Surface display of hemicellulases on Pseudomonas putida: new whole-cell biocatalysts for the degradation of biomass

Schulte M, Sichwart S, Tozakidis IE, Jose J

Abstract

Xylans are polymers of β-1,4 linked β-D-xylopyranosyl units that can be substituted by different side groups like α-D-glucopyranosyl or α-L-arabinofuranosyl units. As a majorcomponent of hemicellulose, xylans are available in large amounts, e.g. in waste material from the agriculture or wood industries. The enzymatic depolymerisation of xylans intotheir monomeric sugars by so-called hemicellulases is of great interest both from the ecological and the economical point of view, however the high costs of these enzymescurrently impede their employment in industrial scales. Here we present the autotransporter mediated surface display of an endo-1,4-β-xylanase (XynA), a 1,4-β-xylosidase (XynB)and two α-L-arabinofuranosidases (Abf2 and AbfCelf) in the Gram-negative soil bacterium Pseudomonas putida (P. putida) KT2440. A western blot analysis of outer membraneproteins revealed that the hemicellulase-autotransporter fusion proteins were located within the outer membrane of P. putida, and the enzymes could be detected on the cellsurface using flow cytometry. All four enzymes retained their functionality with hydrolytic activities of 38.3 mU/mLOD1 for XynA towards beechwood xylan, 6 mU/mLOD1 for XynBtowards 4-nitrophenyl-β-D-xylopyranoside, 8.6 mU/mLOD1 and 6.2 mU/mLOD1 for the two α-L-arabinofuranosidases Abf2 and AbfCelf towards 4-nitrophenyl-α-Larabinofuranoside.A mixture of three strains each displaying one type of hemicellulase was able to degrade 2.5 % (w/v) raw arabinoxylan to D-xylose with a yield of17.8 μg/mLOD1 after 24 hours.