Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Abstract

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km and Kcat for the tested substrates were calculated.

Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829

“…5 ). This is similar to the optimal pH (5–7) of adenosine-phosphate deaminase of A. fumigatus (Yoshimune et al 2005 ), NAD deaminase produced by A. oryzae (Ali et al 2014 ). At pH 8.0, the deaminase loses 40 % of its activity as A. oryzae NAD deaminase, which had its optimum activity at pH 5 and abroad pH stability.…”

“…It was found that the addition of EDTA (10 mM) to the reaction mixture did not inhibit enzyme activity indicating that NAD deaminase is not a metalloenzyme. This property closely resembles that of a nonspecific NAD aminohydrolase produced from A. oryzae (Ali et al 2014 ); whereas the enzyme was slightly activated by addition of Na + and K + , while inhibited by addition of Mn 2+ , Ag + , Hg 2+ , and Cu 2+ . In this concern, Yoshimune et al ( 2005 ) reported that the adenosine-phosphate deaminase produced by A. fumigatus was inhibited by 38 % in the presence of Cu 2+ (1 mM), while no significant enhancement of the enzyme activity was reported by Ba 2+ , Ca 2+ , Co 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Sn 2+ , Zn 2+ and Fe 3+ .…”

“…3 ). In this respect, the molecular masses of 94 and 85 kDa were reported for the enzymes produced by A. oryzae and A. fumigatus 4 as investigated by Ali et al ( 2014 ) and Yoshimune et al ( 2005 ), respectively. On the other hand, the purified enzyme from A. oryzae was found to exhibit a smaller molecular mass of 14.5 kDa as reported by Rosinova et al ( 1978 ).…”

“…6 ). This is about 10–20 °C higher than optimum temperature of the adenosine-phosphate deaminase of A. fumigatus (Yoshimune et al 2005 ) and NAD deaminase A. oryzae (Ali et al 2014 ), respectively. The enzyme was found to thermostable up to 70 °C; the results also showed that 80 and 88 % of its original activity still remained at 50 and 60 °C, respectively, after 30 min of incubation (Data not shown).…”

“…The evidence of high ADA activity during rapid and stimulated growth of normal tissues is of importance in making a fully functional purine salvage pathway possible (Seiler 2004 ). Adenosine deaminase of Aspergillus oryzae (Ali et al 2012 , 2014 ) has been characterized in our laboratory. Both the adenosine and by-products of the NAD-consuming enzymes such as NMN and Nm can be recycled back to NAD (Sorcil et al 2010 ; Gazzaniga et al 2009 ).…”