Mechanistic Enzymology of Bacterial Lignin Degradation

The chemicals that are used to make plastics, drugs, household chemicals & industrial solvents come primarily from oil, so one of the greatest scientific challenges for the 21st century is how we can find renewable sources for these important chemicals. One obvious place to look is plant lignocellulose, the major component of plant cell walls, found in biomass such as agricultural waste. One component of plant lignocellulose is lignin: an aromatic polymer that is naturally hard to break down, since it is held together by C-O ether linkages and C-C bonds that are not broken down by acid or base treatment. We have isolated bacteria from soil that are able to break down lignin, we have studied in detail the first bacterial lignin-degrading enzyme, an enzyme called peroxidase DypB from Rhodococcus jostii RHA1, and we are beginning to understand the biochemical pathways used by bacteria to break down lignin.

We have recently identified a second type of enzyme that can break down lignin,a manganese-dependent superoxide dismutase from Sphingobacterium sp. T2, This is the first time that an enzyme of this kind has been shown to have activity towards lignin. We wish to study in detail the reaction mechanism of this enzyme, to determine exactly what is the reactive species that is responsible for attacking lignin. We have also found another bacterial dyp peroxidase from Pseudomonas fluorescens that can attack lignin, and so we wish to determine the structure of the product that it releases, and determine the mechanism of this reaction. We have also recently determined the three-dimensional structure of a Dyp peroxidase enzyme from Thermobifida fusca; we wish to use this structure to carry out "directed evolution" on this enzyme, to improve its activity towards lignin.

We believe that, as well as the lignin-oxidising enzymes such as Dyp peroxidases and Mn superoxide dismutase, there must be other accessory enzymes and proteins that are needed for bacterial lignin breakdown. In this project, we will try to identify an esterase enzyme that breaks an ester linkage between lignin and hemi-cellulose; and a dehydrogenase enzyme that assists lignin breakdown by quenching radical intermediates generated during lignin breakdown. We will then test combinations of these accessory enzymes and lignin-degrading enzymes with samples of lignin either from plant biomass, or from industrial sources, to test whether they are effective for lignin breakdown in vitro, and whether we can use these enzymes to produce aromatic chemicals.

We wish to carry out detailed mechanistic and structural studies on two recently characterized bacterial lignin-degrading enzymes, a Mn superoxide dismutase enzyme in Sphingobacterium sp. T2, and Dyp peroxidases from Pseudomonas fluorescens and Thermobifida fusca, and to identify further accessory enzymes required for bacterial lignin breakdown.

For Sphingobacterium Mn superoxide dismutase, we will study the catalytic cycle of this enzyme using pre-steady state kinetic methods, to establish whether the reactive species in the catalytic cycle is Mn(III) or hydroxyl radical. We will also seek to determine the crystal structure of this enzyme.

We have recently found a 35 kDa Dyp peroxidase enzyme from Pseudomonas fluorescens that is active in vitro against lignocellulose, releasing a small molecule product. We will determine the structure of the product, and study the mechanism for its formation, using lignin model compounds. We have also recently determined the crystal structure of a Dyp peroxidase from Thermobifida fusca, which we will use to carry out directed evolution studies on this enzyme, to enhance its activity towards lignin substrates.

As well as the primary lignin-oxidising enzymes, we believe that there are several important accessory enzymes and proteins for bacterial lignin degradation. In this project we will seek to identify an extracellular ferulate esterase enzyme responsible for hydrolysis of lignin-xylan ester linkages in lignocellulose; and we will seek to identify a redox protein responsible for quenching radical intermediates formed during lignin breakdown, for which we propose an extracellular dihydrolipoamide dehydrogenase as a likely candidate. We will then test combinations of accessory enzymes with Dyp peroxidases and Mn superoxide dismutase enzymes for bioconversion of lignin-containing feedstocks in vitro, and examine whether they can produce useful yields of small molecular aromatic products.

The production of useful chemicals from lignin breakdown in good yield would be of considerable interest to companies involved in commodity chemicals production, plastics & bio-plastics manufacture, and fine chemicals production, and would be of great interest to companies working in biofuel & biorefinery applications, who could use these discoveries to add value to waste streams. TDHB has a current TSB-funded pilot study with Biome Bioplastics Ltd, who are interested in the production of aromatic chemicals from lignin that could be used to make bioplastics. Novel enzymes for lignin breakdown would also be of considerable interest to biocatalysis & enzymes companies, and TDHB has had discussions in March 2013 with Ingenza Ltd in this area.

This research could therefore lead to intellectual property in areas of biotechnology, that would be protected with the advice of Warwick Ventures. The outputs of the research project would also be disseminated to local schools and the general public through an active schools outreach programme run by the Department of Chemistry, and through popular science articles.