Study On Production, Purification, Cloning And Expression Of The Phytase From The Marine Yeast Kodamaea Ohmeri BG3

Statistical experimental designs were applied for the optimization of phytase production by a marine yeast Kodamaea ohmeri BG3 in a cost-effective oats medium. Using Plackett–Burman design, oats, ammonium sulfate and initial pH were identified as significant factors and these factors were subsequently optimized using a central composite design. The optimum variables that supported maximum enzyme activity were with oats 1.0%(w/v), ammonium sulfate 2.3%(w/v), glucose 2.0% (w/v), NaCl 2.0% (w/v) and initial pH 6.3. The validity of the optimized variables was verified in shake-flasks level. An overall 9-fold enhancement in phytase activity (62.0–575.5 U/mL) was attained due to optimization.The extracellular phytase in the supernatant of cell culture of the marine yeast K. ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadexi G-75), and anion-exchange chromatog- raphy (DEAE Sepharose Fast Flow Anion-Exchange).According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65oC, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline(at concentrations of 1.0 mM and 5.0 mM). The Km and Vmax, values of the purified enzyme forphytate were 1.45 mM, 0.083 mM / min, respectively.The extracellular phytase structural gene was isolated from cDNA of marine yeast K. ohmeri BG3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1389 bp long encoding a phytase. The coding region of the gene had no intron. It encoded 462 amino acid residues of a protein with a putative signal peptide of 15 amino acids. The protein sequence deduced from the extracellular phytase structural gene contained the consensus motifs (RHG XRX P and HD) which are conserved among histidine acid phosphatase and six conserved putative N-glycosylation sites. According to the phylogenetic tree of the phytase, the phytase from K. ohmeri BG3 was closely related to Candida albicans (XP₇13452) and Pichia stipitis (XP₀01385108) phytase protein and more distantly related to other phytases, respectively.The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21 (DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 51.9 kDa was found. Enzyme activity assay verified the recombinant protein as a phytase. A maximum activity of 16.5 U/mg was obtained from cellular extract of E. coli BL21 (DE3) harboring pET-24a (+)PHY1. Optimal pH and temperature of the crude recombinant lipase were 5.0 and 65°C, respectively and the crude recombinant phytase had the hydrolytic activity towards phytate.