Autotransporter‐Based Surface Display of Hemicellulases on Pseudomonas putida: Whole‐Cell Biocatalysts for the Degradation of Biomass

The enzymatic depolymerization of xylans into their monomeric sugars by hemicellulases is of great interest from both the ecological and the economical point of view; however, the high costs of these enzymes impede their employment on industrial scales. The utilization of whole cells displaying the enzymes on their surface could reduce costs by allowing a direct employment of the cells after cultivation and their reuse in multiple reaction cycles. Here, we present the 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 KT2440 by fusing the enzymes to the EhaA autotransporter unit from Escherichia coli. The surface display of the enzymes was verified by flow cytometry. All four enzymes retained their functionality with hydrolytic activities of 48.5 mU mL−1 for XynA towards beechwood xylan, 6 mU mL−1 for XynB towards 4‐nitrophenyl‐β‐d‐xylopyranoside, and 8.6 mU mL−1 and 6.2 mU mL−1 for the two α‐l‐arabinofuranosidases Abf2 and AbfCelf towards 4‐nitrophenyl‐α‐l‐arabinofuranoside, respectively. Measurements were done with cell suspensions of an OD578=1. A mixture of strains displaying the three types of hemicellulases could degrade 2.5 % (w/v) raw arabinoxylan from rye bran to d‐xylose with a yield of 133.5 mg L−1 cell suspension after 24 h. Surface Scissors: A whole‐cell catalytic approach for the enzymatic conversion of complex hemicellulose substrates into xylose employs the robust soil bacterium Pseudomonas putida with surface‐displayed hemicellulases. The reaction can be performed at moderate temperature.