- Bdellovibrio bacteriovorus 100 and B. stolpii UKi-2 each produce
two extracellular proteases which attack Azocoll (collagen)
while B. starrii A3.12 produces at least four such proteolytic
enzymes. Under standard growth and assay conditions each Bdellovibrio
species produces a unique amount of proteolytic activity. The
decreasing order of enzyme units was B. starrii > B. stolpii > B.
bacteriovorus in a ratio of 10:5:1.
When ammonium sulfate precipitated (ASP) partially purified
Bdellovibrio proteases are electrophoresed, bands of proteolytic
activity can be developed on casein agar mounts. Each species produced
unique and different zyrnogram patterns at each pH of 7.3,
8.3, and 9.0. The electrophoretic elution profile (pH 8.3) of the
proteases from each species is also unique. There are two types of proteolytic enzymes produced by the
Bdellovibrio. One enzyme (metallo protease) is inhibited by 10 mM
EDTA, and the other (serine protease) is inhibited by 1 mM phenlymethylsulfonylflouride
(PMSF). Both the metallo and the serine proteases
are stimulated or inhibited by a variety of charged molecules.
These include Tris-glycine (5mM tris, 38 mM glycine), EDTA (10
mM), PMSF (1 mM), Tris (10 mM), Ca⁺⁺ (2mM), Mg⁺⁺ (3mM), and
various amino acids (glycine, alanine, cysteine, glutamic acid, and
arginine, 20 mM each).
When the purified H-I B. bacteriovorus 100 metallo and serine
proteases are dialyzed against Tris-HC1, 49 and 12 percent loss of
activity is observed respectively. A complete recovery in proteolytic
activity is obtained upon the additon of both Ca⁺⁺ and Mg⁺⁺. No
loss in activity occurs when these same enzymes are dialyzed
against pH 7.75 Tris-glycine (5 mM tris, 38 mM glycine). Dialysis
against double distilled water results in an 80 and 49 percent loss in
activity of the metallo and serine enzymes, and upon the addition of
Tris, Ca⁺⁺ and Mg⁺⁺ only a partial recovery in proteolytic activity
Cysteine and Tris-glycine were found to be stimulatory for
both the H-I B. bacteriovorus 100 proteases. Hydrogen peroxide
(5 mM) does not cause any inhibition in proteolytic activity.
The serine proteases of both H-I B. bacteriovorus 100 and H-I B. stolpii UKi-2 have an optimum pH of about 8.0 - 8.1. The
metallo enzymes differ in that the H-I B. bacteriovorus 100 has an
optimum pH at 7.5, while the H-I B. stolpii UKi-2 has an optimum
The H-I B. bacteriovorus 100 metallo protease has an optimum
temperature at 43.5 - 44°C and a Km of 4.8 x 10⁻⁵ M using
Azocoll as substrate, while the serine protease has a temperature
optimum of about 41.5 - 42°C and a Km value of 3.8 x10⁻⁵ M.
The molecular weights of the H-I B. bacteriovorus 100 proteases
were also determined. The metallo enzyme has a molecular
weight of about 50,000 while the molecular weight of the serine protease