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
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2011
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Massey University
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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.
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Keywords
Crosslinking, Invertase, Lysozyme, Thermostabilization, Enzymes