacology; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D004852:Epoxy Compounds; D009596:Nitrophenols; D011480:Protease Inhibitors; C000909:N-diazoacetylnorleucine methyl ester; D009646:Norleucine; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane; D016282:Aspartic Acid Endopeptidases