2024 AIChE Annual Meeting

Cloning, Expression, and Purification of Laccase Enzymes for Biocatalytic Reactions

Laccase, a multi-copper oxidoreductase with the ability of oxidizing a wide range of substrates, such as ascorbic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-Dimethoxyphenol (2,6-DMP), guaiacol, and hydroquinone, is found in multiple species including plants, fungi, and bacteria. While it is known that laccase enzymes are capable of naturally oxidizing lignin, a bio polymer found in plant cell walls and provides stability and stiffness to the plant, the reaction between each strain of laccase and lignin will be different. This study aims to study a library of over 40 different laccase, including cloning and optimization of expression and purification, followed by in vitro characterization of its reaction with substrates.

To begin, we codon-optimized four different laccases from Arabidopsis thaliana, Bacillus stratosphericus, Bacillus subtilis, and Thermothelomyces thermophilus to fit the bacterial expression system. PCR and Gibson Assembly were employed to produce the laccase coding gene and insert the targeted gene into the bacterial expression vector pET28, which contains an IPTG-inducible T7 promoter and a hexahistidine tag for further in vitro. Sequencing confirmed correct construction of laccase-containing plasmids, which were then transformed into E. coli expression host BL21(DE3).

Next, BscotA1 from Bacillus stratosphericus was picked for the pilot study. In order to maximize the gene expression, we first looked into the study of various cultivation media (LB vs TB) with multiple induction temperature (18C, 30C, & 37C). All of the cultures were pelleted and lysed with sonication, followed by gel electrophoresis to determine the expression level. We observed the most soluble protein using TB medium with 18C induction. We then employed affinity chromatography with a Ni+ column to purify the targeted laccase. With the confirmation of the target laccase protein band in the elution column on the SDS protein page, the project will move to the next phase: 1). further optimize the purification protocol for more pure protein. 2). Apply the pilot kinetic study for such purified laccase to finalize the characterization protocol. 3). Provide both purification and characterization for the other laccase.