Extraordinary activation of CALB by alkylammonium ions: a new paradigm for activity enhancement of enzymes

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Abstract
Candida antarctica lipase B (CALB) is widely used in biocatalysis with applications in plastics degradation and chemical synthesis. CALB is activated by hydrophobic matrices and, enigmatically, shows striking activation in polar, choline-based, Deep Eutectic Solvents (DES). Herein, we show that CALB activation and stabilisation by TAAs is caused by binding to choline’s tetraalkylammonium (TAA) moiety. Several related TAA salts also caused CALB activation which was proportional to the hydrophobicity of their alkyl substituents. Remarkably, tetraoctylammonium bromide showed activation of ∼500% even at low micromolar levels. These TAA salts represent a new class of enzyme activator. Molecular modelling identified the alkylammonium binding location as a hydrophobic patch centred around Asp-145 of CALB. Binding at this site explains lipase activation in polar DES solvents and its relationship to other pathways of CALB activation. Herein, we also demonstrate that CALB, like many lipases, is activated by calcium. Intriguingly, mixed soluble activator experiments showed that calcium and choline bind to different CALB sites, suggesting a two-site model for CALB activation. These observations, along with previous findings, show that TAA activation is a widespread property of enzymes and constitutes a novel and potent means to enhance enzyme turnover and stability. Highlights: CALB is activated by choline Several tetraalkylammonium salts cause activation of CALB Hyperactivation of CALB (5-fold) by tetraoctylammonium ions occurs at low micromolar concentrations. Two independent sites for CALB activation, by calcium and TAA ions, are identified Activation at the choline binding site stabilises CALB while calcium binding destabilises the enzyme A soluble activator is demonstrated, that can be used to probe the activation mechanism of CALB or other enzymes.

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