ERA-IB5 14-071 OXYPOL Optimised laccase systems for high-value bio-plastics from biomass

Lead Research Organisation: Aberystwyth University
Department Name: IBERS

Abstract

With a global production > 300 mill ton/year , plastics are an important contributor to increased greenhouse gas emissions. However the use of plastics contributes to e.g. lighter vehicles and better preservation of food and water and is as such a good alternative to meet the demand from the growing population.

Bio-plastics will bring the best from two worlds, combining the material properties of petrochemical-based materials with a better eco-footprint. The bio-based plastics market has grown tremendously in recent years, with global production expected to reach more than 1.5 million tons/year by 2018.

Plastics are made up of what are known as 'building block molecules'. This project aims to develop enzymes that will break down lignin (e.g. from plants, or from the pulp and paper industry) into the building blocks required to produce bio-plastics. This is a pan-European project, with partners from 6 European countries, all with valuable expertise in different parts of the lignin to plastic production cycle.

Technical Summary

These days, the bio-based plastics market is in rapid growth, and bio-based plastic products will more and more replace petroleum-based plastic products, leading to reduced CO2 emissions and herewith a positive environmental impact. The use of biocatalysts (enzymes) in the bio-plastics production value chain has the potential to add largely to this positive impact as it may substitute toxic chemical catalysts in processes using to produce polymers from bio-based feedstock. Lignin is the largest available source of aromatic building blocks for bio-based aromatic derivatives, and its abundance, e.g. from the pulp and paper industry, makes it a highly interesting potential source for novel aromatic chemicals and polymer precursors that may be polymerized by chemical and biocatalysts.

The OXYPOL project addresses the bio-based plastics value chain using biocatalysis for the degradation of lignin into aromatic building blocks and the subsequent controlled polymerization of these aromatic precursors into bio-based polymers. Highly efficient laccase mediator systems (LMS) based on abundant potential low-cost mediator compounds directly derivable from selected lignin preparations will be developed, applying enzyme engineering strategies. In combination with improving enzyme-recycling approaches for LMS involving immobilization on polymer particles and whole-cell biocatalysis, we aim at breaking crucial bottlenecks in the application of LMS based
biocatalysis towards large scale industrial applications in bio-plastics production.

OXYPOL is a pan-European endeavour integrating complementaryscientific excellence and market driven R&D competence of the totally 8 academic, institutional and commercial project partners, aiming at making an impact on the biocatalysis-based bio-plastics production, with positive implications for the European bio-economy.

Planned Impact

In particular, OXYPOL will enable more efficient, scalable, safer and more environmentally friendly bio-plastics manufacturing processes by targeting optimized, specific and cost-efficient biocatalysis both for precursor production and subsequent polymerization.

Central to OXYPOL is the development of new, highly efficient low-cost laccase/mediator systems (LMS) for the controlled biocatalytic depolymerisation of lignin.OXYPOL will access and develop further, previously described as well as new to discover microbial laccases for their optimal function in innovative LMS, feeding from abundant natural low-cost mediator compounds. Specially treated lignin fractions from the crop Miscanthus x giganteus will be the cheap and scalable source of mediators in new LMS for lignin depolymerisation, thus representing a conceptual internal feedback loop in the project. Optimized LMS will, along with established chemical routes, in addition be deployed in the subsequent polymerisation into new bio-polymers of high aromatics content for bioplastics production. Through enzyme recycling and low-cost scalable enzyme production, R&D on laccase immobilization and whole cell biocatalysis included in OXYPOL,will contribute to the definition of more economical processes for lignin and LMS-based bio-polymer product generation up to the large industrial scale.
 
Description The aim of this project is on the deconstruction of lignin, a highly abundant, under-utilised resource, for the production of new polymers. The focus was on developing laccases and laccase mediators to optimise the production of lignin monomers. Nine potential enzymes have been identified from bioinformatic interrogation of databases and genomic libraries (20) that have the required activity and kinetic profile for use in our process. These enzymes were expressed and characterized based on activity, thermostability, halotolerance and resistance to organic solvents. A number of these enzymes were modified to improve their catalytic activity and tolerance to pH and temperature based on structural rules identified from database sequences. An improved, modified enzyme was identified and two additional variants were constructed which showed additional improvement. These modified enzymes were successfully immobilized on to beads (for enzyme recovery during processing) and were also successfully displayed on the surface of bacteria and shown to be functionally active.
To further facilitate the activity of the laccases developed above in the deconstruction of lignin, a number of compounds from miscanthus lignin material were isolated and show to be good mediators for lignin degradation. Additionally, a number of synthetic mediators were designed based on the structure of the best naturally occurring mediators, from experimental data. These were tested and one in particular was identified that showed increased release of monomers from Kraft lignin following incubation with a range of laccase enzymes. One of the main applications of laccase mediator systems is their use in the decolorization of dyes from the textile industry. For this project, an assay based on decolorization of reactive black was used for screening mediators using the laccase from Trametes versicolor, Cot A and the new laccases developed as part of the project. Mediators were selected based on naturally occurring phenolic mediators described in the literature, potential mediators identified in lignin samples and also different mediators synthesized by the University of Oviedo group. A set of 40 mediators in total were screened and around 10 showed notable activity in the decolorization of reactive black 5, including syringaldehyde, acetosyringone. There was a correlation between the mediators which presented a higher activity and the presence of certain substituent groups on the phenyl ring. This data will be used not only to identify the type of mediator which is more reactive with these laccases but also for the analysis of putative amino acids in the laccase protein which are involved in their interaction with mediator molecules. Mediators showing different activity with different laccases can indicate which amino acids are important for the oxidation of these molecules.
Exploitation Route Development of novel lignin-based polymers offer the opportunity to replace those from the petro-chemical industry. Modified enzymes can be used in a range of industries that require laccases including the dye industry. Surface displayed technology can be further developed and used as a cheap method of producing enzymes for microbial based processes.
Sectors Chemicals,Construction,Energy,Manufacturing, including Industrial Biotechology

 
Description European regional development fund
Amount € 2,884,534 (EUR)
Organisation Welsh Assembly 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2023
 
Description Newton Bhabha Industrial Waste BIOREVIEW
Amount £1,001,109 (GBP)
Funding ID BB/S011994/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 08/2021
 
Description Separation and purification of organic condensate output streams from superheated steam torrefaction of forestry and agricultural residues
Amount £19,902 (GBP)
Funding ID BB1150A-BIV-T 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 09/2018
 
Description Value added materials from organic waste sugars VAMOS Horizon 20:20 BBI
Amount £11,872,746 (GBP)
Funding ID H2020-BBI-JTI-2018-837771 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 09/2019 
End 08/2022
 
Description 2nd LBNet International Conference: "Challenges and Opportunities in Lignocellulosic biorefining: Science, Policy and Economics" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact LBNet's 2 annual conference cut across discipline boundaries to bring varied perspectives from plant scientists, biochemists, microbial scientists, chemists, chemical engineers, process engineers, modellers, economists, environmental scientists and policy-makers. The focus of the conference was on:

Biorefineries, biofuels and business
Biorefineries, economics and policy
High value products from lignocellulose biomass
Understanding and improving plant biomass
Enzymes and catalysis for biomass processing
Year(s) Of Engagement Activity 2017
 
Description AIEC opening 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Opening of AU new Biorefining centre. Presentations were made on current biorefining projects along with equipment available to academics and industry.
Year(s) Of Engagement Activity 2019
 
Description Azerbaijan Ambassador visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presentation on biorefining research and potential opportunities for international collaboration.
Year(s) Of Engagement Activity 2018
 
Description Barroness Eluned Morgan Welsh Assembly Member discussion on bio-economy policy and research 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact Presentation on work on biorefining projects and discussion on our contribution to driving the rural strategy for Wales.
Year(s) Of Engagement Activity 2017
 
Description Ben Lake MP visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Presentation on our biorefining research
Year(s) Of Engagement Activity 2018
 
Description BioTech Research & Innovation Hack 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The BioTech Research & Innovation Hack 2017 was a public status seminar and exchange day of projects funded within ERA-IB-2, ERASysAPP, ERASynBio and ERA-NET Bioenergy. The event took take place in Brussels, Belgium, on 5 and 6 December 2017.

The BioTech Research & Innovation Hack 2017 brought together 53 research projects with stakeholders from academia and industry, funding organisations, decision makers and general public. This event provided an opportunity to discuss the achievement of the transnational ERA-NET projects, to network with excellent professionals in the area of industrial biotechnology, systems and synthetic biology etc. and to spark novel interdisciplinary research collaborations!
Year(s) Of Engagement Activity 2017
URL https://www.cobiotech.eu/events?event=BioTech_Research_amp_Innovation_Hack_2017
 
Description Chief Scientific Officer for Wales Julie Williams 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact Presentation of research in biorefining and discussions regarding future direction of science in Wales
Year(s) Of Engagement Activity 2016
 
Description Coleg Cambria visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Presentation and discussion on aspects of biorefining and implications for the future farming community.
Year(s) Of Engagement Activity 2017
 
Description Farming Group - Diversification 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation and discussion with Farmers on new opportunities to diversity into new crops or look for new markets for existing crops in the context of biorefining.
Year(s) Of Engagement Activity 2017
 
Description Functional Genomics Conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Functional Metagenomics 2016 conference taking place in Inderøy, Norway, on September 25-28, 2016 is the 2nd open conference of 'Functional Metagenomics International', and aims at bringing together interested researchers working in the field of Functional Metagenomics for three intense days of scientific presentations, as well as vivid discussions leading to exchange of knowledge and latest research results, in the inspiring surrounding of Mid-Norwegian nature. We presented our work entitled: "Discovery of novel laccases combining in silico screening of metagenomic libraries and functional characterization"
Year(s) Of Engagement Activity 2016
URL https://www.sintef.no/projectweb/fmg2016/program/
 
Description Newtown rotary Club Visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented current and post research in the area of biorefining.
Year(s) Of Engagement Activity 2019
 
Description Oxypol presentation at scientific meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation titled 'High Performance Counter Current Chromatography as a tool for separating potentially high-value chemicals from waste products' at the event; 'Renewable chemicals from waste - securing the molecular value from waste streams' organised by the Royal Society of Chemistry
Year(s) Of Engagement Activity 2015
 
Description Presentation at 4th LB net Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Poster presentation of recent work on lignin deconstruction.
Year(s) Of Engagement Activity 2019
 
Description Presentation at LB net Conference 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation on research in the area of lignin valorsiation at the LBnet International Conference. Title: "Novel enzymatic tools for cell-wall deconstruction using metagenomic screening and protein modelling" 5-7 April 2017. Inform both academics and industrialists on developments in lignin deconstruction from lignocellulosic materials.
Year(s) Of Engagement Activity 2017
 
Description Regional Stakeholder Partnership for Land-based Goods 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Presentation of production of platform chemicals/fuels/fine chemicals and potential health care products as well as integrated biorefining from a range of land-based feedstock.
Year(s) Of Engagement Activity 2018
 
Description Taiwan Ambassador visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presentation on current research activities and opportunity to collaboration and/or student exchanges
Year(s) Of Engagement Activity 2018
 
Description Visit from UTM 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Return Visit by UTM team for further discussion on potential collaborative research and studentships etc.
Year(s) Of Engagement Activity 2020
 
Description Visit to Universiti Teknologi Malaysia (UTM) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Major discussions with Staff at both of the UTM campuses on areas of collaboration in Industrial biotechnology, bio-circular economy, engagement with Business, possible studentships and available equipment.
Year(s) Of Engagement Activity 2019
 
Description Wales Festival of Innovation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presentation on biorefining capability and discussion of relevant projects undertaken by our research group with members of the public, students and farming community to inform on potential opportunities.
Year(s) Of Engagement Activity 2017
 
Description Welsh Gov. innovation team 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact Presentation and discussion with Welsh Government Innovation Team, current projects, research strategy and future directions in the area of Industrial Biotechnology. This included our strategy for interacting with the business community, both in Wales and beyond.
Year(s) Of Engagement Activity 2017
 
Description Welshpool Earth Science group visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation and discussions on future farming and the potential of biorefining, creating new markets for agricultural outputs.
Year(s) Of Engagement Activity 2017