Identification of an extracellular bacterial flavoenzyme that can prevent re-polymerisation of lignin fragments.

A significant problem in the oxidative breakdown of lignin is the tendency of phenolic radical fragments to re-polymerise to form higher molecular weight species. In this paper we identify an extracellular flavin-dependent dehydrolipoamide dehydrogenase from Thermobifida fusca that prevents oxidative dimerization of a dimeric lignin model compound, which could be used as an accessory enzyme for lignin depolymerisation.

Esterase EstK from Pseudomonas putida mt-2: An enantioselective acetylesterase with activity for deacetylation of xylan and poly(vinylacetate).

An extracellular esterase gene estK was identified in Pseudomonas putida mt-2 and overexpressed at high levels in Escherichia coli. The recombinant EstK enzyme was purified and characterized kinetically against p-nitrophenyl ester and other aryl-alkyl ester substrates and found to be selective for hydrolysis of acetyl ester substrates with high activity for p-nitrophenyl acetate (kcat 5.5 Sec-1 , KM 285 µM). Recombinant EstK was found to catalyze deacetylation of acetylated beech xylan, indicating a possible in vivo function for this enzyme, and partial deacetylation of a synthetic polymer (poly(vinylacetate)). EstK was found to catalyze enantioselective hydrolysis of racemic 1-phenylethyl acetate, generating 1R-phenylethanol with an enantiomeric excess of 80.4%.

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds.

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a ß-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.

Enzymatic conversion of lignin into renewable chemicals.

The aromatic heteropolymer lignin is a major component of plant cell walls, and is produced industrially from paper/pulp manufacture and cellulosic bioethanol production. Conversion of lignin into renewable chemicals is a major unsolved problem in the development of a biomass-based biorefinery. The review describes recent developments in the understanding of bacterial enzymes for lignin breakdown, such as DyP peroxidases, bacterial laccases, and beta-etherase enzymes. The use of pathway engineering methods to construct genetically modified microbes to convert lignin to renewable chemicals (e.g. vanillin, adipic acid) via fermentation is discussed, and the search for novel applications for lignin (e.g. carbon fibre).

Characterisation of Dyp-type peroxidases from Pseudomonas fluorescens Pf-5: Oxidation of Mn(II) and polymeric lignin by Dyp1B.

Members of the DyP family of peroxidases in Gram-positive bacteria have recently been shown to oxidise Mn(II) and lignin model compounds. Gram-negative pseudomonads, which also show activity for lignin oxidation, also contain dyp-type peroxidase genes. Pseudomonas fluorescens Pf-5 contains three dyp-type peroxidases (35, 40 and 55kDa), each of which has been overexpressed in Escherichia coli, purified, and characterised. Each of the three enzymes shows activity for oxidation of phenol substrates, but the 35kDa Dyp1B enzyme also shows activity for oxidation of Mn(II) and Kraft lignin. Treatment of powdered lignocellulose with Dyp1B in the presence of Mn(II) and hydrogen peroxide leads to the release of a low molecular weight lignin fragment, which has been identified by mass spectrometry as a ß-aryl ether lignin dimer containing one G unit and one H unit bearing a benzylic ketone. A mechanism for release of this fragment from lignin oxidation is proposed.

Assembly in vitro of Rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nanocompartment.

Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is cotranscribed with a gene encoding an encapsulin protein, which has been shown in Thermotoga maritima to assemble to form a 60-subunit nanocompartment, and DypB contains a C-terminal sequence motif that is thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein was overexpressed in R. jostii RHA1, and purified as a high-Mr assembly (Mr > 10(6)). The purified nanocompartment could be disassembled to form a low-Mr species by treatment at pH 3.0, and reassembled to form an assembly of similar size and shape, as assessed by dynamic light scattering. Recombinant DypB could be assembled in vitro with monomeric encapsulin to form an assembly of similar size to the encapsulin-only nanocompartment, as assessed by gel filtration. The assembled complex showed enhanced lignin degradation activity per milligram of DypB present as compared with native DypB, as determined with a nitrated lignin UV-visible assay method. The measured stoichiometry of 8.6 µmol encapsulin/µmol DypB in the complex was similar to the value of 10 predicted from the crystal structure.

Pathways for degradation of lignin in bacteria and fungi.

Lignin is a heterogeneous aromatic polymer found as 10-35% of lignocellulose, found in plant cell walls. The bio-conversion of plant lignocellulose to glucose is an important part of second generation biofuel production, but the resistance of lignin to breakdown is a major obstacle in this process, hence there is considerable interest in the microbial breakdown of lignin. White-rot fungi are known to break down lignin with the aid of extracellular peroxidase and laccase enzymes. There are also reports of bacteria that can degrade lignin, and recent work indicates that bacterial lignin breakdown may be more significant than previously thought. The review will discuss the enzymes for lignin breakdown in fungi and bacteria, and the catabolic pathways for breakdown of the ß-aryl ether, biphenyl and other components of lignin in bacteria and fungi. The review will also discuss small molecule phenolic breakdown products from lignin that have been identified from lignin-degrading microbes, and includes a bioinformatic analysis of the occurrence of known lignin-degradation pathways in Gram-positive and Gram-negative bacteria.

Histone H1 structural changes and its interaction with DNA in the presence of high glucose concentration in vivo and in vitro.

Histone H1 has an important role in the packing of chromatin and controlling gene expression. The effect of nonenzymatic glycation on the structure of histone H1 (a basic protein), the inhibitory role of spermine on this phenomenon and interaction of H1 with DNA is investigated here. H1 was extracted from the liver of normal and diabetic rats with or without receiving spermine as a chemical chaperone. Rat liver H1 was also incubated with glucose (50 mM) and its structure was compared with the protein obtained from diabetic rat. The results indicated a lower fluorescence emission and alpha-helical content of glycated H1; i.e., alteration in its folding; and reduced DNA (high molecular weight or specific sequences) binding in comparison with normal protein. Spermine partially (not completely) returns the studied parameters on glycated H1 toward the normal values. The changes in the structure and function of histone H1 is suggested as one of the possible mechanisms involved in diabetic complications.