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