Gene Cloning, Identification And Characterization Of A Novel Phytase From Neurospora Crassa

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC3.1.3.8) hydrolyzes phytic acid (myo-insitol hexakisphosphate), which is the primary storage form of phosphorus in cereal grains, legumes, and oil seeds etc, to the myo-insitol and phosphoric acid. The phytase has been widely applied in the animal feeds to improve the absorption phytate phosphorus to animals. Consequently, the amount can be decreased of supplemental inorganic phosphate and the phytic acid excreted to the environment by the animals. This has a positive contribution to mitigating the environment pollution. The research firstly identified N. crassa phytase gene and the biochemical characterization of the purified recombinant phytase. By sequence alignment, a fragment of 1507bp encoding a peptide was found in the whole genome sequence of N. crassa similar to the precursors of phytases from Aspergillus niger Aspergillus ficuum, Aspergillus terrus. The peptide was found to possess the conserved sequence motif RHG×R×P of histine acid phosphates, which was the enzyme activity site of microbial phytases. The intronless N. crassa phy was cloned as an EcoR I fragment downstream of the TPI promoter of pYX212. The resulting plasmid pYX212-phy was transformed into S. cerevisiae W303-1A. The positive recombinant expressed phytase constitutively. The N. crassa phy was also cloned as an EcoR I fragment into the corresponding site of the expression vector pPIC9K downstream of the AOX1 promoter. The resulting plasmid pPIC9K-phy was linearized with Nco I endoenzyme and subsequently electrotransformed into P. pastoris KM71. The recombinant phytase was purified as follows. The supernatant was collected from the culture broth by centrifugation to remove the cell, dialyzed and concentrated by Amico ultrafitration-15 (Biomax 10K; Millipore). The concentrate was fractionized on a Superdex 200 column (Pharmacia Biotech) and an anion-exchange column (DEAE-52;Whatman). The purified recombinant enzyme was revealed to be a 60 kD protein and the specific activity reached 125 U/mg. It has enzyme activity between pHs 2.5 and 7.5 with two pH optima of 3.5 and 5.5, a temperature optimum of 60℃, Km and Vmax values of 228 ?M and 128 ?mol/mg pro.min-1, respectively. The enzyme displayed good pH stability during pH 3.5-9.5. After exposure to 70 to 90℃ for 20min, the recombinant enzyme retained 39 to 58 % residual activity. It was revealed that enzyme activity was greatly inhibited by Cu2+ and not significantly affected by Mg2+, Ca2+, Al3+, Fe2+, Co2+, Zn2+.