3.2 Optimization of the ratio of modifiers and surfactants
Although different modification degrees of modifiers
(-SO3H and -C18) can result in distinct
properties of the carrier and thereby impact its immobilization
efficiency, quantitatively analyzing the modification degree can be
exceedingly challenging. Thus, to further investigate the effect of the
modification degree on the immobilization efficiency and to design a
more efficient immobilization carrier, we used the immobilization effect
of CRL as an indicator to optimize the modification degree. As shown inTable 2 , the results suggested that the enzyme loading and
activity recovery of modified diatomite modified with
-SO3H or
-C18 alone was considerably lower than that of
-SO3H/-C18 (1:1) group, which
demonstrated that the -SO3H and -C18groups may have a synergistic effect. It can be attributed to several
factors, on the one side, the long-chain -C18 provided a
suitable hydrophobic microenvironment for the lipase via an interfacial
activation mechanism, and on the other hand, the -SO3H
groups changed the charge distribution of the lipase surface, resulting
in a stronger binding interaction between lipase and carrier, which
could reduce the enzyme leakage. In addition, the ratio of the two
modifiers had a significant effect on the immobilization effect, and
OSMD had the best immobilization effect with an enzyme loading of 74.40
mg·g-1 and an activity recovery of 63.35% at the
ratio of -SO3H/-C18(1:1.5). The specific
mechanism needs to be further investigated to find out.
Given the high hydrophobicity of OSMD, the addition of surfactant
enhances the dispersion of the support in the phosphate buffer[42,
43]. In addition, the addition of surfactants can also prevent the
aggregation of lipase[44]. However, it also should be noted that the
surfactants may also play a negative role during immobilization due to
the inactivation of lipases. Hence, it becomes crucial to screen the
type and concentration of surfactant (Figure S4 ). The results
showed that SDS has a detrimental effect on lipase activity. Nonionic
surfactants (Triton X-100, Tween 80) yielded favorable results but were
slightly inferior to the cationic surfactant (CTAB). Moreover, the
impact of different CTAB concentrations on immobilization efficacy was
assessed, revealing that at a concentration of 0.50 mM, CRL loading
reached 76.3 mg·g-1, with an enzyme activity recovery
of 74.7%. The presence of the cationic surfactant may alter the surface
charge of the lipase and enhance its electrostatic interactions with the
sulfonic acid groups while preventing lipase dimer formation and thus
beneficial to the immobilization processes[45, 46].
Table 2 . Effect of
modifier ratio on the immobilization effect of OSMD