Aspergillus FicuumExtracellular pH 6.0 Optimum Acid Phosphatase: Purification, N-Terminal Amino Acid Sequence, and Biochemical Characterization

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Abstract
An extracellular acid phosphatase, pH optimum 6.0 from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using cation exchange chromatography and chromatofocusing steps. SDS-PAGE of the purified enzyme exhibited two stained bands at approximately 82-KDa and 70-KDa. The mobility of the active enzyme in gel permeation chromatography indicated the molecular mass to be about 85-KDa. In the concentrated form the enzyme appeared to be purple, the visible absorption spectrum shows a λmax at 580 nm. On the basis of molecular mass of 82-KDa, the molar extinction coefficient of the enzyme at 280 nm and 580 nm was estimated to be 1.2 × 105 M−1 cm−1 and 1.3 × 103 M−1 cm−1respectively. Judging by chromatofocuslng, the isoelectric point of the enzyme was about 4.9. The purified enzyme was unstable at 70°C. The enzyme was catalytically very active from 55° to 65°C with a maximum activity at 63°C. The Michael 1s constant of the enzyme for p-nitrophenylphosphate was 200 μM with a computed Kcat of 260 per sec. Although the enzyme was insensitive to fluoride, tartrate, and N-ethylmaleimide (NEM), it was competitively inhibited by phosphomycin (Ki=1.00 mM) and inorganic orthophosphate (Ki=165 μM). While the enzyme was relatively insensitive to Mn±±, Cu±± and Zn±± inhibited the activity 540 fold at a concentration of 100 μM. The enzyme showed positive PAS staining and hence is a glycoprotein (28% glycosylation); the sugar composition suggests the presence of N-linked high mannose-oligosaccharides and galactose. A partial N-terminal amino acid sequence up to the thirty-fourth residue was elucidated.