Cyclohexanedione modification of arginine at the active site of Aspergillus ficuum phytase.

Reaction of Aspergillus ficuum phytase with the arginine specific modifier 1,2-cyclohexanedione causes a rapid loss of activity. The inactivation can be partially reversed by 0.2 M hydroxylamine and exhibits pseudo-first order kinetics. The reaction order and second order rate constant of inactivation were 0.87 and 6.72 M-1 Min-1, respectively. Amino acid analysis of modified phytase indicates that about 7 arginine of the total 19 were modified. While the chymotryptic maps of treated and untreated phytase wer virtually identical, the tryptic maps had 4 peaks of altered mobility. An Arg containing tripeptide was identified in the phytase which is also present in other phosphohydrolases and may represent one of the labile Arg involved in the formation of the active site.

Extracellular PH 2.5 optimum acid phosphatase from Aspergillus ficuum: immobilization on modified fractogel.

Aspergillus ficuum pH 2.5 optimum acid phosphatase (orthophosphoric monoesters phosphohydrolase, E.C.3.1.3.2) was covalently immobolized on 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP)-activated Fractogel TSK HW-50F. The catalytic parameters and stability of the immobilized enzyme were compared with those of the free enzyme. While the Km and the temperature optima were unchanged, the Ki for orthophosphate was changed from 185 microM to 422 microM and greater stability was observed against heat treatment.

Aspergillus ficuum extracellular pH 6.0 optimum acid phosphatase: purification, N-terminal amino acid sequence, and biochemical characterization.

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 lambda max 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 x 10(5) M-1 cm-1 and 1.3 x 10(3) M-1 cm-1 respectively. Judging by chromatofocusing, the isoelectric point of the enzyme was about 4.9. The purified enzyme was unstable at 70 degrees C. The enzyme was catalytically very active from 55 degrees to 65 degrees C with a maximum activity at 63 degrees C. The Michaelis constant of the enzyme for p-nitrophenylphosphate was 200 microM 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 microM). While the enzyme was relatively insensitive to Mn++, Cu++ and Zn++ inhibited the activity 540 fold at a concentration of 100 microM. 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.

Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum.

An acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme in gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 3.4 x 10(5) M-1 cm-1. The isoelectric point of the enzyme, as judged by chromatofocusing, was about 4.0. The purified enzyme is highly stable at 0 degree C. Thermal inactivation studies have indicated that the enzyme is unstable at 70 degrees C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63 degrees C. The Km of the enzyme for p-nitrophenylphosphate is about 270 microM with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-oligosaccharides. A partial N-terminal amino acid sequence up to the twenty-third residue was obtained. The enzyme was inhibited competitively by inorganic orthophosphate (Ki = 185 microM) and non-competitively by phosphomycin (Ki = 600 microM).