Periodic Reporting for period 2 - EnXylaScope (Mining Microbes and Developing Advanced Production Platforms for Novel Enzymes To Rapidly Unleash Xylans’ Potential In a Scope Of Products For the Consumer Market)
Période du rapport: 2022-11-01 au 2024-04-30
Enzymes have significant potential to support sustainability, reduce environmental pollution, lower processing costs and enhance product performance and functionalities due to their unique selectivity and catalytic activity. The EnXylaScope project is discovering novel enzymes for debranching xylan, a highly abundant polymer in plants. Production systems for these enzymes are being optimised and the enzymes are applied to produce a debranched (water insoluble) form of xylan.
This xylan has unique functional properties (such as reduced water solubility and enhanced viscosity) and is suitable for direct incorporation in consumer products or for further modifications to confer the functional properties for more demanding applications.
In total, three types of enzymatically modified xylan will be produced and will be tested for six consumer products. These products belong to three different sectors (cosmetics, personal care and nutraceuticals).
Over the past 18 months, the EnXylaScope consortium partners have made significant progress. The candidate list of enzymes of interest using sequence mining approaches has been obtained. These enzymes have been tested for establishing an efficient production system and production optimization. In total, more than 100 strains have been screened giving a shortlist of 48 bacterial and 15 fungal strains. Also, considerable work has been done related with verifying the hosting organism. The work has been focused on two production systems and other back-up systems to use reported gene candidates for the targeted enzymes for the design and selection of production systems.
Three feedstocks of xylan have been studied. In total, 90 different conditions have been tested; the xylan fractions are being analysed to select the feedstock which gave higher purity xylan and easy extraction in mild conditions.
Information about the process and about the enzymes and substrates available has been prepared as a guideline to analyse the data of the xylan extraction experiments, establishing significant fractions and correlations. The experimental design to develop the enzyme cocktail has been provided.
This xylan has unique functional properties (such as reduced water solubility and enhanced viscosity) and is suitable for direct incorporation in consumer products or for further modifications to confer the functional properties for more demanding applications.
In total, three types of enzymatically modified xylan will be produced and will be tested for six consumer products. These products belong to three different sectors (cosmetics, personal care and nutraceuticals).
Over the past 18 months, the EnXylaScope consortium partners have made significant progress. The candidate list of enzymes of interest using sequence mining approaches has been obtained. These enzymes have been tested for establishing an efficient production system and production optimization. In total, more than 100 strains have been screened giving a shortlist of 48 bacterial and 15 fungal strains. Also, considerable work has been done related with verifying the hosting organism. The work has been focused on two production systems and other back-up systems to use reported gene candidates for the targeted enzymes for the design and selection of production systems.
Three feedstocks of xylan have been studied. In total, 90 different conditions have been tested; the xylan fractions are being analysed to select the feedstock which gave higher purity xylan and easy extraction in mild conditions.
Information about the process and about the enzymes and substrates available has been prepared as a guideline to analyse the data of the xylan extraction experiments, establishing significant fractions and correlations. The experimental design to develop the enzyme cocktail has been provided.
SO1: Establishing experimentally driven cross-functional process map and guidance platform to expand the use of xylan debranching enzymes and functionalising (grafting) enzymes. The work done has delivered a material transfers flow chart (Ms3) and the preliminary process flow diagram (PFD) with unit operations presented in the deliverable D4.3. In addition, the protocol for xylan extraction and purification from three different feedstocks (D4.1) and 50g Xylan extracted and purified from three feedstock samples (D4.2) were achieved. Finally, the prediction algorithms for designing feedstock and product specific enzyme cocktails, Ms 15, was reached (M18).
SO2: Accelerated discovery and functional characterisation of 4 novel high-performance xylan debranching enzymes by using multi-omics and advanced high-throughput screening (HTPS) methods. Significant work has been done on the discovery of different novel xylan debranching enzymes with superior catalytic activity and wide operation potential and reported in D2.2 and D2.1 leading to the accomplishment of the milestone Ms4 about establishing efficient screening for target enzymes. Also, it was produced the deliverable D2.4 that contained the sequence data and cloned libraries from newly processed environmental samples.
SO3: Establishing efficient production systems and techno-economically viable production strategies. During this first period, extensive work has been done to find an optimised production platform suitable for each of the xylan debranching enzymes. The work has been focused on reaching Ms10 “At least one gene encoding known enzymes of each class cloned and successfully expressed” and all the work done was reported at the deliverable D3.3.
SO4: 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. Considerable work has already been done in the prediction models for chemical constituents of process streams in the xylan extraction and enzymatic modification process. Further work has been devoted to establishing a process benchmark for enzymatic conversion of extracted xylan to WIS-xylan and developing feedstock and product specific enzyme cocktails using EnXylaScope-discovered enzymes for WIS-xylan production.
SO5: Pilot process demonstration of 4 enzymes and application testing of 6 different enzymatically derived xylan-based consumer products. Comprehensive environmental and social assessments, including life cycle assessment (LCA), will be performed for the production of WIS-xylan, WIS-Phe, and WIS-Phe-FA and their consumer products. Until now, the progress has been monitored by the preparation and final accomplishment of the milestone Ms 22 “Internal workshop on definitions and settings” (M19).
SO6: Regulatory compliance and market acceptance. As a first step to obtaining more knowledge on how European consumers view the role of substances such as xylans in sustainable and responsible consumption, a multinational online survey (complying with the milestone Ms20 Online study designed & deployed) targeting these issues was launched.
SO7: Effective communication and dissemination procedures. Period 1 has been marked by the COVID-19 pandemic, however the project results has been communicated in an excellent manner thanks to the DCP (D8.1). EnXylascope has participated at the Horizon Results Booster programme. The results were considered at the Report on exploitable results (D8.5) and Business plan (D8.7). Contact with the other FNR-16 projects were made and periodic meetings are held (Ms 24). The projects have created a cluster called “Enzymes for greener products”. The first Policy and regulatory framework report (D8.9) has been submitted.
SO2: Accelerated discovery and functional characterisation of 4 novel high-performance xylan debranching enzymes by using multi-omics and advanced high-throughput screening (HTPS) methods. Significant work has been done on the discovery of different novel xylan debranching enzymes with superior catalytic activity and wide operation potential and reported in D2.2 and D2.1 leading to the accomplishment of the milestone Ms4 about establishing efficient screening for target enzymes. Also, it was produced the deliverable D2.4 that contained the sequence data and cloned libraries from newly processed environmental samples.
SO3: Establishing efficient production systems and techno-economically viable production strategies. During this first period, extensive work has been done to find an optimised production platform suitable for each of the xylan debranching enzymes. The work has been focused on reaching Ms10 “At least one gene encoding known enzymes of each class cloned and successfully expressed” and all the work done was reported at the deliverable D3.3.
SO4: 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. Considerable work has already been done in the prediction models for chemical constituents of process streams in the xylan extraction and enzymatic modification process. Further work has been devoted to establishing a process benchmark for enzymatic conversion of extracted xylan to WIS-xylan and developing feedstock and product specific enzyme cocktails using EnXylaScope-discovered enzymes for WIS-xylan production.
SO5: Pilot process demonstration of 4 enzymes and application testing of 6 different enzymatically derived xylan-based consumer products. Comprehensive environmental and social assessments, including life cycle assessment (LCA), will be performed for the production of WIS-xylan, WIS-Phe, and WIS-Phe-FA and their consumer products. Until now, the progress has been monitored by the preparation and final accomplishment of the milestone Ms 22 “Internal workshop on definitions and settings” (M19).
SO6: Regulatory compliance and market acceptance. As a first step to obtaining more knowledge on how European consumers view the role of substances such as xylans in sustainable and responsible consumption, a multinational online survey (complying with the milestone Ms20 Online study designed & deployed) targeting these issues was launched.
SO7: Effective communication and dissemination procedures. Period 1 has been marked by the COVID-19 pandemic, however the project results has been communicated in an excellent manner thanks to the DCP (D8.1). EnXylascope has participated at the Horizon Results Booster programme. The results were considered at the Report on exploitable results (D8.5) and Business plan (D8.7). Contact with the other FNR-16 projects were made and periodic meetings are held (Ms 24). The projects have created a cluster called “Enzymes for greener products”. The first Policy and regulatory framework report (D8.9) has been submitted.
EnXylaScope is an innovative process that uses biomass from regional sources, ensuring agriculture and processing jobs in rural areas and production and commercial jobs in urban areas.
In many petrochemical-based processes employment is inclined towards males, either due to the choice of degree and employment by women or to undesired gender bias that exist in traditional industries. However, the biotechnology and bioprocessing industries are inclusive to all genders/sectors of people. Hence, EnXylaScope’s innovations, through the utilisation of regional resources and fostering inclusiveness of stakeholders of all genders, sectors, and ages, will enhance the socio-economic fabric of biobased cosmetic and personal care products.
EnXylaScope uses agriculture, forestry, and agro-processing residues which are otherwise used inefficiently and poorly valorised (e.g. burnt, landfilled, composted, or anaerobically digested). In addition, all processes used are environmentally friendly, first, a benign process to extract xylan (no chlorinated compounds) and enzymatically modifying xylan to hydrogels is carried out without using harsh conditions (e.g. periodate oxidation, chemical esterification), secondly, the obtained polymers are biodegradable with very low eutrophication problems and zero environment toxicity and the wastewater generated from the use of these polymers can be effectively treated by existing waste-water plants.
In many petrochemical-based processes employment is inclined towards males, either due to the choice of degree and employment by women or to undesired gender bias that exist in traditional industries. However, the biotechnology and bioprocessing industries are inclusive to all genders/sectors of people. Hence, EnXylaScope’s innovations, through the utilisation of regional resources and fostering inclusiveness of stakeholders of all genders, sectors, and ages, will enhance the socio-economic fabric of biobased cosmetic and personal care products.
EnXylaScope uses agriculture, forestry, and agro-processing residues which are otherwise used inefficiently and poorly valorised (e.g. burnt, landfilled, composted, or anaerobically digested). In addition, all processes used are environmentally friendly, first, a benign process to extract xylan (no chlorinated compounds) and enzymatically modifying xylan to hydrogels is carried out without using harsh conditions (e.g. periodate oxidation, chemical esterification), secondly, the obtained polymers are biodegradable with very low eutrophication problems and zero environment toxicity and the wastewater generated from the use of these polymers can be effectively treated by existing waste-water plants.