Transesterification with CE15 glucuronoyl esterase from Cerrena unicolor reveals substrate preferences.

PURPOSE: Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from Cerrena unicolor (CuGE) can reveal the enzyme's preference for the alcohol-part of the ester-bond. METHODS: This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-O-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme's alcohol preferences. CONCLUSION: CuGE preferentially forms the γ-ester from the methyl ester of 4-O-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme's substrate preferences are primarily dictated by the presence of the 4-O-methylation on the glucuronoyl donor, and secondly on the type of alcohol.

Substrate specificity and transglycosylation capacity of α-L-fucosidases across GH29 assessed by bioinformatics-assisted selection of functional diversity.

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N- and O-linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison.

Enzymatic production of a suite of human milk oligosaccharides directly in milk.

Human milk oligosaccharides (HMOs) denote specific glycans in human breast milk. They function as prebiotics, immune modulating, and antimicrobial agents in the gut of breastfed infants, and certain HMOs even promote the cognitive development of the baby. HMOs are virtually absent in cow's milk and hence in infant formula, which provides a huge incentive for identifying ways in which HMOs can be produced to improve infant formulas. Here, we show that different sialylated and fucosylated HMOs can be generated in cow's milk via different simultaneous enzymatic transglycosylation reactions catalyzed by an engineered sialidase (EC 3.2.1.18, from Trypanosoma rangeli) and an 1,2-α-L-fucosidase (EC 3.2.1.63, from Tannerella forsinthia) acting on the lactose in the milk and on casein glycomacropeptide, two types of commercially available HMOs, i.e. 2'-fucosyllactose and lacto-N-neotetraose, added to the milk. We also outline the details of the individual reactions in aqueous systems, demonstrate that the enzymatic reactions can be accomplished at 5 °C, and validate the products formed by LC-MS and NMR analysis. Enzymatic production of HMOs directly in milk provides opportunities for enriching milk and infant formulas and extends the use of enzymatic transglycosylation reactions to synthesis of HMOs in milk and eventually in other beverages.

A new FTIR assay for quantitative measurement of endo-fucoidanase activity.

Endo-fucoidanases, including EC 3.2.1.211 endo-α-1,3-L-fucanase and EC 3.2.1.212 endo-α-1,4-L-fucanase activities, catalyze depolymerization of fucoidans - a group of bioactive, sulfated fucosyl-polysaccharides found primarily in brown macroalgae (brown seaweeds). Quantitative assessment of endo-fucoidanase activity is critical for characterizing endo-fucoidanase kinetics and for comparing the action of different endo-fucoidanases on different types of fucoidans. However, the current state-of-the-art endo-fucoidanase assay consists of a qualitative assessment based on Carbohydrate-Polyacrylamide Gel Electrophoresis. Here, we report a new quantitative endo-fucoidanase assay based on real time spectral evolution profiling of changes in substrate and product during endo-fucoidanase action using Fourier Transform InfraRed spectroscopy (FTIR) combined with Parallel Factor Analysis (PARAFAC). The FTIR-PARAFAC assay was validated by monitoring the reaction progress of three different microbial endo-fucoidanase enzymes, FcnAΔ229, FFA2 and Fhf1Δ470, on two different fucoidan substrates. The substrates were purified from the brown macroalgae Fucus evanescens and Fucus vesiculosus, respectively. The evolution profiling showed that the strongest spectral change of the fucoidans during enzymatic depolymerization occurred in the spectral range 1220-1260 cm-1, but the profiles differed depending on the substrate and the enzyme used. Spectral changes within 1220-1260 cm-1 are in agreement with the enzymatic depolymerization inducing signature changes in the mid-infrared absorption of sulfated fucosyls as sulfate ester bonds and C-O stretching vibrations absorb in this spectral region. Based on the data obtained, we also introduce an activity unit for endo-fucoidanases: One endo-fucoidanase Unit, Uf, is the amount of enzyme able to catalyze a change in the FTIR-PARAFAC score by 0.01 during 498 s of reaction (8.3 min) on 20 g/L pure fucoidan from F. evanescens at 42 °C, pH 7.4, 100 mM NaCl and 10 mM CaCl2. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

May standard basal echocardiogram allow to obtain predictors of asymptomatic cardiac dysfunction in alcoholics?

BACKGROUND: Long-lasting heavy alcohol intake has been progressively recognized as a leading cause of nonischemic dilated cardiomyopathy, involving 10% of all people who use alcohol. It is of huge importance to identify the earliest markers of this dysfunction and it is known that the newest echocardiographic techniques such as speckle tracking may allow to do it. In this study we investigated if standard basal echocardiogram features allow to obtain predictors of asymptomatic cardiac dysfunction in alcoholics. METHODS: A population of 80 consecutive asymptomatic alcoholics was enrolled. None presented history, signs or symptoms of cardiovascular disease. All of them underwent a conventional transthoracic monobidimensional and doppler echocardiography. RESULTS: Our cohort did not present echocardiographic findings of increased left ventricular sizes, mass or relative wall thickness. Hence, a significant rate of systolic dysfunction was not found. Furthermore, statistical analysis displayed an inverse relationship between alcohol consumption and systolic pulmonary arterial pressure as well as between alcohol abuse and left atrium enlargement. This may be explained by a potential vasodilator mechanism occurring in the earliest stages of alcohol intake. On the contrary, a positive correlation with the E/A ratio was found, and this might be ascribed to state of high cardiac output determined by alcohol abuse. There were modes sex-related differences. CONCLUSIONS: This study has demonstrated that standard echocardiography may allow to predict cardiac dysfunction in asymptomatic alcoholics, and sex-related differences may be identified in this regard. These data need to be confirmed by further studies involving larger population.

Enzymatic production of 3'-sialyllactose in milk.

Human milk oligosaccharides (HMOs) are lactose-based glycan molecules present in human breast milk. HMOs are essentially not present in cow's milk and hence not naturally available in infant formulas. HMOs possess several health and developmentally beneficial properties, and the sialylated HMOs are thought to play a particularly important role for infant brain development. Enzymatic transsialylation directly in cow's milk, involving enzyme catalyzed transfer of sialic acid from a sialic acid donor to an acceptor, is a novel route for producing sialylated HMOs for e.g. infant formulas. The transsialidase (EC 2.4.1.-) of Trypanosoma cruzi is linked to trypanosomatid pathogenicity, but certain hydrolytic sialidases (neuraminidases), EC 3.2.1.18, from non-pathogenic organisms, can actually catalyze transsialylation. Here, we report enzymatic production of the HMO compound 3'-sialyllactose directly in cow's milk using engineeredsialidases, Tr15 and Tr16, originating from the nonpathogenic Trypanosoma rangeli. Both Tr15 and Tr16 readily catalyzed transsialylation in milk at 5 °C-40 °C using κ(kappa)-casein glycomacropeptide (cGMP) as sialyl donor substrate. Tr15 was the most efficient as this enzyme produced 1160 mg/L (1.8 mM) 3'-sialyllactose in whole milk during 10 min of reaction at 5 °C. The activation energy values, Ea, of the enzymatic transsialylation reactions were similar in milk and in buffer solutions containing cGMP and lactose. The Ea of the Tr15 catalyzed transialylation reaction in milk was 16.5 kJ/mol, which was three times lower than the Ea of Tr16 (66 kJ/mol) and of T. cruzi transsialidase (50 kJ/mol), corroborating that Tr15 was the fastest of the three enzymes and a promising candidate for potential industrial production of 3'-sialyllactose in milk. 3'sialyllactose was stable during pasteurization (30 min. at 62.5 °C) and freeze-drying.

Laccase activity measurement by FTIR spectral fingerprinting.

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis. We employed FTIR to monitor enzymatic oxidation of monolignols (sinapyl, coniferyl and p-coumaryl alcohol), sinapic acid, and sinapic aldehyde by four different laccases: three fungal laccases from Trametes versicolor, Trametes villosa and Ganoderma lucidum, respectively, and one bacterial laccase from Meiothermus ruber. By coupling the FTIR measurements with Parallel Factor Analysis (PARAFAC) we established a quantitative assay for assessing laccase activity. By combining PARAFAC modelling with Principal Component Analysis we show the usefulness of this technology as a multivariate tool able to compare and distinguish different laccase reaction patterns. We also demonstrate how the FTIR approach can be used to create a reference system for laccase activity comparison based on a relatively low number of measurements. Such a reference system has potential to function as a high-throughput method for comparing reaction pattern similarities and differences between laccases and hereby identify new and interesting enzyme candidates in large sampling pools.

Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS.

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.

On the Origins and Dissemination of Domesticated Sorghum and Pearl Millet across Africa and into India: a View from the Butana Group of the Far Eastern Sahel.

Four decades have passed since Harlan and Stemler (1976) proposed the eastern Sahelian zone as the most likely center of Sorghum bicolor domestication. Recently, new data on seed impressions on Butana Group pottery, from the fourth millennium BC in the southern Atbai region of the far eastern Sahelian Belt in Africa, show evidence for cultivation activities of sorghum displaying some domestication traits. Pennisetum glaucum may have been undergoing domestication shortly thereafter in the western Sahel, as finds of fully domesticated pearl millet are present in southeastern Mali by the second half of the third millennium BC, and present in eastern Sudan by the early second millennium BC. The dispersal of the latter to India took less than 1000 years according to present data. Here, we review the middle Holocene Sudanese archaeological data for the first time, to situate the origins and spread of these two native summer rainfall cereals in what is proposed to be their eastern Sahelian Sudan gateway to the Red Sea and the Indian Ocean trade.

Quatre décennies se sont écoulées depuis que Harlan et Stemler ont proposé la zone sahélienne orientale comme le centre le plus probable de la domestication du sorgho bicolore. Récemment, de nouvelles données sur les impressions de semences sur les poteries du groupe Butana du IVe millénaire avant JC dans la région sud d'Atbai dans la ceinture sahélienne d'Afrique orientale montrent des preuves d'activités de culture du sorgho présentant certains traits de domestication. Pennisetum glaucum pourrait être en cours de domestication peu après dans l'ouest du Sahel, puisque le millet perlé entièrement domestiqué est. présent dans le sud-est du Mali vers la seconde moitié du troisième millénaire avant J.-C. et présent dans l'est du Soudan au début du deuxième millénaire avant notre ère. La dispersion de ce dernier en Inde a pris moins de mille ans selon les données actuelles. Nous examinons ici pour la première fois les données archéologiques soudanaises de l'Holocène moyen pour situer les origines et la propagation de ces deux céréales de pluie estivales indigènes dans ce qui est. proposé comme leur porte d'entrée soudanienne du Sahel oriental à la mer Rouge et à l'océan Indien.

Quantitative enzymatic production of sialylated galactooligosaccharides with an engineered sialidase from Trypanosoma rangeli.

Sialylated galactooligosaccharides (GOS) represent a potential infant formula ingredient, which is believed to contribute with a combination of the beneficial properties of the prebiotic GOS as well as of sialylated human milk oligosaccharides. Sialylated GOS do not exist in natural milk, but can be produced from κ(kappa)-casein glycomacropeptide (CGMP), a sialylated side stream component from cheese-making, by sialidase-catalyzed transsialylation. Using a rationally designed mutant of the sialidase from Trypanosoma rangeli, Tr13, with enhanced transsialylation activity, six different GOS preparations with a varying degree of polymerization (DP) were effectively sialylated with molar yields of 20-30% on the CGMP sialyl in batch reactions. The rate of sialylation of the individual DPs was largely dependent on the DP distribution in each GOS preparation, and Tr13 catalysis did not discriminate against large GOS molecules, providing the novelty point that GOS molecules are sialylated independently of their size by Tr13. Using CGMP, GOS, and Tr13, the production of gram-scale quantities of sialyl-GOS was achieved in 20L volume reactions. Compared to the benchmark transsialidase from pathogenic Trypanosoma cruzi, the Tr13 was significantly more thermostable. By employing an enzymatic membrane reactor, Tr13 could be recycled and after seven consecutive 1-h reaction cycles, the biocatalytic productivity of the enzyme was increased 7-fold compared to the batch reaction. Assuming that the enzyme may be specific for α-2,3-bound sialyl moieties only, and that only 50% of sialyl linkages in CGMP are α-2,3-linked, the molar yield of sialyl-GOS on the available α-2,3-bound sialyl moieties in CGMP reached 80% in the enzymatic membrane reactor system.