Stabilization of enzymes by chemical modifications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, New Zealand

Abstract

This study focused on thermostabilization of enzymes in solution by intramolecular crosslinking of the specific functional groups within an enzyme molecule. Three model enzymes were used: α-amylase of Aspergillus oryzae (EC 3.2.1.1), β-galactosidase of Aspergillus oryzae (EC 3.2.1.23) and extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae. Crosslinking was examined using the following homobifunctional reagents: diisocyanates (O=C=N(CH2)nN=C=O, n = 4, 6, 8), diimidoesters (CH3O(=NH)C(CH2)nC(=NH)OCH3, n = 4, 5, 6) and diamines (NH2(CH2)nNH2, n = 0, 2, 4, 6, 8, 10, 12). The concentration of the enzymes was kept low at 0.9 μM in attempts to promote intramolecular crosslinking as opposed to intermolecular crosslinking. Only invertase could be stabilized relative to controls by crosslinking with diisocyanates. Invertase (0.9 μM) crosslinked with 1,4-diisocyanatobutane (n = 4; or butamethylene diisocyanate, BMDC) and 1,6-diisocyanatohexane (n = 6) showed enhanced thermostability. Stability was improved dramatically by crosslinking invertase with 20-30 μM of the reagent. Molecular engineering of invertase by crosslinking reduced its first-order thermal denaturation constant at 60 °C from 1.232 min-1 for the native enzyme to 0.831 min-1 for the stabilized enzyme. Similarly, the best crosslinking treatment increased the activation energy for thermal denaturation from 372 kJ mol-1 for the native invertase to 517 kJ mol-1 for the stabilized enzyme. Values of the Michaelis- Menten parameters (Km and νmax) showed a reduced efficiency of invertase after the crosslinking treatment. The nature of the crosslinking was examined using size exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and multiple angle laser light scattering (MALLS). Depending on the conditions used, both intermolecular and intramolecular crosslinking occurred. The estimated molecular weight of the intermolecularly crosslinked invertase appeared to be much higher compared to the intramolecularly crosslinked invertase and the native invertase. In attempts to simplify certain analyses, attempts were made to remove the carbohydrate moiety from crosslinked invertase (a glycoprotein) molecule. Deglycosylation with PNGase F achieved a significant reduction of carbohydrate for the native invertase but not for the intra- and intermolecularly crosslinked invertase. Circular dichroism (CD) measurements showed that crosslinking with BMDC affected slightly the secondary structure of invertase. The nature of the crosslinking that might be occurring in invertase molecule was further studied using small model oligopeptides, small nonglycosylated enzymes (hen egg white lysozyme and pepsin) and glycoprotein models (ovalbumin). Crosslinking of the model pentapeptide (0.9 μM) suggested that crosslinking with BMDC involved reaction between BMDC and the amino group of lysine or the carboxylate at C-terminal of the pentapeptide. Using a heptapeptide (1 mM) in crosslinking with BMDC showed a changed hydrophobicity of the crosslinked peptide. The crosslinking treatment of lysozyme (3.5 mM) with BMDC clearly produced an intermolecularly crosslinked lysozyme as evidenced by SEC and SDS-PAGE. A changed net charge of lysozyme after the crosslinking treatment was demonstrated using native PAGE. Mass spectrometry was used to then prove the intramolecular crosslinking of lysozyme with BMDC. CD spectra of the intramolecularly crosslinked lysozyme showed it be more resistant to thermal unfolding relative to native lysozyme.