Concerns of phosphorus pollution and its impact on environments have driven the biotechnological development of phytases. Phosphoric acid, inositol phosphate, or inositols are produced after hydrolysis of phosphate from phytate, initiated by phytase. Research over the last two decades on microbial phytases has deepened our understanding of their production, optimization, and characterization. Despite the wide availability of phytase producing microorganisms, only a few have been commercially exploited. The current high cost of phytases, inability to withstand high temperatures (>85 C), a limited pH range, and poor storage stability are a major bottleneck in the commercialization of phytases. The development of novel phytases with optimal properties for various applications is a major research challenge. In this paper, recent advances in microbial phytase production, application of tools to optimize higher enzyme production, and characterization of phytases along with potential biotechnological applications are reviewed. Additionally the development of phytase assay methods and functions of phytate and phytate degradation products are discussed.

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40-80 °C) and pH (2.0-6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K m, V max, K cat, and K cat/K m of the pure phytase were 0.21 mM, 131.58 nmol mg(-1) s(-1), 1.64 × 10(3) s(-1), and 7.81 × 10(6) M(-1) s(-1), respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca(+2), Mg(+2) and Mn(+2), but inhibited by Zn(+2), Cu(+2), Fe(+2), Pb(+2), Ba(+2) and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.

A bacterial strain producing an extracellular phytase was identified as Enterobacter sp. ACSS. Optimization of process parameters using statistical methods such as Plackett-Burman design (PBD), the steepest ascent method, and response surface methodology (RSM) significantly improved phytase production by 4.6–fold in shake-flasks. In addition, an overall 1.9-fold increase in phytase production was attained in fed-batch fermentations in a 5 l laboratory fermenter, respectively. The purified 62 kDa phytase from Enterobacter sp. ACSS was active between 40 to 80°C and an acidic pH range of 2.0 to 6.0 with half-life of 693 and 577.5 min at 60°C and pH 2.0, respectively. Additionally, the enzyme is fairly stable with proteolytic enzymes under physiological conditions. It was activated by Ca+2, Mg+2 and Mn+2 while inhibition was caused by Zn+2, Cu+2, Fe+2, Pb+2, Co+2, Ba+2 and surfactants. The Km, Vmax and Kcat observed were 0.21 mM, 131.58 nmol mg-1s-1 and 1.64 × 103 s-1, respectively. The enzyme released inorganic phosphate from animal feed (4.0-6.62 mg/g of diet) and insoluble metal-phytates (45-219 µg/ml) and was effective in improving the characteristics of brown bread. Overall, this study shows that Enterobacter sp. ACSS has the potential to produce significant titres of a thermo- and acid-stable phytase and can be applied in dephytinizing animal feeds, and the baking industry.