A PHYTASE FROM RHIZOPUS OR YZA E: PRODUCTION, CHARACTERIZATION AND APPLICATION STUDIES

Abstract

Phytase is an important enzyme in the food/feed industry. It catalyzes the hydrolysis of phytate, an antinutrient compound present in cereals and grains, thereby, releasing orthophosphate and myo-inosito1-6-phosphate with lower degrees of phosphorylation. Phytic acid is a strong chelator capable of complexing with a variety of metal ions under neutral and alkaline conditions, as well as with proteins and starch under acidic condition. Treatment with phytase increases not only the bioavailability of inorganic phosphorus but also the digestibility of proteins and the absorption of minerals from food/feed. The action of phytase also contributes towards reducing the pollution in surface and ground water caused by the phytate and phosphorus run-off from manure in intensive livestock regions. This thesis presents the studies of a phytase from Rhizopus sp. The focus has been directed towards identification of a potential phytase producing microorganism, biochemical characterization of phytase, and optimization of parameters for the production of phytase, large volume production studies and evaluation of phytase potential in terms of phytate degradation. A novel phytase from mesophilic fungal strain Rhizopus oryzae MTCC 1987 was identified. The phytase showed acid stable and pepsin resistant properties, with its optimum activity at 45°C and a dual pH profile at 1.5 and 5.5. Besides superior activity at physiological temperature (37-39°C), it demonstrated high thermostability with t1127 0°C of 8.25 h, and a broad substrate specificity. All the tested metal ions (at 5 mM) exerted significant stimulatory effect except Fee, Niz+ and Cue, however, the interesting feature of this phytase was insensitivity to heavy metal ions like Bat+ and Ag+. Phytase was purified by using fractional ammonium sulphate precipitation and subsequent ion-exchange and gel filtration chromatography. Molecular size determination was carried out by SDS-PAGE and Gel filtration, and it was found to be —34 kDa and —34-36 kDa, respectively. Zymogram analysis displayed a single enzyme responsible for the phytase activity corresponding to —34 kDa. Kinetic studies demonstrated a significant 1 ABSTRACT low K,,, value (2.42 x 10-4mM), high catalytic efficiency (kcat/Km) (2.38 x 106 M-1 s 1) and low activation energy value (28.99 kJ/mol). An uncompetitive inhibition was observed in presence of tested inhibitors (Fluoride > Phosphate > Vanadate). Present study investigated the feasibility of various agro-industrial residues as solid substrate for phytase production, followed by optimization of culture parameters (substrate particle size, inoculum age, inoculum size, effect of supplementation of various surfactants and metal ions) under solid state fermentation (SSF). Linseed oilcake (LOC) and wheat bran (WB) (1:1) was found to be the most effective substrate with maximum phytase activity (37.43 U/gds) under optimal culture conditions. Present study also includes the statistical optimization of culture variables for the production of phytase under SSF. The study also aimed at enhancing the phytase production using an improved strain of Rhizopus oryzae followed by statistical optimization of the medium components for submerged fermentation. Higher titer and higher productivity of phytase by improved strain can contribute to a significant reduction of the cost of phytase production from R. oryzae thereby, increasing its industrial utilization. The ability of this phytase to completely degrade the phytate to its lower derivative, inositol tris-phosphate, was assessed by an improved method of HPLC analysis. It demonstrated the phytate degrading potency of this phytase using simple, precise and reproducible method using reversed-phase high performance liquid chromatography (RP-HPLC), equipped with variable wavelength detector (VWD). The feasibility of determining phytic acid and IP3 using this method was successfully demonstrated with minimal sample preparation and lower retention time at 246 nm. The results show that VWD system with UV spectrum specifically detects the phytate and its lower derivatives with a good resolution. Furthermore, an in vitro study in milieu of physiological conditions revealed an unusual stability of this phytase at low pH with a degradation of 50% of phytate content. In other way, it can be stated that even under low pH condition (and at physiological temperature), this phytase has the ability to break three phosphomonoester bonds of phytate. These findings, taken together, demonstrate the new enzyme from R. oryzae as a novel phytase and render it of potential industrial interest. ii ABSTRACT A novel strain improvement procedure has also been developed to increase the yields of phytase production through adaptation of R. oryzae cells to subsequent heat and cold shock. The outcome suggests that an enhancement in phytase production by R. oryzae could be obtained by the combination of heat and cold treatment of spore suspension. The study was further extended to a comparative study on efficacy of wild and mutant strains pertaining to ash content and mineral extractability. The results confirm the effectiveness of this procedure as well as the enhanced efficacy of mutant strain in term of mineral extractability....