Integration of stability and activity for Candida antarctica lipase B and its covalent immobilization on a modified sol-gel matrix for biodiesel production

Abstract

Lipases play a vital role in various processes. Lipase-mediated processes can be done in mild reaction conditions requiring less energy, faster reaction rates, a cheaper starting materials can be utilized since lipases are selective and wastewater treatment/downstream processing pose no problem for lipases are biodegradable. Even though the use of lipase provides many advantages, their industrial application is hindered due to their expensive cost. In addition, lipases have low activity with long chain fatty acids as substrate and they are easily inhibited by short chain alcohol which leads to their shorter life span when applied as biocatalyst. A lipase with improved characteristics is in demand for a biochemical process to be economically feasible.

In this study, structural flexibility modulation and immobilization were carried out in order to enhance the functionality of Candida antarctica lipase B (CALB). The dynamics, structure configuration and functional groups of CALB were used to obtain an enzyme that shows higher activity and robustness. The dynamics of CALB in terms of its flexibility was modulated to improve the activity and stability. Structure configuration was also considered to maximize the effect and impact of mutation. Functional groups specifically exposed lysine residues were exploited to immobilize CALB on a modified sol-gel matrix.

Both stability and activity improvements were incorporated in Candida antarctica lipase B (CALB) through multiple-site mutagenesis. CALB was divided into two different regions to optimize its performance. Modulating the flexibility within the substrate-binding region and the hydrophilic solvent-affecting region enhanced the catalytic activity and organic solvent stability of CALB, respectively. Combining the mutation sites from the substrate-binding region and from the hydrophilic solvent-affecting region yielded an enzyme (V139E,A92E) with improved functionality.

The use of modified sol-gel matrix to immobilize CALB was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol-gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88% attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol-gel supports yielded higher thermal stability with 2.7 times higher half-life compared with soluble enzymes at 60oC. This enzyme immobilization system retained the enzyme residual activity even for repetitive use.

The resultant enzyme with enhanced functionality was covalently immobilized on the modified sol-gel matrix and its performance on biodiesel production was tested. The biodiesel production was approximately 3.7 times higher using the immobilized double mutant (V139E,A92E) enzyme compared with the immobilized wild type CALB. Optimization on the immobilized enzyme system using the double mutant (V139E,A92E) can be further investigated to further increase the biodiesel production. The technique applied in this study can also be extended to other lipases.