Identification, Characterization And Gene Cloning Of A Phytase With Potential Industrial Interest

Phytase (myo-inositol hexakisphate phosphohydrolase, EC 3.1.3.8) catalyze the hydrolysis of phytate to myo-inositol and inorganic phosphate. A high phytase-producing strain of Aspergillus niger N-3 was identified. The enzyme was purified, the enzymatic characterization was studied and its gene was cloned. Furthermore, kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP were studied by using Aspergillus sp. phytase expressed by pichia pastoris as material. By the investigation, the following results were achieved:1. Screening of the strains that produce extracellular phytase. A total of 104 microbial strains isolated from soil samples, catsup and lees locally were screened for their ability to produce extracellular phytase, and 76 strains were found to produce a clear zone of hydrolysis surrounding the growing mycelium. The enzyme activities of most strains were between 8 U/mL and 50 U/mL, and the highest one measuring 495 U/mL, given the name Aspergillus niger N-3.2. Cloning of the phytase gene The Aspergillus niger N-3 chromosomal DNA was extracted using the benzyl chloride method. The gene sequence showed that the coding region, not including the introns and putative signal sequence, was comprised of 1 347 nucleotides. It encoded a polypeptide of 448 amino acids, which exhibited high amino acid sequence homologies (94.87%) with Aspergillus niger NRRL 3135. The nucleotide sequence homologies of these two kinds of strains were 91.5%. The protein included 10 potential N-glycosylation sites. 3. Purification of phytase from Aspergillus niger N-3 The crude extract sample was first precipitated by ammonium sulfate at the concentration of 30% of its saturation. After 3 h, ammonium sulfate was added to the supernatant to a saturation of 80%. 8 h later, the precipitate was collected by centrifugation, and dissolved in 100 mM NaAc-HAc buffer (pH 5.5). The sample was dialyzed to remove the ammonium sulfate, and the retentate before being loaded onto a 25 mL Sephadex G-75 column. The specific activity of the enzyme increased 1.52 fold by ammonium sulfate precipitation. After gel filtration chromatography, two peaks were observed. Fractions eluted on the peak having phytase activity showed a single diffuse band on SDS-PAGE. The enzyme was shown to be homogenous, and the molecular mass of the purified enzyme was estimated to be 60-80 kDa.4. Enzymatic characterization The enzyme activity reached the highest point at temperature of 55°C for 10 min. The phytase displayed maximum activity at pH 2.0, with an additional significant peak of activity at pH 5.5. Activity at pH 2.0 was about 30% higher than that at pH 5.5. The Km value for sodium phytate and pNPP were 132.3μM and 2.23 mM respectively. After treating at 80, 85 and 90°C for 5 min, the remaining activities were 57%, 49% and 45% respectively.5.Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP Chemical probing of histidine residues using specific modifiers, IAA and DEP resulted in the inactivation of phytase (phy A). The results suggested that histidine residues are involved in the active site of the enzyme. The results also indicated that inactivation reaction of the enzyme by IAA was a complexing type inhibition, and DEP reaction involved a conformation-change step before inactivation.