Organosoluble enzyme conjugates with poly(2-oxazoline)s via pyromellitic acid dianhydride

• Published in: Journal of biotechnology 2012-06, Vol.159 (3), p.195-203
• Main Authors: Konieczny, Stefan, Fik, Christoph P., Averesch, Nils J.H., Tiller, Joerg C.
• Format: Article
• Language: English
• Subjects: Animals; Benzoates - chemistry; Biotechnology; Cattle; Chickens; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Enzyme-conjugate; gel chromatography; Hydrolases - chemistry; Hydrolases - metabolism; Isoelectric Focusing; light scattering; lysozyme; Molecular Weight; Organic solvent; Oxazoles - chemistry; polyacrylamide gel electrophoresis; Polyamines - chemistry; polymers; Polyoxazoline; proteins; Pyromellitic acid dianhydride; ribonucleases; Solubility; solvents
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2012.01.016

[Display omitted] ► POXylation is successful via pyromellitic acid dianhydride. ► Hen-egg white lysozyme and RNase A became fully DMF soluble by POXylation. ► POXylated enzymes partly retain their activity in water. The use of enzymes in organic solvents offers a great opportunity for the synthesis of complex organic compounds and is therefore in focus of current research. In this work we describe the synthesis of poly(2-methyl-1,3-oxazoline) (PMOx) and poly(2-ethyl-1,3-oxazoline) (PEtOx) enzyme conjugates with hen-egg white lysozyme, RNase A and α-chymotrypsin using a new coupling technique. The POXylation was carried out reacting pyromellitic acid dianhydride subsequently with ethylenediamine terminated POx and then with the NH2-groups of the respective enzymes. Upon conjugation with the polymers, RNase A and lysozyme became fully soluble in DMF (1.4mg/ml). These are the first examples of fully POXylated proteins, which become organosoluble. The synthesized enzyme conjugates were characterized by SDS-PAGE, isoelectric focusing, dynamic light scattering and size exclusion chromatography, which all indicated the full POXylation of the enzymes. The modified enzymes even partly retained their activity in water. With α-chymotrypsin as example we could demonstrate that the molecular weight of the attached polymer significantly influences the activity.