ABSTRACT
Optimized acetyl esterase enzyme production conditions using Aspergillus niger ATCC 10864 in 14-L fermentation jars were determined to be 33 degrees C, 1.5 vvm aeration, and 300 rpm agitation without pH control. The acetyl esterase was purified by precipitation in 60-80% saturation in ammonium sulfate. The pellet was applied directly to a Pharmacia high-load Phenyl Sepharose column for hydrophobic interaction chromatography and purified to homogeneity in two steps. Stability and kinetic characteristics of the acetyl esterase were determined over a pH range of 4.0-7.5 and from 4 to 45 degrees C. At temperatures > 25 degrees C, stability was superior at pH values < 5.0. The temperature activity optimum was 35 degrees C, and the pH optimum was 7.0. The Vmax was determined to be 46,700 U/mg protein, and the Km was 0.023M p-nitrophenyl acetate at pH 6.5 in 0.2M phosphate buffer at 35 degrees C. The mol wt of the enzyme was 35,000 dalton by size-exclusion chromatography and SDS gel electrophoresis. The N-terminal amino acid sequence and the glycosylation composition were also determined.
