title: Single-step purification and immobilization of MBP-phytase fusion on starch agar beads: application in dephytination of soy milk.
abstract: Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60 °C. Cleavage of 87 kDa fusion protein using factor Xa released 45 kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50 °C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.
authors.affiliation: Biotechnology Division, National Institute for Interdisciplinary Science and Technology-NIIST, CSIR, Trivandrum 695019, Kerala, India. ushusbt@gmail.com
authors.forename: Mrudula Vasudevan
authors.initials: MV
authors.lastname: Ushasree
authors.forename: Paramasamy
authors.initials: P
authors.lastname: Gunasekaran
authors.forename: Ashok
authors.initials: A
authors.lastname: Pandey
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000362:Agar / Q000737:chemistry*; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D058619:Maltose-Binding Proteins / Q000235:genetics*; D008863:Microspheres; D010833:Phytic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D041942:Soy Milk; D013213:Starch / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D010833:Phytic Acid; D000362:Agar; D013213:Starch; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli