Purification and characterization of an acetyl esterase from Aspergillus niger

Journal Article · · Applied Biochemistry and Biotechnology; (United States)

DOI:https://doi.org/10.1007/BF02941813· OSTI ID:6904190

Linden, J; Samara, M; Decker, S; Johnson, E; Boyer, M ^[1]; Pecs, M ^[2]; Adney, W; Himmel, M ^[3]

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1. Colorado State Univ., Fort Collins, CO (United States)
2. Technical Univ. of Budapest (Hungary)
3. National Renewable Energy Lab., Golden, CO (United States)

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 V[sub max] was determined to be 46,700 U/mg protein, and the K[sub m] 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. 14 refs., 6 figs. 1 tab.

• OSTI ID: 6904190
• Journal Information: Applied Biochemistry and Biotechnology; (United States), Journal Name: Applied Biochemistry and Biotechnology; (United States) Vol. 45-46; ISSN ABIBDL; ISSN 0273-2289
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
550700 -- Microbiology
59 BASIC BIOLOGICAL SCIENCES
ENZYME ACTIVITY
ENZYMES
ESTERASES
FRACTIONATION
HYDROLASES
MOLECULAR WEIGHT
ORGANIC COMPOUNDS
PROTEINS
SEPARATION PROCESSES