Title page for ETD etd-09062000-11440034

Type of Document Master's Thesis
Author Jewell, Sally Nicole
URN etd-09062000-11440034
Title Purification and characterization of a novel protease form Burkholderia strain 2.2N
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Falkinham, Joseph O. III Committee Co-Chair
Potts, Malcolm Committee Co-Chair
Hagedorn, Charles III Committee Member
  • Pseudomonas
  • protease
  • protein purification
  • Burkholderia
Date of Defense 2000-08-23
Availability unrestricted
The bacterium Burkholderia strain 2.2 N is a soil isolate and a member of a group of non-obligative predator bacteria that can prey on other microorganisms or grow saprophytically. The bacterium has anti-bacterial, anti-fungal, anti-yeast and anti-protozoan activities. Burkholderia strain 2.2 N culture shows hydrolysis on Milk Casein Agar, indicating the bacterium also produces a protease.

Azocoll hydrolysis was used to detect and measure protease activity. Protease activity was two-fold higher at pH of 7.5 than pH 9.0 and 25-fold at pH 4.0. Cultures grown in media containing 1.0 % yeast extract (YE), tryptic soy, tryptone or beef extract had protease activity, whereas activity was absent in cultures grown in media containing peptone, soytone, casitone, or tryptone as sole protein source. Addition of 1.0 % sucrose or glucose to 1.0 % YE medium increased protease activity 1.8-fold and 1.4-fold, respectively. Protease activity was 2-fold higher in cultures grown in media containing 1.0 % YE and 10 mM MgCl2 or FeCl2, than in 1.0 % YE medium lacking metals or containing 10 mM MnCl2 or CaCl2. The 1.0 % YE medium containing either ZnCl2 or CuCl2 lacked protease activity (< 5.0 %). In cultures grown in 1.0 % YE at 30°C with rotation at 120 rpm, protease activity was higher in stationary phase (0.38 units /mg protein) than in exponential phase (0.04 units/mg protein). The Burkholderia strain 2.2 N protease is evidently exported from cells because 86 % of the total proteolytic activity of cells was found in the cell-free culture medium.

The cell free filtered culture supernatant medium assayed at 4°C had protease activity, however at a three-fold lower specific activity compared to the same supernatant assayed at 3°C. Protease activity was lower in filtered culture supernatants stored at 4°C, room temperature, and 30°C. Protease activity in samples stored at 4°C was only 40 % (24 hours) and 15 % (48 hours) of activity at time zero. Protease activity in samples stored at room temperature was only 45 % (24 hours) and 35 % (48 hours) of activity at time zero. Protease activity in samples stored at 30°C was only 78 % (24 hours) and 9 % (48 hours) of activity at time zero.

Purification of the protease from filtered culture supernatant medium by ammonium sulfate precipitation, increased the protease activity 20-fold. An eluted protein fraction from DEAE-Sepharose column chromatography had 50-fold higher protease activity. Protease activity was inhibited by 10 mM 1-10-phenathroline, EDTA and EGTA, all metalloprotease inhibitors. Purified protease activity inactivated with 10 mM 1-10-phenanthroline or 10 mM EGTA was regained through the addition of Ca2+ or Mg2+. Protease activity was reduced by exposure to dithiothreitol (29 % with 1 mM and 84 % with 10 mM), a disulfide bond inhibitor. Protease activity was not inhibited by leupeptin or phenylmethylsulphonyl flouride.

Casein polyacrylamide zymography revealed a band of hydrolysis at approximately 60,000 Da. SDS-PAGE resolved a doublet band present at 60,000 Da present in both the filtered culture supernatant sample and the ammonium sulfate / DEAE-Shepharose column chromatography purified protease sample.

Burkholderia strain 2.2 N protease is a metalloprotease with a broad temperature range of activity. It has a molecular weight of approximately 60,000 Da.

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