EnXylaScope aims to optimize production systems that allow industrially relevant production of the novel xylan debranching enzymes discovered in the project.

The EnXylaScope project has highlighted four different xylan debranching enzyme classes; i) GH115-α-glucuronidase, ii) α-1,2-L-arabinofuranosidase, iii) acetyl xylan esterase and iv) ferulic esterase, as target catalysts for enzymatic processing of Xylan polymers, with a focus to modify substituents on the polymer.

The objective is to establish production systems for these target enzymes, with yields and productivities that allow upscaling for industrial production. Selection of the production system from the positive results will depend not only on enzyme yield and activity of the enzyme but also on process conditions required for large-scale production of the targeted enzyme using that system. For each enzyme, a set of suitable expression systems will be defined, including Pichia pastoris and Escherichia coli, as production hosts.

The selected production system for each of the targeted enzymes will be optimized for better production rates, e.g. by further codon optimisation and/or minimising GC content at the 5’-end and/or addition of a highly expressive fusion partner. The production system will also be optimized by varying the time of induction and concentration of inducer. The function of the produced candidates will be verified (specific activity), as well as characterised for thermal and pH stability.

Each enzyme class will be produced in 2 L bench-scale bioreactors, for quantification of enzyme production, growth characteristics and substrate and oxygen demand in relation to the enzyme production rate. Steady state chemostat and/or accelerostat cultivations will be carried out to determine the relation between growth rate and enzyme production rate and to obtain basic stoichiometric and kinetic parameters for each production system.

Selected conditions will be scaled-up for all four targeted enzymes at m3 pilot scale, with the E. coli production platform as main target. The enzymes produced in the scale-up runs will be characterised for activity and desired functional properties and will be compared with the results obtained at lab scale (2L).

During the first year of the EnXylaScope project, work on the following tasks have started:

• Selection of genes for production using genes deposited in databases (e.g. CaZy)
• Expression of genes encoding arabinofuranosidases (from GH43) and glucuronidases (from GH115) in Escherichia coli, applying different strategies
• Preparation of constructs for production in Pichia pastoris using GH43 and GH115 targets.
• Synthesis of genes encoding feruloyl and acetylxylanesterases for production trials.

These work package activities will in the nearest future focus on:

• Creating a shortlist of the currently successfully produced targets using the E. coli system.
• Producing corresponding enzymes in the P. pastoris system.
• Setting up production trials for the selected feruloyl- and acetylxylanesterase candidates.
• Introducing novel candidates from the screening in WP2, when available.