Isolation and Characterization of Glutamyl Endopeptidase 2 from Bacillus intermedius 3-19

Abstract

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

“…The maximum activity was detected for substrates that had Glu in position P1 and Leu or Phe in position P2. If P2 was occupied by Trp, the enzyme activity was significantly reduced (Balaban et al, 2003). Similar results were previously reported for glutamyl endopeptidase Staphylococcus aureus and B. subtilis, both of which were 10 times more active towards substrates with Leu in position P2 (Niidome et al, 1990).…”

“…Glutamyl endopeptidases from Staphylococcus aureus and B. licheniformis hydrolyse synthetic substrates with Glu 1000 times faster than those with Asp in position P1 (Breddam and Meldal, 1992). Moreover, after 30 min of incubation early glutamyl endopeptidase from B. intermedius hydrolyses 100% of Glu-containing substrate, but only 5.6% of substrates with Asp (Leshchinskaya et al, 1997a), while late glutamyl endopeptidase from B. intermedius has a 100 times faster hydrolysis of peptide bonds formed by glutamic acid (Balaban et al, 2003). The amino acids in position P2 usually also have an effect, albeit less critical, on the rate of substrate hydrolysis.…”

“…Both fractions contain 274 and 272 amino acids, correspondingly, with minor changes in the number of particular amino acids. Since both enzymes from B. amyloliquefaciens and glutamyl endopeptidase from B. intermedius are recognised by the same antiserum, we also included B. intermedius enzyme in our com- A B parison (Leshchinskaya et al, 1997a;Balaban et al, 2003). The amino acid composition of the proteins is mostly different for Ile, Gly, and Tyr.…”

“…The cysteic acids are likely formed by oxidation of corresponding cysteines. Glutamyl endopeptidases from B. subtilis (Okamoto et al, 1997) and B. intermedius (Leshchinskaya et al, 1997a;Balaban et al, 2003) display similar substrate specificity. We conclude that glutamyl endopeptidase, purified on different stage of bacterial growth B. amyloliquefaciens have identical specificity on B-chain of oxidised insulin.…”

“…These values lie in the same range as pH-optima reported for glutamyl endopeptidases from Acitomyces sp. (Mosolova et al, 1987), Streptomyces fradiae (Kitadokoro et al, 1993), B. intermedius (Leshchinskaya et al, 1997a;Balaban et al, 2003). Interestingly, the range of pHstability for early glutamyl endopeptidase (pH 7.2-9.5) is broader than that of late glutamyl endopeptidase (pH 7.2-8.5).…”