Specific Objective 1: Establishing experimentally driven cross-functional process map and guidance platform to expand the use of xylan debranching enzymes and functionalising (grafting) enzymes. The work successfully created a final Process Flow Diagram (PFD) and confirmed all mass and energy balances for the biorefinery. A transferable decision platform software tool, including a graphical user interface (GUI), was developed and validated to efficiently design feedstock and product-specific enzyme cocktails, drastically reducing the required number of experiments.
Specific Objective 2: Accelerated discovery and functional characterisation of 4 novel high-performance xylan debranching enzymes by using multi-omics and advanced High-Throughput Screening (HTPS) methods. The project successfully implemented and utilized advanced screening methods. Comprehensive lists of over 40 enzyme candidates from four targeted classes (Alpha-Glucuronidase, Alpha-L-Arabinofuranosidase, Acetyl Xylan Esterase, and Feruloyl Esterase) were produced via sequence mining and functional screening, achieving and surpassing discovery targets.
Specific Objective 3: Establishing efficient production systems and techno-economically viable production strategies. Optimized fed-batch processes for four key enzymes were successfully validated at 20-liter scale, and industrial protocols and designs were established for full-scale production. Production strains were cloned and expressed, confirming superior yields compared to initial benchmarks. Large-scale batches were successfully produced and purified.
Specific Objective 4: Targeted enzymatic modification to introduce xylan in consumer products by using substrate and product specific enzyme cocktails developed using a combination of debranching and grafting enzymes. Kilogram-scale production of Water-Insoluble Xylan (WIS-xylan) and its derivatives (xylan-phenolic ester, xylan-phenolic-fatty acid ester) was achieved using optimized enzyme cocktails. The sequential enzymatic modification process was successfully optimized to produce a high-performing xylan ingredient.
Specific Objective 5: Pilot process demonstration of 4 enzymes and application testing of 6 different enzymatically derived xylan-based consumer products. Application testing confirmed that the modified xylans were highly effective oil stabilizers for cosmetics and demonstrated improved functional properties for food and feed applications. Successful pilot production of three key enzymes (Alpha-Glucuronidase, Alpha-L-Arabinofuranosidase, and Acetyl Xylan Esterase) at 400-liter scale was achieved.
Specific Objective 6: Regulatory compliance and market acceptance. An extensive online survey of consumers across nine European countries confirmed high acceptance for xylan-based ingredients when adequate information is provided. Due to unforeseen microbial contamination in final batches, the full in-vitro toxicology testing and subsequent consumer sensory evaluation could not be completed, preventing full compliance with this objective.
Specific Objective 7: Effective communication and dissemination procedures. Communication targets were fully achieved. Activities included generating joint policy briefs, implementing an online e-learning course, designing a robust Business Plan, and leading the "Enzymes for greener products" cluster, ensuring wide-ranging dissemination and exploitation planning.
