Semi-rational protein engineering to improve the activity of surface displayed exocellulase CelK

Gesing K, Lenz F, Tozakidis IEP, Jose J

Abstract

We recently developed a whole-cell catalytic approach for the saccharification of lignocellulosic biomass employing the soil bacterium Pseudomonas putida with surface displayed cellulases1. In this work wefocused on the improvement of exocellulase CelK from Ruminiclostridium thermocellum, which is the current bottleneck of the hydrolysis process. Therefore a semi-rational approach was chosen by using sitesaturation mutagenesis (SSM) on six positions (Y543, S547, A475, R683, T797, W791) that were selected based on a low conservation and their location at the entrance of the substrate binding pocket. Thevariants of CelK were expressed as autotransporter fusion proteins on the surface of Pseudomonas putida KT2440. A 96-well plate activity assay was developed to test the variants against the synthetic substrate4-nitrophenyl-β-d-cellobioside (pNPC) and the more natural substrate carboxymethylcellulose (CMC). Overall, the mutation at two of the six positions led to an increase of the activity with pNPC. The mutation ofT797A led to an 1.7 fold increase of the activity to 5.49 mU*ml-1*OD-1, whereas variant A475W even showed an activity increase of 2.9 fold to 9.35 mU*ml-1*OD-1 in comparison to the not mutated CelK(3.24 mU*ml-1*OD-1).