The Engineering of Tannase for Industrial Application

Abstract

Enzymes are effective and efficient biocatalysts that when applied can greatly improve production outputs in industrial settings and create more environmentally friendly processes when compared with chemical options. However, the application of enzymes is often hindered due their functionality having evolved naturally against moderate environmental conditions. This generally results in them being relatively unstable and unfit for usage under industrial conditions. This study applied a combination of computational prediction and experimentation with the aim of optimizing the enzyme tannase from Lactobacillus plantarum (LpTan) for industrial usage. Two different methods were explored. The first method described in detail in Chapter 2 of this thesis applied genetic code expansion in the active site of the enzyme to improve catalytic function. In particular, the impact of replacing histidine 451 in the esterase’s catalytic triad with its non-canonical counterpart 3-methyl-histidine (NmH) was evaluated. A decreased reliance on the Asp of the catalytic triad, while shortening the distance between the His and the Ser was hypothesized to create a more efficient catalytic dyad. However, when the activity of the derived NmH451 tannase was compared to wild type, it showed a decrease in activity. The second method described in detail in Chapter 3 of this thesis applied combinatorial multi-site-directed mutagenesis toward improving thermal stability and total turnover number of the enzyme. Protein Repair One Stop Shop (PROSS) was used to generate stabilizing mutation predictions for a ‘flapless (FLT)’ version of LpTan, including a list of 62 possibilities that were narrowed down to 8 through a set of selection criteria. These mutations included Q63T, A65T, A184Y, A257D, V276Y, T321G, G421D and G439D. Combinatorial screening of the impact of these site changes on functionality has, to date, yielded 4 variants that were characterized for catalytic efficiency (kcat/Km), melting temperature (Tm), inactivation constant (kinact) and total turnover numbers (TTN; kcat / kinact). Variant P8E5, with 6 of 8 site variations showed the highest significant Tm value, by an increase of 6.5 ˚C compared to FLT. In conclusion, while the genetic code expansion strategy was not successful, the combinatorial PROSS-predicted site-mutagenesis method did yield a more thermostable variant of the FLT, highlighting the value of computational prediction. Overall, we provide evidence that a tannase enzyme can be stabilized for potential industrial applications.