title: Molecular and biochemical characteristics of recombinant β-propeller phytase from Bacillus licheniformis strain PB-13 with potential application in aquafeed.
abstract: Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase 'rPhyPB13' was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other β-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0-6.5 and 60 °C in the presence of 1 mM Ca(2+) and was highly active over a wider pH range (pH 4.0-8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 μmol/min/mg and 27.46 s(-1), respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca(2+) and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a β-propeller phytase that has application potential in aquaculture feed.
authors.affiliation: Department of Biochemistry, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar, India, 263145.
authors.forename: Vinod
authors.initials: V
authors.lastname: Kumar
authors.forename: Punesh
authors.initials: P
authors.lastname: Sangwan
authors.forename: A K
authors.initials: AK
authors.lastname: Verma
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Agrawal
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000821:Animal Feed; D017756:Aquaculture; D001205:Ascorbic Acid / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D004063:Digestion; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
chemical_list: D008670:Metals; D010618:Phenanthrolines; D011994:Recombinant Proteins; D004492:Edetic Acid; D010832:6-Phytase; D014357:Trypsin; D001205:Ascorbic Acid; C025205:1,10-phenanthroline