title: Microbial Transglutaminase Separation by pH-Responsive Affinity Precipitation with Crocein Orange G as the Ligand.
abstract: A microbial transglutaminase (MTG) was efficiently purified by using pH-responsive affinity precipitation with Crocein orange G (COG) as the affinity ligand. The docking method was used to identify the appropriate ligand. The molecular simulation results were compared with the label-free detection data analyzed by ForteBio's Octet. A pH-responsive polymer, PMMDN, was polymerized and subsequently coupled with COG as the ligand. The isoelectric point (pI) and recovery of PMMDN and PMMDN-COG were 4.51, 99.8,% and 4.78, 98.1 %, respectively. The optimal adsorption conditions were found to be a ligand density of 60.0 μmol/g, pH 7.0, and 0.2 mol/L NaCl. The adsorption isotherm showed that the maximum adsorption capacity was 91.32 mg/g polymer and the dissociation constant was 0.021 mg/mL. Interaction information between PMMDN-COG and MTG in the whole process of affinity precipitation were obtained by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). PB (0.02 mol/L pH 7.0) in 20.0 % glycol was used to elute the binding MTG from PMMDN-COG. Under these conditions, electrophoretically pure MTG was obtained in only one step with elution recoveries of 98.96 % (protein) and 95.09 % (activity).
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Ding
authors.forename: Sipeng
authors.initials: S
authors.lastname: Li
authors.forename: Xuejun
authors.initials: X
authors.lastname: Cao
mesh_terms: D000327:Adsorption; D001391:Azo Compounds / Q000378:metabolism*; D011232:Chemical Precipitation; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009282:Naphthalenesulfonates / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D001391:Azo Compounds; D008024:Ligands; D009282:Naphthalenesulfonates; D011108:Polymers; C000607925:crocein orange G; C008710:Orange G; D011503:Transglutaminases