Abstract
Microbial transglutaminase (MTG) has been widely used in the food and pharmaceuticals industries. In this study, MTG was purified using affinity precipitation with an affinity polymer (PMMDN-T), which was synthesized using a pH-responsive polymer (PMMDN) coupled with L-thyroxin as an affinity ligand. Interactions between MTG and PMMDN-T were investigated using turbidimetric titration, zeta potential measurements, and low-field nuclear magnetic resonance (LF-NMR). We found different behaviors, architectures, and phase states of pH-dependent interactions between MTG and PMMDN-T interactions. Binding energetics between MTG and PMMDN-T were determined by isothermal titration calorimetry (ITC). The isoelectric point (pI) of the affinity polymer was 4.65 and was recovered with 96.7% efficiency after recycling the polymer three times. The optimal adsorption condition was 0.02 mol/L phosphate buffer (pH 6.0) with 1.0 mol/L NaCl at 30.0°C and a ligand density of 50.0 μmol/g. The maximum elution recoveries of total MTG were 98.44% (protein) with 92.19% (activity) using 0.02 mol/L pH 10.0 Gly-NaOH as the eluent.
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Cuatrecasas, P. and C. B. Anfinsen (1971) Affinity chromatography. Annu. Rev. Biochem 40: 259–278.
Vunnum, S., S. R. Gallant, Y. J. Kim, and S. M. Cramer (1995) Immobilized metal affinity chromatography: Modeling of nonlinear multicomponent equilibrium. Chem. Eng. Sci. 50: 1785–1803.
Subramanian, S. and P. D. Ross (1984) Dye-ligand affinity chromatography: The interaction of Cibacron Blue F3GA® with proteins and enzyme. Crit. Rev. Biochem. Mol. Biol. 16: 169–205.
Porath, J., J. A. N. Carlsson, I. Olsson, and G. Belfrage (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598–599.
Freitag, R., I. Schumacher, and F. Hilbrig (2007) Affinity precipitation an option for early capture in bioprocessing. Biotechnol. J. 2: 685–690.
Teotia, S., R. Lata, S. K. Khare, and M. N. Gupta (2001) Onestep purification of glucoamylase by affinity precipitation with alginate. J. Mol. Recognit. 14: 295–299.
Gupta, M. N., R. Kaul, D. Guoqiang, U. Dissing, B. Mattiasson, and W. H. Scouten (1996) Affinity precipitation of proteins. J. Mol. Recognit. 9: 356–359.
Mattiasson, B., A. Kumar, and I. Y. Galaev (1998) Affinity precipitation of proteins: Design criteria for an efficient polymer J. Mol. Recognit. 11: 211–216.
Eggert, M., T. Baltes, F. Garret-Flaudy, and R. Freitag (1998) Affinity precipitation–an alternative to fluidized bed adsorption. J. Chromatogr. A 827: 269–280.
Arnold, L. and R. Chen (2014) Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation. Biotechnol. Bioeng. 111: 413–417.
Chern, C. S., C. K. Lee, C. Y. Chen, and M. J. Yeh (1996) Characterization of pH-sensitive polymeric supports for selective precipitation of proteins. Colloids Surf. B: Biointer. 6: 37–49.
Bulmus, V., Z. Ding, C. J. Long, P. S. Stayton, and A. S. Hoffman (2000) Site-specific polymer-streptavidin bioconjugate for pHcontrolled binding and triggered release of biotin. Bioconjug. Chem. 11: 78–83.
Hoffman, A. S. and P. S. Stayton (2007) Conjugates of stimuliresponsive polymers and proteins. Prog. Polym. Sci. 32: 922–932.
Mattiasson, B., A. Kumar, A. E. Ivanov, and I. Y. Galaev (2007) Metal-chelate affinity precipitation of proteins using responsive polymers. Nat. Protoc. 2: 213–220.
Ding, Z., L. Kang, J. Liu, X. Zhang, and X. Cao (2017) Preparation of pH-responsive metal chelate affinity polymer for adsorption and desorption of insulin. J. Chem. Technol. Biotechnol. 92: 1590–1595.
Ding, Z., S. Li, and X. Cao (2014) Separation of lysozyme from salted duck egg white by affinity precipitation using pHresponsive polymer with an l-thyroxin ligand. Sep. Purif. Technol. 138: 153–160.
Ding, Z., S. Li, and X. Cao (2015) Microbial transglutaminase separation by pH-responsive affinity precipitation with Crocein Orange G as the ligand. Appl. Biochem. Biotechnol. 177: 253–266.
Li, S., Z. Ding, and X. Cao (2016) Separation of transglutaminase by thermo-responsive affinity precipitation using l-thyroxin as ligand. SpringerPlus. 5: 37.
Ando, H., M. Adachi, K. Umeda, A. Matsuura, M. Nonaka, R. Uchio, H. Tanaka, and M. Motoki (1989) Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric. Biol. Chem. 53: 2613–2617.
Gaspar, A. L. C. and S. P. de Góes-Favoni (2015) Action of microbial transglutaminase (MTGase) in the modification of food proteins: A review. Food Chem. 171: 315–322.
Oteng-Pabi, S. K., C. Pardin, M. Stoica, and J. W. Keillor (2014) Site-specific protein labelling and immobilization mediated by microbial transglutaminase. Chem. Commun. 50: 6604–6606.
Chen, P.-Y., K.-C. Yang, C.-C. Wu, J.-H. Yu, F.-H. Lin, and J.-S. Sun (2014) Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase. Acta Biomater. 10: 912–920.
Ding, Z. and X. Cao (2013) Affinity precipitation of cellulase using pH-response polymer with Cibacron Blue F3GA. Sep. Purif. Technol. 102: 136–141.
Grossowicz, N., E. Wainfan, E. Borek, and H. Waelsch (1950) The enzymatic formation of hydroxamic acids from glutamine and asparagine. J. Biol. Chem. 187: 111–125.
Kayitmazer, A. B., D. Seeman, B. B. Minsky, P. L. Dubin, and Y. Xu (2013) Protein–polyelectrolyte interactions. Soft Matt. 9: 2553–2583.
Freyer, M. W. and E. A. Lewis (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Method Cell Biol. 84: 79–113.
Velazquez-Campoy, A. and E. Freire (2006) Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nat. Protoc. 1: 186–191.
Freire, E. (2004) Isothermal titration calorimetry. Curr. Protoc. Cell Biol. 17: 1–17.18.
Wang, S., K. Chen, Y. Xu, X. Yu, W. Wang, L. Li, and X. Guo (2013) Protein immobilization and separation using anionic/ cationic spherical polyelectrolyte brushes based on charge anisotropy. Soft Matt. 9: 11276.
Karg, M., I. Pastoriza-Santos, B. Rodriguez-González, R. Von Klitzing, S. Wellert, and T. Hellweg (2008) Temperature, pH, and ionic strength induced changes of the swelling behavior of PNIPAM-poly(allylacetic acid) copolymer microgels. Langmuir. 24: 6300–6306.
Wang, H., Y. Wang, L. Yuan, L. Wang, W. Yang, Z. Wu, D. Li, and H. Chen (2013) Thermally responsive silicon nanowire arrays for native/denatured-protein separation. Nanotechnol. 24: 105101.
Wang, S., K. Chen, A. B. Kayitmazer, L. Li, and X. Guo (2013) Tunable adsorption of bovine serum albumin by annealed cationic spherical polyelectrolyte brushes. Colloids Surf. B: Biointer. 107: 251–256.
Hou, H. and X. Cao (2015) pH recycling aqueous two-phase systems applied in extraction of Maitake β-Glucan and mechanism analysis using low-field nuclear magnetic resonance. J. Chromatogr. A 1405: 40–4
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Yan, B. and X. Cao (2012) Preparation of aqueous two-phase systems composed of two pH-response polymers and liquid–liquid extraction of demeclocycline. J. Chromatogr. A 1245: 39–45.
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Li, S., Chen, J., Zhang, X. et al. Preparation and Characterization of a pH-responsive Polymer that Interacts with Microbial Transglutaminase during Affinity Precipitation. Biotechnol Bioproc E 23, 31–38 (2018). https://doi.org/10.1007/s12257-017-0366-y
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DOI: https://doi.org/10.1007/s12257-017-0366-y