Hyperactivation Behavior of Site-Specifically Immobilized Fusion Protein of Lipase with α-Synuclein and Silica-Binding Peptide.

Bacillus thermocatenulatus lipase 2 (BTL2) is a highly versatile enzyme for catalyzing the hydrolysis and synthesis of various esters, but the practical application of the enzyme is limited by its poor operational stability and difficulty in recovery. To address these limitations, we have herein proposed a dual fusion strategy that combines the chaperone-like protein α-synuclein (αS) at the C-terminus and silica-binding peptide (SiBP) at the N-terminus, making a fusion enzyme (SiBP-BTL2-αS) for enhanced enzymatic performance and site-specific immobilization on mesoporous silica nanoparticles (MSNs) for repeated use. The catalytic activity of the enzymes was evaluated using a colorimetric p-nitrophenyl palmitate (pNPP) assay at 30 °C in 50 mM HEPES buffer (pH 8.0), and relative activity was expressed as the ratio to the wild-type BTL2. It was found that free SiBP-BTL2-αS showed a 1.8-fold higher activity than BTL2 due to the chaperone effect of αS, and immobilization on MSNs brought out a further 1.4-fold increase in activity at an enzyme loading of 194 mg/g. Thus, SiBP-BTL2-αS@MSNs presented 3.3-fold higher activity than BTL2. Moreover, SiBP-BTL2-αS@MSNs exhibited significantly improved thermostability and broad pH tolerance over the free counterpart and BTL2. In repeated uses, SiBP-BTL2-αS@MSNs retained 82.1% of its initial activity after seven consecutive reaction cycles. In the synthesis of vitamin E succinate, SiBP-BTL2-αS@MSNs showed 32% and 78% higher yields over SiBP-BTL2-αS and BTL2, respectively, verifying the superiority of SiBP-BTL2-αS@MSNs in enzymatic catalysis. This work not only offers a highly efficient, robust, and recyclable enzyme preparation, but also provides a promising way to design immobilized lipase with hyperactivation behavior.