Record Details
Physical and chemical properties of some Bdellovibrio extracellular proteases
ScholarsArchive at Oregon State University
| Field | Value |
|---|---|
| Title | Physical and chemical properties of some Bdellovibrio extracellular proteases |
| Names |
Klubek, Brian Paul
(creator) Seidler, Ramon J. (advisor) |
| Date Issued | 1973-09-11 (iso8601) |
| Note | Graduation date: 1974 |
| Abstract | 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 is observed. 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 at 7.75. 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 is 32,000. |
| Genre | Thesis/Dissertation |
| Topic | Proteolytic enzymes |
| Identifier | http://hdl.handle.net/1957/46091 |
