Showing papers by "Novozymes published in 2013"
TL;DR: In this paper, the authors present a review of the findings of these studies and recommend further developments regarding environmental assessment and implementation of enzymatic processes and further support the hypothesis that enzyme technology is a promising means of moving toward cleaner industrial production.
306 citations
TL;DR: The high chemical consumption for alkaline pretreatment technology indicates that the main challenge for commercialization is chemical recovery, however, repurposing or co-locating a biorefinery with a paper mill would be advantageous from an economic point of view.
Abstract: Previous research on alkaline pretreatment has mainly focused on optimization of the process parameters to improve substrate digestibility. To achieve satisfactory sugar yield, extremely high chemical loading and enzyme dosages were typically used. Relatively little attention has been paid to reduction of chemical consumption and process waste management, which has proven to be an indispensable component of the bio-refineries. To indicate alkali strength, both alkali concentration in pretreatment solution (g alkali/g pretreatment liquor or g alkali/L pretreatment liquor) and alkali loading based on biomass solids (g alkali/g dry biomass) have been widely used. The dual approaches make it difficult to compare the chemical consumption in different process scenarios while evaluating the cost effectiveness of this pretreatment technology. The current work addresses these issues through pretreatment of corn stover at various combinations of pretreatment conditions. Enzymatic hydrolysis with different enzyme blends was subsequently performed to identify the effects of pretreatment parameters on substrate digestibility as well as process operational and capital costs. The results showed that sodium hydroxide loading is the most dominant variable for enzymatic digestibility. To reach 70% glucan conversion while avoiding extensive degradation of hemicellulose, approximately 0.08 g NaOH/g corn stover was required. It was also concluded that alkali loading based on total solids (g NaOH/g dry biomass) governs the pretreatment efficiency. Supplementing cellulase with accessory enzymes such as α-arabinofuranosidase and β-xylosidase significantly improved the conversion of the hemicellulose by 6–17%. The current work presents the impact of alkaline pretreatment parameters on the enzymatic hydrolysis of corn stover as well as the process operational and capital investment costs. The high chemical consumption for alkaline pretreatment technology indicates that the main challenge for commercialization is chemical recovery. However, repurposing or co-locating a biorefinery with a paper mill would be advantageous from an economic point of view.
287 citations
TL;DR: Analysis by ESR spectroscopy revealed increased radical intensities in sausages added plant extracts, which was ascribed to originate from protein-bound phenoxyl radicals, which may protect against other oxidatively induced protein modifications.
178 citations
TL;DR: Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production.
Abstract: Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.
169 citations
TL;DR: The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact.
Abstract: The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6–8 months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild Saccharomyces cerevisiae strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and baker's strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.
149 citations
TL;DR: The proposition that an ascorbate/oxygen biofuel cell could be a suitable power source for glucose-sensing contact lenses to be used for continuous health monitoring by diabetes patients is supported.
Abstract: A microscale membrane-less biofuel cell, capable of generating electrical energy from human lachrymal liquid, was developed by utilizing the ascorbate and oxygen naturally present in tears as fuel and oxidant. The biodevice is based on three-dimensional nanostructured gold electrodes covered with abiotic (conductive organic complex) and biological (redox enzyme) materials functioning as efficient anodic and cathodic catalysts, respectively. Three-dimensional nanostructured electrodes were fabricated by modifying 100 μm gold wires with 17 nm gold nanoparticles, which were further modified with tetrathiafulvalene-tetracyanoquinodimethane conducting complex to create the anode and with Myrothecium verrucaria bilirubin oxidase to create the biocathode. When operated in human tears, the biodevice exhibited the following characteristics: an open circuit voltage of 0.54 V, a maximal power density of 3.1 μW cm–2 at 0.25 V and 0.72 μW cm–2 at 0.4 V, with a stable current density output of over 0.55 μA cm–2 at 0.4 ...
145 citations
TL;DR: In this paper, an initial study of the implementation of two new enzymes, an endoglucanase and a concoction of hemicellulases and pectinases to obtain cellulosic nanoparticles was conducted.
Abstract: This paper is an initial study of the implementation of two new enzymes, an endoglucanase and a concoction of hemicellulases and pectinases to obtain cellulosic nanoparticles. In this study, curaua and sugarcane bagasse were dewaxed and bleached prior to enzymatic action for 72 h at 50 °C, and then followed by sonication. The concentration between these two enzymes was varied, and for the concentrations and time of enzymatic treatment used, subsequent sonication was necessary for cellulose nanoparticle release. It was easier to extract cellulose nanofibers from sugarcane bagasse which resulted in nanoparticles without damage of cellulose chains. On the other hand, curaua fibers needed a higher concentration of enzymes and the nanofibers obtained displayed a decrease of crystallinity suggesting that the cellulose structure was compromized. For both fibers, cellulose nanocrystals (single crystals) and larger diameter nanofibers were attained after the sonication.
107 citations
TL;DR: In this paper, the performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production in order to reduce the costs of the industrial enzymatic biodiesel production process.
92 citations
TL;DR: Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.
Abstract: Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.
88 citations
TL;DR: It is shown that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry, and the key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product.
84 citations
TL;DR: This work invented a novel method, using Dextran as carrier, for applying the water insoluble chemicals to the apical surface of the airway epithelia, and successfully tested some reference chemical compounds known to cause respiratory sensitisation in human beings, including MDI, TMA and HCPt.
TL;DR: The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported per oxygengenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production.
Abstract: The goal of this study is the selective oxy- functionalization of aliphatic compounds under mild and environmentally friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advan- tage from a new peroxidase type that catalyzes monoox- ygenase reactions with H2O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxyge- nase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identifiedbymassspectrometry.Regioselectivehydroxylation of saturated/unsaturated fatty acids was observed at the v-1 and v-2 positions (only at the v-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also v-1 or v-2 hydroxylated, but di- and tri-glycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the v-1 or v-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or di-hydroxyl- ated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production. Biotechnol. Bioeng. 2013;110: 2323-2332. 2013 Wiley Periodicals, Inc.
TL;DR: A deterministic model is applied to real-time kinetic data with high temporal resolution and finds that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.
Abstract: Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling...
TL;DR: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis and shows that RoBE preferentially creates new branches by intermolecular mechanism, a novel case for the thin border that exists between enzymes of the GH13 family.
TL;DR: A binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic Acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response.
Abstract: Malic acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity malic acid producers like the malic acid producer Aspergillus flavus have so far been disqualified because of special growth requirements or the production of mycotoxins. As A. oryzae is a very close relative or even an ecotype of A. flavus, it is likely that its high malic acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the malic acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on malic acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 +/- 1.05 g liter(-1), using peptone than the one of 22.27 +/- 0.46 g liter(-1) obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.
TL;DR: The particle formation process is mainly governed by the PLGA precipitation rate, which is solvent-dependent, and the migration rate of celecoxib molecules during drying, which can therefore be tailored by adjusting the solvent composition.
Abstract: Purpose
It is imperative to understand the particle formation mechanisms when designing advanced nano/microparticulate drug delivery systems. We investigated how the solvent power and volatility influence the texture and surface chemistry of celecoxib-loaded poly (lactic-co-glycolic acid) (PLGA) microparticles prepared by spray-drying.
TL;DR: In this article, the authors proposed a new method (baseline time accounting) that takes global land use dynamics into account that is consistent with the global warming potential, that is applicable to any phenomenon causing land use change, and that is independent of production period assumptions.
Abstract: Purpose Current estimations of the climate impact from indirect land use change (ILUC) caused by biofuels are heavily influenced by assumptions regarding the biofuel production period. The purpose of this paper is to propose a new method (baseline time accounting) that takes global land use dynamics into account that is consistent with the global warming potential, that is applicable to any phenomenon causing land use change, and that is independent of production period assumptions. Methods We consider ILUC in two forms. The first is called “accelerated expansion” and concerns ILUC in regions with an expanding agricultural area. The second is called “delayed reversion” and concerns ILUC in regions with a decreasing agricultural area. We use recent trends in international land use and projections of future land use change to assess how ILUC from biofuels will alter the development in global agricultural land use dynamics compared to the existing trend (i.e., the baseline development). We then use the definition of the global warming potential to determine the CO2 equivalence of the change in land use dynamics. Results and discussion We apply baseline time accounting to two existing ILUC studies in the literature. With current trends in global agricultural land use, the method significantly reduces the estimated climate impact in the previous ILUC studies (by more than half). Sensitivity analyses show that results are somewhat sensitive to assumptions regarding carbon sequestration and assumptions regarding postreversion ecosystems. Conclusions The global dynamic development in land use has important implications for the time accounting step when estimating the climate impact of ILUC caused by biofuel production or other issues affecting land use. Ignoring this may lead to erroneous conclusions about the actual climate impact of ILUC. Several land use projections indicate that the global agricultural area will keep expanding up to and beyond 2050. We therefore recommend to apply the baseline time accounting concept as an integrated part of future ILUC studies and to update the results on a regular basis.
TL;DR: In this paper, a biological pretreatment method of the high yielding grass variety Festulolium Hykor was tested as a biological pre-treatment method for cellulosic conversion.
Abstract: Grass biomass is a prospective type of lignocellulosic biomass for bioenergy and fuel production, but the low dry matter in grass at harvest calls for new pretreatment strategies for cellulosic conversion In this study, ensiling was tested as a biological pretreatment method of the high yielding grass variety Festulolium Hykor The biomass was harvested in four cuts over a growing season Three important factors of ensiling: biomass composition, dry matter (DM) at ensiling, and inoculation of lactic acid bacteria, were assessed in relation to subsequent enzymatic cellulose hydrolysis The organic acid profile after ensiling was dependant on the composition of the grass and the DM, rather than on the inocula High levels of organic acids, notably lactic acid, produced during ensiling improved enzymatic cellulose convertibility in the grass biomass Ensiling of less mature grass gave higher convertibility Low DM at ensiling (
TL;DR: A deterministic kinetic model that relies on a processive set of enzyme reactions and a quasi steady-state assumption is suggested, and it is shown that this approach is practicable in the sense that it leads to mathematically simple expressions for the steady‐state rate, and only requires data from standard assay techniques as experimental input.
Abstract: Processive enzymes perform sequential steps of catalysis without dissociating from their polymeric substrate. This mechanism is considered essential for efficient enzymatic hydrolysis of insoluble cellulose (particularly crystalline cellulose), but a theoretical framework for processive kinetics remains to be fully developed. In this paper, we suggest a deterministic kinetic model that relies on a processive set of enzyme reactions and a quasi steady-state assumption. It is shown that this approach is practicable in the sense that it leads to mathematically simple expressions for the steady-state rate, and only requires data from standard assay techniques as experimental input. Specifically, it is shown that the processive reaction rate at steady state may be expressed by a hyperbolic function related to the conventional Michaelis-Menten equation. The main difference is a 'kinetic processivity coefficient', which represents the probability of the enzyme dissociating from the substrate strand before completing n sequential catalytic steps, where n is the mean processivity number measured experimentally. Typical processive cellulases have high substrate affinity, and therefore this probability is low. This has significant kinetic implications, for example the maximal specific rate (V(max)/E₀) for processive cellulases is much lower than the catalytic rate constant (k(cat)). We discuss how relationships based on this theory may be used in both comparative and mechanistic analyses of cellulases.
TL;DR: The optimization of anode biocatalysts showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.
Abstract: After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 μW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.
TL;DR: The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase, exceeding usual physiological ranges of oxygen in human tissues and fluids.
Patent•
09 Dec 2013TL;DR: In this paper, a detergent composition comprising one or more anionic surfactants; an enzyme selected from the group consisting of: a protease, a lipase, cutinase, an amylase, carbocalase, carbohydrase, a cellulase, pectinase and an oxidase; and a deoxyribonuclease (DNase).
Abstract: The present invention concerns a detergent composition comprising one or more anionic surfactants; an enzyme selected from the group consisting of: a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, and an oxidase; and a deoxyribonuclease (DNase).
TL;DR: Identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate are reported.
Abstract: Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.
TL;DR: A kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst found that methanolysis of refined oil by CALB is slow, because triglycerides are the least reactive substrates.
Abstract: We have designed a kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst. The scheme assumed reversibility of all reaction steps and imitated phase effects by introducing various molecular species of water and methanol. The global model was assembled from separate reaction blocks analyzed independently. Computer simulations helped to explore behavior of the reaction system under different conditions. It was found that methanolysis of refined oil by CALB is slow, because triglycerides (T) are the least reactive substrates. Conversion to 95% requires 1.5–6 days of incubation depending on the temperature, enzyme concentration, glycerol inhibition, etc. Other substrates, free fatty acids (F), diglycerides (D) and monoglycerides (M), are utilized much faster (1–2 h). This means that waste oil is a better feedstock for CALB. Residual enzymatic activity in biodiesel of standard quality causes increase of D above its specification level because of the reaction 2M ↔ D + G. Filtration or alkaline treatment of the product prior to storage resolves this problem. The optimal field of Nz435 application appears to be decrease of F, M, D in waste oil before the conventional alkaline conversion. Up to 30-fold reduction of F-content can be achieved in 1–2 h, and the residual enzyme (if any) does not survive the following alkaline treatment.
TL;DR: This work reports the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast and shows that S. Cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol.
Abstract: Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–13C6 glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol.
TL;DR: Two in vitro, human cell based assays, based on the use of human keratinocytes, may be able to identify contact sensitizers and also to quantify sensitizer potency when used in a tiered strategy.
TL;DR: The results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.
Abstract: The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.
TL;DR: The data demonstrate that LP5 may have a dual mode of action against S. aureus, where at MIC concentrations, LP5 binds DNA and inhibits macromolecular synthesis and growth, whereas at concentrations above the MIC,LP5 targets the bacterial membrane leading to disruption of the membrane.
Abstract: Background: The increase in antibiotic resistant bacteria has led to renewed interest in development of alternative antimicrobial compounds such as antimicrobial peptides (AMPs), either naturally-occurring or synthetically-derived. Knowledge of the mode of action (MOA) of synthetic compounds mimicking the function of AMPs is highly valuable both when developing new types of antimicrobials and when predicting resistance development. Despite many functional studies of AMPs, only a few of the synthetic peptides have been studied in detail. Results: We investigated the MOA of the lysine-peptoid hybrid, LP5, which previously has been shown to display antimicrobial activity against Staphylococcus aureus. At concentrations of LP5 above the minimal inhibitory concentration (MIC), the peptoid caused ATP leakage from bacterial cells. However, at concentrations close to the MIC, LP5 inhibited the growth of S. aureus without ATP leakage. Instead, LP5 bound DNA and inhibited macromolecular synthesis. The binding to DNA also led to inhibition of DNA gyrase and topoisomerase IV and caused induction of the SOS response. Conclusions: Our data demonstrate that LP5 may have a dual mode of action against S. aureus. At MIC concentrations, LP5 binds DNA and inhibits macromolecular synthesis and growth, whereas at concentrations above the MIC, LP5 targets the bacterial membrane leading to disruption of the membrane. These results add new information about the MOA of a new synthetic AMP and aid in the future design of synthetic peptides with increased therapeutic potential.
TL;DR: The α-glucans synthesized from sucrose by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes.
TL;DR: The DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.
Abstract: Plant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow. A method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products. The DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.