A pepstatin-insensitive aspartic proteinase from a thermophilic Bacillus sp.
Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q. The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum proteolytic activity was observed at pH 3.5. The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c. Substrate inhibition was observed with both these substrates. The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3. The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.
Toogood
H S
HS
Thermophile Research Unit, University of Waikato, Hamilton, New Zealand.
Prescott
M
M
Daniel
R M
RM
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006454:Hemoglobins / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010436:Pepstatins / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D006454:Hemoglobins; D007328:Insulin; D046911:Macromolecular Substances; D008670:Metals; D010436:Pepstatins; D010455:Peptides; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin