Enzymatic Breakdown Research Articles

Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy

A novel time-lapse synchrotron deep-UV microscopy methodology was developed that made use of the natural tryptophan fluorescence of proteins. It enabled the monitoring in situ of the microstructural changes of protein gels during simulated gastric digestion. Two dairy gels with an identical composition, but differing by the coagulation mode, were submitted to static in vitro gastric digestion. The kinetics of gel particle breakdown were quantified by image analysis and physico-chemical analyses of digesta. The results confirm the tendency of rennet gels, but not acid gels, to form compact protein aggregates under acidic conditions of the stomach. Consequently, the kinetics of proteolysis were much slower for the rennet gel, confirming the hypothesis of a reduced pepsin accessibility to its substrate. The particle shapes remained unchanged and the disintegration kinetics followed an exponential trend, suggesting that erosion was the predominant mechanism of the enzymatic breakdown of dairy gels in these experimental conditions.

The Adenosinergic System as a Therapeutic Target in the Vasculature: New Ligands and Challenges.

Adenosine is an adenine base purine with actions as a modulator of neurotransmission, smooth muscle contraction, and immune response in several systems of the human body, including the cardiovascular system. In the vasculature, four P1-receptors or adenosine receptors—A1, A2A, A2B and A3—have been identified. Adenosine receptors are membrane G-protein receptors that trigger their actions through several signaling pathways and present differential affinity requirements. Adenosine is an endogenous ligand whose extracellular levels can reach concentrations high enough to activate the adenosine receptors. This nucleoside is a product of enzymatic breakdown of extra and intracellular adenine nucleotides and also of S-adenosylhomocysteine. Adenosine availability is also dependent on the activity of nucleoside transporters (NTs). The interplay between NTs and adenosine receptors’ activities are debated and a particular attention is given to the paramount importance of the disruption of this interplay in vascular pathophysiology, namely in hypertension., The integration of important functional aspects of individual adenosine receptor pharmacology (such as in vasoconstriction/vasodilation) and morphological features (within the three vascular layers) in vessels will be discussed, hopefully clarifying the importance of adenosine receptors/NTs for modulating peripheral mesenteric vascular resistance. In recent years, an increase interest in purine physiology/pharmacology has led to the development of new ligands for adenosine receptors. Some of them have been patented as having promising therapeutic activities and some have been chosen to undergo on clinical trials. Increased levels of endogenous adenosine near a specific subtype can lead to its activation, constituting an indirect receptor targeting approach either by inhibition of NT or, alternatively, by increasing the activity of enzymes responsible for ATP breakdown. These findings highlight the putative role of adenosinergic players as attractive therapeutic targets for cardiovascular pathologies, namely hypertension, heart failure or stroke. Nevertheless, several aspects are still to be explored, creating new challenges to be addressed in future studies, particularly the development of strategies able to circumvent the predicted side effects of these therapies.

How important are terrestrial organic carbon inputs for secondary production in freshwater ecosystems?

Abstract Many freshwater systems receive substantial inputs of terrestrial organic matter. Terrestrially derived dissolved organic carbon (t‐DOC) inputs can modify light availability, the spatial distribution of primary production, heat, and oxygen in aquatic systems, as well as inorganic nutrient bioavailability. It is also well‐established that some terrestrial inputs (such as invertebrates and fruits) provide high‐quality food resources for consumers in some systems. In small to moderate‐sized streams, leaf litter inputs average approximately three times greater than the autochthonous production. Conversely, in oligo/mesotrophic lakes algal production is typically five times greater than the available flux of allochthonous basal resources. Terrestrial particulate organic carbon (t‐POC) inputs to lakes and rivers are comprised of 80%–90% biochemically recalcitrant lignocellulose, which is highly resistant to enzymatic breakdown by animal consumers. Further, t‐POC and heterotrophic bacteria lack essential biochemical compounds that are critical for rapid growth and reproduction in aquatic invertebrates and fishes. Several studies have directly shown that these resources have very low food quality for herbivorous zooplankton and benthic invertebrates. Much of the nitrogen assimilated by stream consumers is probably of algal origin, even in systems where there appears to be a significant terrestrial carbon contribution. Amino acid stable isotope analyses for large river food webs indicate that most upper trophic level essential amino acids are derived from algae. Similarly, profiles of essential fatty acids in consumers show a strong dependence on the algal food resources. Primary production to respiration ratios are not a meaningful index to assess consumer allochthony because respiration represents an oxidised carbon flux that cannot be utilised by animal consumers. Rather, the relative importance of allochthonous subsidies for upper trophic level production should be addressed by considering the rates at which terrestrial and autochthonous resources are consumed and the growth efficiency supported by this food. Ultimately, the biochemical composition of a particular basal resource, and not just its quantity or origin, determines how readily this material is incorporated into upper trophic level consumers. Because of its highly favourable biochemical composition and greater availability, we conclude that microalgal production supports most animal production in freshwater ecosystems.

Optimizing isothiocyanate formation during enzymatic glucosinolate breakdown by adjusting pH value, temperature and dilution in Brassica vegetables and Arabidopsis thaliana

Consumption of glucosinolate-rich Brassicales vegetables is associated with a decreased risk of cancer with enzymatic hydrolysis of glucosinolates playing a key role. However, formation of health-promoting isothiocyanates is inhibited by the epithiospecifier protein in favour of nitriles and epithionitriles. Domestic processing conditions, such as changes in pH value, temperature or dilution, might also affect isothiocyanate formation. Therefore, the influences of these three factors were evaluated in accessions of Brassica rapa, Brassica oleracea, and Arabidopsis thaliana. Mathematical modelling was performed to determine optimal isothiocyanate formation conditions and to obtain knowledge on the kinetics of the reactions. At 22 °C and endogenous plant pH, nearly all investigated plants formed nitriles and epithionitriles instead of health-promoting isothiocyanates. Response surface models, however, clearly demonstrated that upon change in pH to domestic acidic (pH 4) or basic pH values (pH 8), isothiocyanate formation considerably increases. While temperature also affects this process, the pH value has the greatest impact. Further, a kinetic model showed that isothiocyanate formation strongly increases due to dilution. Finally, the results show that isothiocyanate intake can be strongly increased by optimizing the conditions of preparation of Brassicales vegetables.

Chewing bread: impact on alpha-amylase secretion and oral digestion.

During chewing, saliva helps in preparing the food bolus by agglomerating the formed particles, and it initiates enzymatic food breakdown. However, limited information is actually available on the adaptation of saliva composition during the oral processing of complex foods, especially for foods that are sensitive to salivary enzymes. We addressed this question in the context of starch-based products and salivary alpha-amylase. The objectives were two-fold: (1) to determine if salivary alpha-amylase secretion can be modulated by the bread type and (2) to evaluate the contribution of the oral phase in bread enzymatic breakdown. Mouthfuls of three different wheat breads (industrial, artisan and whole-meal breads) were chewed by twelve subjects. Saliva samples were collected at rest and at different times corresponding to 33, 66 and 100% of the individual's chewing sequence. Alpha-amylase activity and total protein content were determined for all saliva samples that were collected. Additionally, the salivary maltose concentration was measured as a marker of bread enzymatic digestion. Boluses were collected at the swallowing time to evaluate the saliva uptake. Chewing industrial bread induced higher saliva uptake than the other breads despite a similar chewing duration. The evolution of salivary amylase activity tended to depend on the type of bread and was highly influenced by a large degree of inter- and intra-subject variability. The protein and maltose concentration steadily increased during chewing as a result of bread breakdown. The salivary protein concentration was mainly affected by the release of the water-soluble proteins of the bread. The salivary maltose concentration was found to be significantly lower for the whole-meal bread. When considering the weight of the mouthful, enzymatic breakdown was found to be most efficient for the breads ranking from industrial > artisan > whole-meal.

Enzymology of Microbial Dimethylsulfoniopropionate Catabolism.

The biochemistry of dimethylsulfoniopropionate (DMSP) catabolism is reviewed. The microbes that catalyze the reactions central to DMSP catabolic pathways are described, and the focus is on the enzymology of the process. Approximately 109tons of DMSP is released annually by marine eukaryotes as an osmolyte. A vast majority of DMSP is assimilated by bacteria through either a demethylation or lyase pathways, producing either the methane thiol or the volatile dimethylsulfide (DMS), respectively. Enzymatic breakdown of DMSP generates ~107tons of DMS annually, which may have impact on global climate. DMS also acts as a chemoattractant for zooplanktons and seabirds. Both DMSP and DMS play a key role in the global sulfur cycle and are key nutrients for marine microbial growth. Important enzymes in the biochemical pathways of DMSP catabolism are covered in this review, with a focus on the latest developments in their mechanism.

Multiple pathways for elevating extracellular adenosine in the rat hippocampal CA1 region characterized by adenosine sensor cells.

Extracellular adenosine in the brain, which modulates various physiological and pathological processes, fluctuates in a complicated manner that reflects the circadian cycle, neuronal activity, metabolism, and disease states. The dynamics of extracellular adenosine in the brain are not fully understood, largely because of the lack of simple and reliable methods of measuring time-dependent changes in tissue adenosine distribution. This study describes the development of a biosensor, designated an adenosine sensor cell, expressing adenosine A1 receptor, and a genetically modified G protein. This biosensor was used to characterize extracellular adenosine elevation in brain tissue by measuring intracellular calcium elevation in response to adenosine. Placement of adenosine sensor cells below hippocampal slices successfully detected adenosine releases from these slices in response to neuronal activity and astrocyte swelling by conventional calcium imaging. Pharmacological analyses indicated that high-frequency electrical stimulation-induced post-synaptic adenosine release in a manner dependent on L-type calcium channels and calcium-induced calcium release. Adenosine release following treatments that cause astrocyte swelling is independent of calcium channels, but dependent on aquaporin 4, an astrocyte-specific water channel subtype. The ability of ectonucleotidase inhibitors to inhibit adenosine release following astrocyte swelling, but not electrical stimulation, suggests that the former reflects astrocytic ATP release and subsequent enzymatic breakdown, whereas the latter reflects direct adenosine release from neurons. These results suggest that distinct mechanisms are responsible for extracellular adenosine elevations by neurons and astrocytes, allowing exquisite regulation of extracellular adenosine in the brain.

236 - Hydrogen Sulfide Increases GSH Biosynthesis, Glucose Uptake and Utilization in Mouse Myotubes

Diabetes is a leading cause of chronic diseases, such as heart disease. Diabetic patients have lower blood concentrations of H 2 S and L-Cysteine (LC). LC undergoes enzymatic breakdown to produce hydrogen sulphide (H 2 S), a gasotransmitter that regulates glucose and lipid homeostasis. Using C2C12 mouse myoblast (differentiated into myotubes) as a model, this study investigates the hypothesis that beneficial effects of LC supplementation are mediated by the upregulation of H 2 S status in diabetic patients. Results show that exogenous administration of Sodium hydrosulfide (NaHS 10µM; 6 hours), a H 2 S donor, significantly ( p 2 S production induced by using CSE siRNA caused an increase in ROS, and down regulation of potent antioxidant GSH biosynthesis enzymes (GCLC, Glutathione Synthetase) but not GGLM. Further, CSE knockdown downregulate Glucose Transporter Type 4 (GLUT4) mRNA level and reduces glucose uptake in C2C12 myotubes. Taken together, these data suggest that the upregulation of physiological levels of H 2 S can have beneficial effects on glucose homeostasis and redox balance via GSH pathway. Further mechanistic studies are under investigation on how H 2 S regulate GSH biosynthesis and L-Cysteine transporter (SLC7A11) in the maintenance of glucose homeostasis. Supported by NIH/NCCIH RO1 AT007442

Glycoside Hydrolase Family 51 α‐l‐Arabinofuranosidases from Clostridium thermocellum and Cellvibrio japonicus Release O–5‐Feruloylated Arabinose

Arabinofuranosidases act synergistically with other enzymes to depolymerize arabinoxylans by cleaving arabinofuranose substituents from the β‐(1→4)‐linked d‐xylopyranose backbone. Because arabinose feruloylation is a barrier to some, but not all, arabinofuranosidases, we investigated the actions of three α‐l‐arabinofuranosidases from the glycoside hydrolase (GH) family 51 on feruloylated arabinoxylan‐oligosaccharide standard compounds with and without feruloyl esterase. GH51 α‐l‐arabinofuranosidases from Clostridium thermocellum and Cellvibrio japonicus both partially released feruloylated arabinose (up to 59% for C. thermocellum). Simultaneous incubation with arabinofuranosidases and feruloyl esterase quantitatively released arabinose from feruloylated standard compounds. Therefore, although feruloylation does not completely obstruct GH51 arabinofuranosidases, synergistic approaches utilizing multiple enzymes remain the most effective tactic for enzymatic breakdown of feruloylated compounds.

Doping control container for urine stabilization: a pilot study.

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd.

Chronic wasting disease of deer – is the battle to keep Europe free already lost?

CHRONIC wasting disease (CWD) is a prion disease affecting many species of deer, which was, until recently, confined to North America. However, confirmed diagnoses in Norway in both reindeer (...

Subsecond Regulation of Synaptically Released Dopamine by COMT in the Olfactory Bulb.

The efficacy of neurotransmission depends on multiple factors, including presynaptic vesicular release of transmitter, postsynaptic receptor populations and clearance/inactivation of the transmitter. In the olfactory bulb (OB), short axon cells (SACs) form an interglomerular circuit that uses GABA and dopamine (DA) as cotransmitters. Selective optical activation of SACs causes GABA and DA co-release, resulting in a fast, postsynaptic GABA inhibitory response and a slower G-protein-coupled DA rebound excitation. In most systems, vesicular release of DA is cleared by the dopamine transporter (DAT). However, in the OB, high levels of specific DA metabolites suggest that enzymatic catalysis by catechol-O-methyl-transferase (COMT) predominates over DAT re-uptake. To assess this possibility we measured the amount of the DA breakdown enzyme, COMT, present in the OB. Compared with the striatum, the brain structure richest in DA terminals, the OB contains 50% more COMT per unit of tissue. Furthermore, the OB has dramatically less DAT compared with striatum, supporting the idea that COMT enzymatic breakdown, rather than DAT recycling, is the predominant mechanism for DA clearance. To functionally assess COMT inactivation of vesicular release of DA we used fast-scan cyclic voltammetry and pharmacological blockade of COMT. In mice expressing ChR2 in tyrosine hydroxylase-containing neurons, optical activation of SACs evoked robust DA release in the glomerular layer. The COMT inhibitor, tolcapone, increased the DA signal ∼2-fold, whereas the DAT inhibitor GBR12909 had no effect. Together, these data indicate that the OB preferentially employs COMT enzymatic inactivation of vesicular release of DA. In the olfactory bulb (OB), odors are encoded by glomerular activation patterns. Dopaminergic short axon neurons (SACs) form an extensive network of lateral connections that mediate cross talk among glomeruli, releasing GABA and DA onto sensory nerve terminals and postsynaptic neurons. DA neurons are ∼10-fold more numerous in OB than in ventral tegmental areas that innervate the striatum. We show that OB has abundant expression of the DA catalytic enzyme catechol-O-methyl-transferase (COMT), but negligible expression of the dopamine transporter. Using optogenetics and fast-scan cyclic voltammetry, we show that inhibition of COMT increases DA signals ∼2-fold. Thus, in contrast to the striatum, which has the brain's highest proportion of DAergic synapses, the DA catalytic pathway involving COMT predominates over re-uptake in OB.

Risk and prevention strategies can help avert tumor lysis syndrome

Risk and prevention strategies can help avert tumor lysis syndrome

Effect of Pulp Concentration during Cellulase Pretreatment on Microfibrillated Cellulose and Its Film Properties

Pulp concentration was increased, during preparation of microfibrillated cellulose (MFC), in an effort to improve the efficiency of cellulase pretreatment. It was hypothesized that increased pulp concentration could possibly increase the interactions between cellulase and cellulose, therefore improving the cellulase pretreatment efficiency and benefiting MFC and its film properties. Results showed that higher pulp concentration enhanced the cellulase adsorption ratio from 70% to 90% for pulp concentrations of 2% and 10%, during the pretreatment process. While pulp concentration was changed from 2% to 10% during cellulase pretreatment, the specific area rose from 30.1 m2/g to 35.5 m2/g. Compared with the original eucalyptus pulp, the crystallinity degree of different pulp concentration was increased, presumably due to the enzymatic breakdown of amorphous cellulose. In addition, the aspect ratio of MFC rose from 19.1 to 35.5. Concurrently, MFC film properties showed better performance, as the elongation at break increased from 0.75% to 1.95%, tensile strength increased from 15.3 MPa to 33.5 MPa, and oxygen permeability coefficient decreased from 111×10-14 cm3·cm/cm2·s·Pa to 89.7×10-14 cm3·cm/cm2·s·Pa, reflecting the oxygen barrier properties of MFC film.

Insights into the functionality and stability of designer cellulosomes at elevated temperatures.

Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50°C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6h at 60°C. Finally, within this time frame and conditions, the full complex appeared over 50% more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

Identification and Overexpression of a Knotted1-Like Transcription Factor in Switchgrass (Panicum virgatum L.) for Lignocellulosic Feedstock Improvement.

High biomass production and wide adaptation has made switchgrass (Panicum virgatum L.) an important candidate lignocellulosic bioenergy crop. One major limitation of this and other lignocellulosic feedstocks is the recalcitrance of complex carbohydrates to hydrolysis for conversion to biofuels. Lignin is the major contributor to recalcitrance as it limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars. Therefore, genetic manipulation of the lignin biosynthesis pathway is one strategy to reduce recalcitrance. Here, we identified a switchgrass Knotted1 transcription factor, PvKN1, with the aim of genetically engineering switchgrass for reduced biomass recalcitrance for biofuel production. Gene expression of the endogenous PvKN1 gene was observed to be highest in young inflorescences and stems. Ectopic overexpression of PvKN1 in switchgrass altered growth, especially in early developmental stages. Transgenic lines had reduced expression of most lignin biosynthetic genes accompanied by a reduction in lignin content suggesting the involvement of PvKN1 in the broad regulation of the lignin biosynthesis pathway. Moreover, the reduced expression of the Gibberellin 20-oxidase (GA20ox) gene in tandem with the increased expression of Gibberellin 2-oxidase (GA2ox) genes in transgenic PvKN1 lines suggest that PvKN1 may exert regulatory effects via modulation of GA signaling. Furthermore, overexpression of PvKN1 altered the expression of cellulose and hemicellulose biosynthetic genes and increased sugar release efficiency in transgenic lines. Our results demonstrated that switchgrass PvKN1 is a putative ortholog of maize KN1 that is linked to plant lignification and cell wall and development traits as a major regulatory gene. Therefore, targeted overexpression of PvKN1 in bioenergy feedstocks may provide one feasible strategy for reducing biomass recalcitrance and simultaneously improving plant growth characteristics.

Concentrations of thiocyanate and goitrin in human plasma, their precursor concentrations in brassica vegetables, and associated potential risk for hypothyroidism

Brassica vegetables are common components of the diet and have beneficial as well as potentially adverse health effects. Following enzymatic breakdown, some glucosinolates in brassica vegetables produce sulforaphane, phenethyl, and indolylic isothiocyanates that possess anticarcinogenic activity. In contrast, progoitrin and indolylic glucosinolates degrade to goitrin and thiocyanate, respectively, and may decrease thyroid hormone production. Radioiodine uptake to the thyroid is inhibited by 194 μmol of goitrin, but not by 77 μmol of goitrin. Collards, Brussels sprouts, and some Russian kale (Brassica napus) contain sufficient goitrin to potentially decrease iodine uptake by the thyroid. However, turnip tops, commercial broccoli, broccoli rabe, and kale belonging to Brassica oleracae contain less than 10 μmol of goitrin per 100-g serving and can be considered of minimal risk. Using sulforaphane plasma levels following glucoraphanin ingestion as a surrogate for thiocyanate plasma concentrations after indole glucosinolate ingestion, the maximum thiocyanate contribution from indole glucosinolate degradation is estimated to be 10 μM, which is significantly lower than background plasma thiocyanate concentrations (40-69 μM). Thiocyanate generated from consumption of indole glucosinolate can be assumed to have minimal adverse risks for thyroid health.

Hyaluronic Acid Suppresses the Expression of Metalloproteinases in Osteoarthritic Cartilage Stimulated Simultaneously by Interleukin 1β and Mechanical Load.

PurposeIn patients with osteoarthritis (OA), intraarticular injection of hyaluronic acid (HA) frequently results in reduced pain and improved function for prolonged periods of time, i.e. more than 6 months. However, the mechanisms underlying these effects are not fully understood. Our underlying hypothesis is that HA modifies the enzymatic breakdown of joint tissues.MethodsTo test this hypothesis, we examined osteochondral cylinders from 12 OA patients. In a bioreactor, these samples were stimulated by interleukin 1β (Il1ß) (2 ng/ml) plus mechanical load (2.0 Mpa at 0.5 Hz horizontal and 0.1 Hz vertical rotation), thus the experimental setup recapitulated both catabolic and anabolic clues of the OA joint.ResultsUpon addition of HA at either 1 or 3 mg/ml, we observed a significant suppression of expression of metalloproteinase (MMP)-13. A more detailed analysis based on the Kellgren and Lawrence (K&L) OA grade, showed a much greater degree of suppression of MMP-13 expression in grade IV as compared to grade II OA. In contrast to the observed MMP-13 suppression, treatment with HA resulted in a suppression of MMP-1 expression only at 1 mg/ml HA, while MMP-2 expression was not significantly affected by either HA concentration.ConclusionTogether, these data suggest that under concurrent catabolic and anabolic stimulation, HA exhibits a pronounced suppressive effect on MMP-13. In the long-run these findings may benefit the development of treatment strategies aimed at blocking tissue degradation in OA patients.

Designer Plants for Biofuels: A Review

Injudicious over-consumption of fossil fuels over the last century has generated an imbalance in the carbon cycle, dramatically increasing atmospheric carbon dioxide and contributing to climate change. Much of the released carbon is ancient, accumulating in the earth’s crust in the form of oil, coal or methane gas over millions of years. In contrast, the carbon in biofuels has only recently been sequestered from the atmosphere as a result of photosynthesis. From this perspective, the release of carbon dioxide from biofuels can be viewed as carbon neutral making biofuels particularly attractive as an energy source. First generation biofuels relied primarily on cereal starch as a source of glucose for biofuel production, a practice that faced scrutiny due to its competition for human food, its impact on biodiversity and reliance on chemical fertilizers and fossil fuels for harvest. Second generation biofuels focused on lignocellulosic waste as a carbon resource for bioenergy production. Cellulosic biomass is the most abundant of the renewable resources and represents an enormous storehouse of sugars. However, plant cell walls are chemically complex, heterogeneous structures that have evolved to counter the process of enzymatic digestion. The layered structure with cross-linkages among cellulose, xylan and lignin are examples of the recalcitrant moieties that resist enzymatic breakdown. The use of exogenous enzymes or chemical treatment to weaken these barriers is expensive and typically achieves only partial hydrolysis. Recent advancements in plant genetic engineering have demonstrated the potential to design plants for improved cell wall deconstruction as well as to produce cell wall carbohydrases within the plant biomass. These technologies offer significant promise to lower the cost of production of biofuels from lignocellulosic biomass. In this review, we provide summary of recent research efforts focused on improving the biomass characteristics of plants for biofuel production through alteration of the major cell wall biosynthesis genes or pathways. Keywords: Biofuel, biomass, cell wall, genetic engineering, lignin, lignocellulose, transcription factor.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.