title: Purification of urease from jack bean (Canavalia ensiformis) with copper (II) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) cryogels.
abstract: Jack bean (Canavalia ensiformis) is the source of interesting proteins that contribute to modern biochemistry, and urease is the primary of these proteins. Owing to its role and occurrence in nature, urease has become a part of extensive studies. In this study, jack bean urease (JBU) was purified by immobilized metal affinity chromatography using Cu(2+) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) [PHEMAH-Cu(2+)]-based cryogels. PHEMAH-Cu(2+) cryogel was synthesized and characterized for swelling degree, morphology (by SEM), N-methacryloyl-(L)-histidine methyl ester and Cu(2+) incorporation (by elemental analysis and atomic absorption spectrophotometry). The binding of JBU to PHEMAH-Cu(2+) cryogel was optimized by examining the effect of pH, flow rate and JBU concentration on binding. The maximal binding of JBU was 23.2 mg/dry gram of adsorbent. The maximal binding of JBU extracted from jack bean meal was 67.8 mg/dry gram of adsorbent. The elution of JBU from cryogel column was accomplished by 1.0 M NaCl in 20 mM phosphate buffer (pH 8.0). Molecular weight and purity of JBU from jack bean meal was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was observed that JBU could be repeatedly bound and eluted from (PHEMAH)-Cu(2+) cryogel with less than 10% loss in column capacity.
authors.affiliation: Molecular Biology Division, Department of Biology, Hacettepe University, Ankara, Turkey.
authors.forename: Pınar
authors.initials: P
authors.lastname: Tekiner
authors.forename: Işik
authors.initials: I
authors.lastname: Perçin
authors.forename: Bahar
authors.initials: B
authors.lastname: Ergün
authors.forename: Handan
authors.initials: H
authors.lastname: Yavuz
authors.forename: Erol
authors.initials: E
authors.lastname: Aksöz
mesh_terms: D002021:Buffers; D032449:Canavalia / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D059985:Cryogels; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D011109:Polymethacrylic Acids / Q000737:chemistry*; D013054:Spectrophotometry, Atomic; D014510:Urease / Q000302:isolation & purification*
chemical_list: D002021:Buffers; D002614:Chelating Agents; D059985:Cryogels; D011109:Polymethacrylic Acids; C579323:poly(hydroxyethyl methacrylate-N-methacryloyl-histidine methyl ester); D006639:Histidine; D003300:Copper; D014510:Urease