Size-exclusion chromatography of large molecules from coal liquids,petroleum residues,soots, biomass tars and humic substances

Size-exclusion chromatography (SEC) using 1-methyl-2-pyrrolidinone (NMP) as eluent has been calibrated using various standard polymers and model compounds and applied to the analysis of extracts of coal, petroleum and kerogens, to petroleum vacuum residues, soots, biomass tars and humic substances. Three separate columns of different molecular mass (MM) ranges were used, with detection by UV absorption; an evaporative light scattering detector was used for samples with no UV absorption. Fractionation was useful to separate signal from the less abundant high-mass material, which was normally masked by the strong signal from the more abundant low-mass material in the absence of fractionation. Fractionation methods used to isolate high-mass materials before SEC analysis included planar chromatography, column chromatography and solvent solubility. The apparently large molecules were concentrated into the fractions not soluble in common solvents and were relatively immobile in planar chromatography. All samples and fractions contained some material excluded from the column porosity. Evidence from other techniques suggests that the excluded material is of different structures from that of the resolved material rather than consisting of aggregates of small molecules. We speculate that the excluded material may elute early because the structures of this material are three-dimensional rather than planar or near planar.     

Origin of fungal biomass degrading enzymes: Evolution,diversity and function of enzymes of early lineage fungi

The aim of this study was to elucidate the evolution of enzyme secretome of early lineage fungi to contribute to resolving the basal part of Fungal Kingdom and pave the way for industrial evaluation of their unique enzymes. By combining results of advanced sequence analysis with secretome mass spectrometry and phylogenetic trees, we provide evidence for that plant cell wall degrading enzymes of higher fungi share a common ancestor with enzymes from aerobic ancient fungi. Sequence analysis (HotPep, confirmed by dbCAN-HMM models) enabled prediction of enzyme function directly from sequence. For the first time, oxidative enzymes are described here in early lineage fungi (Chytridiomycota & Cryptomycota), which supports the conceptually new understanding that fungal LPMOs were also present in the early evolution of the Fungal Kingdom. Phylogenetic analysis of fungal AA9 proteins suggests an LPMO-common-ancestor with Ascomycetes and Basidiomycetes and describes a new clade of AA9s. We identified two very strong biomass degraders, Rhizophlyctis rosea (soil-inhabiting) and Neocallimastix californiae (rumen), with a rich spectrum of cellulolytic, xylanolytic and pectinolytic enzymes, characteristically including several different enzymes with the same function. Their secretome composition suggests horizontal gene transfer was involved in transition to terrestrial and rumen habitats. Methods developed for recombinant production and protein characterization of enzymes from zoosporic fungi pave the way for biotechnological exploitation of unique enzymes from early lineage fungi with potential to contribute to improved biomass conversion. The phyla of ancient fungi through evolution have developed to be very different and together they constitute a rich enzyme discovery pool.     

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.     

From molecular biology to biotics: the development of bio-, info- and nano-technologies.

Important scientific and technological developments have been achieved in biology during the last few years. Through the application of these developments, biotechnology will have a growing influence on the life sciences and their industrial use in the near future. This influence is evident in new drug design and in the development of diagnostic tests, bioelectronic equipment, and services. It has been observed that three fields of study are becoming increasingly interdependent: the molecular, the computational, and the mechanical. This convergence is achieved through the still closer relationships between biotechnologies, infotechnologies, nanotechnologies and microelectronics.     

Proximate analyses and predicting HHV of chars obtained from cocracking of petroleum vacuum residue with coal, plastics and biomass

Higher heating value (HHV) and analysis of chars obtained from cocracking of petroleum vacuum residue (XVR) with coal (SC), biomass (BG, CL) and plastics (PP, PS, BL) are important which define the energy content and determine the clean and efficient use of these chars. The main aim of the present study is to analyze the char obtained from cocracking in terms of their proximate analysis data and determination of the HHV of the chars. The char obtained from XVR+PP cocracking showed a HHV of 32.84 MJ/kg, whereas that from CL cracking showed a HHV of 18.52 MJ/kg. The experimentally determined heating values of the char samples obtained from cocracking have been correlated with the theoretical equation based on proximate analysis data. There exists a variety of correlations for predicting HHV from proximate analysis of fuels. Based upon proximate analysis data, the models were tested. The best results show coefficient of determination (R2) of 0.965 and average absolute and bias error of 3.07% and 0.41%, respectively. The heating values obtained from the model were in good agreement with that obtained by experiment. Proximate analysis of the chars obtained from the cocracking of XVR with coal, biomass and plastics showed that there exists a definite interaction of the reactive species, when they were cocracked together.     

Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides

Background  

High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides.     

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.     

Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.     

Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.     

Microbial arsenic: from geocycles to genes and enzymes

Arsenic compounds have been abundant at near toxic levels in the environment since the origin of life. In response, microbes have evolved mechanisms for arsenic resistance and enzymes that oxidize As(III) to As(V) or reduce As(V) to As(III). Formation and degradation of organoarsenicals, for example methylarsenic compounds, occur. There is a global arsenic geocycle, where microbial metabolism and mobilization (or immobilization) are important processes. Recent progress in studies of the ars operon (conferring resistance to As(III) and As(V)) in many bacterial types (and related systems in Archaea and yeast) and new understanding of arsenite oxidation and arsenate reduction by respiratory-chain-linked enzyme complexes has been substantial. The DNA sequencing and protein crystal structures have established the convergent evolution of three classes of arsenate reductases (that is classes of arsenate reductases are not of common evolutionary origin). Proposed reaction mechanisms in each case involve three cysteine thiols and S-As bond intermediates, so convergent evolution to similar mechanisms has taken place.     

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging ‘omics’-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia.     

Glycollate-pathway enzymes in mitochondria from phototrophic,organotrophic and mixotrophic cells of Euglena

Glycollate dehydrogenase and NADFH-glyoxylate reductase are constitutive enzymes in Percoll-purified mitochondria from phototrophic, mixotrophic and organotrophic cells of Euglena gracilis Klebs strain z Pringsheim. Glycollate oxidation by isolated mitochondria is stimulated four-fold by the addition of glutamate but rates of glycine oxidation are low in mitochondria from all cell types, the ratio of malate to glycine oxidation always being greater than 4:1. Measurement of the rate of NADPH oxidation in intact mitochondria and mitoplasts showed that the outer mitochondrial membrane is impermeable to NADPH and in the absence of NADPH-dehydrogenase activity the oxidation of NADPH by mitoplasts is dependent on the presence of glyoxylate for NADPH-glyoxylate-reductase activity. It is concluded that glycollate oxidation in the mitochondrion provides glyoxylate which, in the presence of a suitable amino-donor, can be converted to glycine by glutamate-glyoxylate amino-transferase so providing essential intermediates for biosynthesis. Glycollate oxidation outside the mitochondrion is concerned with photorespiratory metabolism and the inability of mitochondria to oxidise exogenous glycine at appreciable rates means that the separation of photorespiratory metabolism from the biosynthesis of essential intermediates is effected.     

Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells,bind Tudor and protect from transposable elements

Germ cells give rise to all cell lineages in the next‐generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single‐particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.     

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O₂ demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized. An erratum to this article can be found at