In-Cell Crosslinked Enzymes: Improving Bacillus megaterium whole-cell biocatalyst stability for the decarboxylation of ferulic acid

Abstract

Abstract In-Cell Crosslinked Enzymes (InCLEs) is a procedure to improve the thermal and operational stability of Bacillus megaterium intracellular decarboxylase activity, allowing to obtain enhanced whole-cell biocatalyst to produce 4-vinyl guaiacol (4VG). InCLEs were achieved using a fixed amount of biomass in a two-step procedure: 1) permeation of the membrane cell for reactants diffusion, and 2) intracellular proteins crosslinking. To optimize InCLEs preparation, 7.68 mg of biomass in the presence of different ethanol concentrations (10–50% v/v) were permeated, subsequently, the intracellular proteins were crosslinked with a glutaraldehyde/biomass ratios of 0, 1.63 and 3.26 μmol/mg. At the best conditions (10% v/v ethanol and 1.63 μmol/mg), thermostability increases 4.7-fold against the permeated uncrosslinked biocatalyst (PUCB). To further enhance the stability, the internalization of different molecular weight polyethyleneimine (PEI, 1.09 mg/mL) was evaluated. PEI-InCLEs using 25 kDa PEI, showed a half-life 68-fold higher than PUCB (1 min) at 50 °C. After 7 cycles (26.3 mM ferulic acid, pH 7), PEI-InCLEs retained 83 and 69% of the decarboxylase activity at 40 and 50 °C, producing 32.7 and 29.8 μmol of 4VG, respectively. Here we demonstrated that the InCLEs procedure is an effective strategy to obtain stable whole-cell biocatalysts for the production of high-valued biotechnological molecules.