Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components is not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance, to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biologica...

Enzymatic hydrolysis of cellulosic biomass

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

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In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

Domination of the global biosphere by human beings is unprecedented in the history of the planet, and our impact is such that substantive changes in ecosystems, and the global environment as a whole, are now becoming apparent. Our activity drives the steady increase in global temperature observed in recent decades. The realization of the adverse effects of greenhouse gas emissions on the environment, together with declining petroleum reserves, has ensured that the quest for sustainable and environmentally benign sources of energy for our industrial economies and consumer societies has become urgent in recent years. Consequently, there is renewed interest in the production and use of fuels from plants. The 'first-generation' biofuels made from starch and sugar appear unsustainable because of the potential stress that their production places on food commodities. Second-generation biofuels, produced from cheap and abundant plant biomass, are seen as the most attractive solution to this problem, but a number of technical hurdles must be overcome before their potential is realized. This review will focus on the underpinning research necessary to enable the cost-effective production of liquid fuels from plant biomass, with a particular focus on aspects related to plant cell walls and their bioconversion.

Sustainable liquid biofuels from biomass: the writing's on the walls

http://www.salmone.org/wp-content/uploads/2008/09/transgenics-are-imperative-for-biofuel-crops.pdf

Transgenics are imperative for biofuel crops

Laccases (benzenediol: oxygen oxidoreductase; EC 11032), a multicopper oxidase enzyme, widely distributed in plants, fungi and bacteria have ability to catalyze oxidation of various phenolic and non-phenolic compounds as well as many environmental pollutants The diversified functions of laccases, including the antagonistic ones such as their involvement in lignin biosynthesis (in plants) as well as lignin degradation (in fungi and bacteria), make them an interesting enzyme for study from the point of view of their structure, function and application Important applications of laccases include delignification, pulp bleaching and bioremediation The ability of laccases to polymerize natural phenols helps to develop new cosmetic pigments, hair dyeing materials, deodorants, toothpastes, mouthwashes and other useful products Recently, the utility of enzyme has also been shown in the nanoparticle based biosensor technology as well as in medical fields In the present review, a comparative account of the bacterial, fungal and plant laccases has been presented from these points of views Laccases are dimeric or tetrameric glycoproteins usually containing four copper atoms per monomer To perform catalytic function, laccase depends on Cu atoms that are distributed at the three different copper centers These copper centers in laccases are categorized into three groups: Type-1 or blue copper center, Type-2 or normal copper and Type-3 or coupled binuclear copper centers The four copper atoms are differing in their characteristic electronic paramagnetic resonance (EPR) signals The phylogenetic analysis reveals, laccases from these groups (viz bacteria, fungi and plant) form independent clades, in consistent with that of taxonomical classification From previous experimental evidence and from in silico studies, it is evident that despite their wide taxonomic distribution and substrate diversity, molecular architecture of laccases is common to multicopper oxidases Three dimensional structure predictions, at monomeric level, for all laccases (bacterial, fungal and plant) suggest that they are composed of three sequentially arranged cupredoxin-like domains Multiple alignment of primary sequences of all three modeled laccases shows that, the copper binding motives are highly conserved in all sequences The similarities are more significant in the N- and C-terminal regions, corresponding to Domains 1 and 3, as the copper interacting motif is present in Domains 1 and 3 not in Domain 2 This structural conservation reflects a common reaction mechanism for the copper oxidation and the O2 reduction in these enzymes Putative binding pocket analysis depicts, larger binding cavity for bacterial laccase as compared to those for plants and fungi An in depth analysis of copper binding site, yielded significant differences in conserved residues for laccases of bacteria, fungi and plants which provided the basis for the dual and contrasting functions of laccases

Structure–function relationship among bacterial, fungal and plant laccases

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Improved recovery of active recombinant laccase from maize seed.

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Manganese peroxidase from the white‐rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed

Jeff Lane

Papers

Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

Improved recovery of active recombinant laccase from maize seed.

Manganese peroxidase from the white‐rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed