Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

Biosynthesis and separation of dextran fructose mixtures in a chromatographic reactor

A literature review of work carried out on batch and continuous chromatographic biochemical reactor-separators has been made. The major part of this work has involved the development of a batch chromatographic reactor-separator for the production of dextran and fructose by the enzymatic action of the enzyme dextransucrase on sucrose. In this reactor, simultaneous reaction and separation occurs thus reducing downstream processing and isolation of products as compared to the existing industrial process. The chromatographic reactor consisted of a glass column packed with a stationary phase consisting of cross linked polysytrene resin in the calcium form. The mobile phase consisted of diluted dextransucrase in deionised water. Initial experiments were carried out on a reactor separtor which had an internal diameter of 0.97cm and length of 1.5m. To study the effect of scale up the reactor diameter was doubled to 1.94cm and length increased to 1.75m. The results have shown that the chromatographic reactor uses more enzyme than a conventional batch reactor for a given conversion of sucrose and that an increase in void volume results in higher conversions of sucrose. A comparison of the molecular weight distribution of dextran produced by the chromatographic reactor was made with that from a conventional batch reactor. The results have shown that the chromatographic reactor produces 30% more dextran of molecular weight greater than 150,000 daltons at 20% w/v sucrose concentration than conventional reactors. This is because some of the fructose molecules are prevented as acting as acceptors in the chromatographic reactor due to their removal from the reaction zone. In the conventional reactor this is not possible and therefore a greater proportion of low molecular weight dextran is produced which does not have much clinical use. A theoretical model was developed to describe the behaviour of the reactor separator and this model was simulated using a computer. The simulation predictions showed good agreement with experimental results at high eluent flowrates and low conversions.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Avaliação do potencial da fibra e casca de coco maduro, casca de coco verde e cacto pré-tratados visando à produção de etanol

The present work investigated the potential of different residual lignocellulosic materials generated in rural and urban areas (coconut fibre mature, green coconut shell and mature coconut shell), and vegetable cultivated in inhospitable environments (cactus) aimed at the production of ethanol, being all materials abundant in the Northeast region of Brazil. These materials were submitted to pretreatments with alkaline hydrogen peroxide followed by sodium hydroxide (AHP-SHP), autohydrolysis (AP), hydrothermal catalyzed with sodium hydroxide (HCSHP) and alkali ethanol organosolv (AEOP). These materials pretreated were submitted to enzymatic hydrolysis and strategies of simultaneous saccharification and fermentation (SSF) and saccharification and fermentation semi-simultaneous (SSSF) by Saccharomyces cerevisiae, Zymomonas mobilis and Pichia stipitis. It was also evaluated the presence of inhibitory compounds (hydroxymethylfurfural, furfural, acetic acid, formic acid and levulinic acid) and seawater during the fermentative process. Materials pretreated with AHP-SHP have resulted in delignification of the materials in a range between 54 and 71%, containing between 51.80 and 54.91% of cellulose, between 17.65 and 28.36% of hemicellulose, between 7.99 and 10.12% of lignin. Enzymatic hydrolysis resulted in the conversions in glucose between 68 and 76%. Conversion yields in ethanol using SSF and SSSF for coconut fibre mature pretreated ranged from 0.40 and 0.43 g/g, 0.43 and 0.45 g/g, respectively. Materials pretreated by AP showed yields of solids between 42.92 and 92.74%, containing between 30.65 and 51.61% of cellulose, 21.34 and 41.28% of lignin. Enzymatic hydrolysis resulted in glucose conversions between 84.10 and 92.52%. Proceeds from conversion into ethanol using green coconut shell pretreated, in strategy SSF and SSSF, were between 0.43 and 0.45 g/g. Coconut fibre mature pretreated by HCSHP presented solids yields between 21.64 and 60.52%, with increased in cellulose between 28.40 and 131.20%, reduction of hemicellulose between 43.22 and 69.04% and reduction in lignin between 8.27 and 89.13%. Enzymatic hydrolysis resulted in the conversion in glucose of 90.72%. Ethanol yields using the SSF and SSSF were 0.43 and 0.46 g/g, respectively. Materials pretreated by AEOP showed solid reductions between 10.75 and 43.18%, cellulose increase up to 121.67%, hemicellulose reduction up to 77.09% and lignin reduced up to 78.22%. Enzymatic hydrolysis resulted in the conversion of glucose between 77.54 and 84.27%. Yields conversion into ethanol using the SSF and SSSF with cactus pretreated ranged from 0.41 and 0.44 g/g, 0.43 and 0.46 g/g, respectively. Fermentations carried out in bioreactors resulted in yields and ethanol production form 0.42 and 0.46 g/g and 7.62 and 12.42 g/L, respectively. The inhibitory compounds showed negative synergistic effects in fermentations performed by P. stipitis, Z. mobilis and S. cerevisiae. Formic acid and acetic acid showed most significant effects among the inhibitory compounds, followed by hydroxymethylfurfural, furfural and levulinic acid. Fermentations carried out in culture medium diluted with seawater showed promising results, especially for S. cerevisiae (0.50 g/g) and Z. mobilis (0.49 g/g). The different results obtained in this study indicate that lignocellulosic materials, pretreatments, fermentative processes strategies and the microorganisms studied deserve attention because they are promising and capable of being used in the context of biorefinery, aiming the ethanol production.

Avaliação da influência de surfactantes químico e biológico na hidrólise enzimática de casca de coco verde após pré-tratamento ácido/alcalino e com peróxido de hidrogênio alcalino

In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolipíd for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.

Avaliação da influência de surfactantes químico e biológico na hidrólise enzimática de casca de coco verde após pré-tratamento ácido/alcalino e com peróxido de hidrogênio alcalino

In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolipíd for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine-tuned system for cohesin-dockerin recognition

Funded by United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel Israel Science Foundation (ISF). Grant Number: 1349 Israel Science Foundation Israel Strategic Alternative Energy Foundation (I-SAEF) BBSRC. Grant Number: BB/L009951/1 Scottish Government Food, Land and People program Society for Applied Microbiology

Enzymatic profiling of cellulosomal enzymes from the human gut bacterium, Ruminococcus champanellensis, reveals a fine-tuned system for cohesin-dockerin recognition

Funded by United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel Israel Science Foundation (ISF). Grant Number: 1349 Israel Science Foundation Israel Strategic Alternative Energy Foundation (I-SAEF) BBSRC. Grant Number: BB/L009951/1 Scottish Government Food, Land and People program Society for Applied Microbiology

Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase

We thank EPSRC and the Scottish Imaging Network (SINAPSE) for grants. DO’H thanks the Royal Society for a Wolfson Research Merit Award and ST is grateful to the John and Kathleen Watson Scholarship for financial support. We are grateful to Dr Catherine Botting and Dr Sally Shirran of the St Andrews Mass Spectrometry Service for MALDI-MS acquisitions. We also thank Dr Sally Pimlott of the University of Glasgow for the use of radiochemistry facilities.

Last-step enzymatic [18F]-fluorination of cysteine-tethered RGD peptides using modified Barbas linkers

Acknowledgements We thank the Engineering and Physical Sciences Research Council, UK, for a research grant. Funded by Engineering and Physical Sciences Research Council, UK