Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis

The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

Purification and Interaction of β-amylase from Dioscorea alata (Water Yam) with Epicatechin

β-amylase is an enzyme that hydrolyzes the α-1,4-glucan bonds from the non-reducing ends of starch and other carbohydrate polymers reducing it to maltose units. Maltose has much application with food processing and pharmaceutical industries. The enzyme was purified to apparent homogeneity with a monomeric molecular weight of 30.1 kDa based on SDS-PAGE. The binding Constant (Ka), Kd ΔH, ΔS and ΔG values were 1.53´103Lmol-1 ,3.12x10-4Lmol-1, 19.35kJmol-1, 56.67Jmol-1K-1, and -18.17kJmol-1 respectively. The binding profile of β-amylase with epicatechin was spontaneous with a stoichiometric ratio of 2:1. Hydrophobic bonding played a major role in stabilizing the β-amylase-ligand complex. The mode of reaction was by static quenching. It further dictates that the binding reaction is entropy driven. The inhibitory effect of this plant polyphenols on β-Amylase might contribute to the regulation of β-Amylase activity in plants.

Legislation and Legislative Process in Eastern Europe

The study describes and systemises the constitutional requirements on legislation in Eastern Europe. The comparison reveals that the basic structures of the legislative process live up to the standards of the rule of law. The details, however, are quite frequently deficient or problematic. Laws requiring a qualified majority often cause structural problems, based on poor political culture, and the vague and contradictory regulatory framework. Other problems are a legacy of socialism, e.g. the instrumental perception of the law, or the immature separation of powers. However, the apparent homogeneity of the region and its structural problems that was typical of the socialist era, has given way to a stronger differentiation which often reflects differences that existed prior to the socialist dictatorship. This stronger differentiation concerns, i.a. the extent of executive law-making, the structure of parliament (mono- or bicameral), the majority requirement for the decisions in parliament, and the participation of the people in legislation. In the states that have joined the EU, the European criteria of the rule of law have had their effect, whereas the candidate states on the Wester Balkans are on the way of consolidating their legislative system. Further to the East, the rule of law becomes weaker and weaker.

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production

Background Rhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications. Results An extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was non-specific lipase and the utmost activity was recorded at pH 6, 40 °C with high stability for 30 min. The constants Km and Vmax, calculated from the Lineweaver-Burk plot, are 0.3 mg/mL and 208.3 U/mL, respectively. Monovalent metal ions such as Na+ (1 and 5 mM) and K+ (5 mM) were promoters of the lipase to enhance its activity with 110, 105.5, and 106.5%, respectively. Chitosan was used as a perfect support for immobilization via both adsorption and cross-linking in which the latter method attained immobilization efficiency of 99.1% and reusability of 12 cycles. The partially purified enzyme proved its ability in forming methyl oleate (biodiesel) through the esterification of oleic acid and transesterification of olive oil. Conclusion The partially purified and immobilized lipase from Rhizopus oryzae R1 approved excellent efficiency, reusability, and a remarkable role in detergents and biodiesel production.

Biochemical Characterization of purified β-amylase from Dioscorea alata (water yam)

Water yam (Dioscorea alata) β-amylase was isolated and purified to apparent homogeneity. The enzyme was found to be monomeric with a molecular weight of 30.1 kDa based on SDS-PAGE and 31.0 kDa based on non-denaturation gel filtration. The enzyme readily hydrolyzed soluble starch, amylose, amylopectin and glycogen. The enzyme was stable over a wide range of pHs (4 - 8). The enzyme has a Km, of 2.24 mg/ml for soluble starch. Activation energy (Ea) for catalysis by β-amylase at 25 0C was 6.45 kcal/mol. β-amylase Contains high content of both acidic and hydrophobic amino acid but low in arginine and histidine. The activation energy (Ea), half-life, free energy change (ΔG‡), enthalpy change (ΔH‡), and entropy change (ΔS‡) for inactivation were 13.92 kcal/mol, 41.25 min, 20.89 kcal/mol, 13.30 kcal/mol and -24.25 cal/mol/K, respectively. The binding profile of β-amylase with epicatechin, quercetin, rutin and gallic acid were all spontaneous with stoichiometric ratio of 1:1. Hydrophobic bonding played a major role in stabilizing the β-amylase-ligand complex. Sodium alginate immobilization of the enzyme improved the Optimum temperature from 40 to 50 0C and changed the optimum pH from 5.0 to 6.0 with retainment up to 67 % activity after 5 cycles of usage. The enzyme would be of importance in manufacturing company based on the kinetics and other parameters reported in this study.

Stable isotopic insights into crop cultivation, animal husbandry, and land use at the Linearbandkeramik site of Vráble-Veľké Lehemby (Slovakia)

The plant and animal components of Linearbandkeramik (LBK) subsistence systems were remarkably uniform with cattle, emmer and einkorn wheat providing the primary source of sustenance for Europe’s earliest agricultural communities. This apparent homogeneity in plant and animal use has been implicitly understood to indicate corresponding similarity in the types of husbandry practices employed by LBK farmers across the entire distribution of the LBK culture. Here, we examine the results from the stable (δ13C/δ15N) isotope analysis of animal bone and cereal grains from the site of Vráble-Veľké Lehemby (Slovakia), providing new information about Linearbandkeramik farming practices in the western Carpathians. Moderately high carbon isotope values from animal bone collagen show that all livestock were pastured in open areas with no evidence of forest pasturing, previously associated with LBK settlements in north-western Europe. High δ15N values measured from domesticated cereal grains suggest manuring took place at the site, while 15N enrichment in bone collagen suggest livestock fed on agricultural by-products and possibly grains. An integrated plant-animal management system was in use at Vráble where livestock grazed on cultivation plots post-harvest. Use of such strategy would have helped fatten animals before the lean winter months while simultaneously fertilising agricultural plots with manure. This study contributes to our growing understanding that although the building blocks of LBK subsistence strategies were remarkably similar, diversity in management strategies existed across central and north-western Europe.

Kinetics and Computational Evaluation of Eugenol and Vanillic Acid on Inhibition of a Potential Enzyme of a Nosocomial Pathogen that Promotes Struvite Formation

Background: Struvite/infection stone is one of the major clinical burdens in urinary tract infections that is caused by the ureolytic behavior of pathogenic bacteria. Objective: The current strategy for treating infective stones is mostly antibiotic therapy, which ends in promoting resistance to the organisms. Hence in the present study, we investigated two phytocompounds, eugenol (an allyl-substituted guaiacol) and vanillic acid (a phenolic acid) that are found to be effective in inhibiting the urease enzyme of a nosocomial pathogen Proteus mirabilis. Methods: The enzyme was purified to apparent homogeneity and the kinetic parameters were studied in the presence and in the absence of eugenol and vanillic acid. Molecular docking and simulation were done to understand the level of protein-ligand interactions and the interacting residues. Results: Kinetic parameters obtained from the Michaelis-Menten plot show that both eugenol and vanillic acid exhibit non-competitive inhibition of urease enzyme in a dose-dependent manner. In silico studies showed that eugenol and vanillic acid have almost similar binding affinities to the regulatory pocket of the modeled protein. Dynamics and simulation results indicate that the interaction of ligands with the ARG373 residue of the protein provides a stable bound conformation. Conclusion: Overall, our results suggest that both the phytocompounds eugenol and vanillic acid have a potential application as a new therapy for the inhibition of urease enzyme that could possibly replace the complexions related to struvite stone formation.

A Thermostable and Alkalitolerant Arabinofuranosidase by Streptomyces lividus

Aim: The study aimed at producing and purifying thermostable and alkalitolerant microbial arabinofuranosidase using local Palm Kernel Cake (PKC) as substrate. Study Design: This is an experimental design in which samples were collected thrice and subjected to laboratory analyses from which quantitative data were obtained and analysed. Place and Duration of Study: Ibadan, Nigeria, Five months. Methodology: Bacterial strains were isolated from degrading PKC by serial dilution and pour plate technique on formulated Modified Basal Salt Agar Medium and incubated at 50°C for enzyme activity screening. Plates were afterwards flooded with 1% congo red solution for visualization of hydrolysis zone. Its arabinofuranosidase activity was optimized in solid state fermentation in PKC. Production temperature, pH, moisture content, inoculum size and agitation were studied for optimization test. Optimal production temperature and pH for arabinofuranosidase by isolate was 45°C and pH 9. Produced arabinofuranosidase was purified to apparent homogeneity with ammonium sulphate precipitation, dialysis and column chromatography techniques. Stability of arabinofuranofuranosidase obtained to temperature, pH, substrate concentration and some ions was determined as well as its molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Isolate with highest arabinofuranosidase activity was selected and identified as Streptomyces lividus. Purity level attained was 16.36 fold. Enzyme had a specific activity of 25.4 U/mg, and total enzyme activity of 13.2 U. Molecular weight of enzyme appeared as a band of 30 kDa. Purified arabinofuranosidase enzyme revealed optimum temperature and pH as 60oC and 9 respectively. Enzyme was stable over a broad pH range of 3-11, and temperature of 30-80oC. Residual activity after incubating for 1 hour at 70oC was 64%. Enzyme kinetics studies showed Km and Vmax values for P-nitrophenyl arabinofuranoside were 2.3mM and 0.7U/min respectively. Conclusion: Apart from Solid State Fermentation (SSF) of PKC being a potential fermentation technique for production of arabinofuranosidase by Streptomyces lividus, the enzyme was highly stable.

A new serine protease family with elastase activity is produced by Streptomyces bacteria

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

Analysis of contents of annual sustainability reports of industrial companies participating in the sustainability index of the Brazilian Stock Exchange

The objective of this article is to analyze the content of environmental disclosure of industrial companies participating in the Corporate Sustainability Index (ISE) of the Brazilian Stock Exchange (B3) from 2009 to 2016, based on the Stakeholders’ Theory perspective. We adopt a descriptive approach, with qualitative and quantitative analysis of secondary data that we analyze by means of content analysis and statistical techniques. The data collection took place from the institutional site of each organization to obtain the Annual Sustainability Reports. The reports went through text analysis, which resulted in an inventory of words related to the concept of sustainability. The results indicate that the analyzed companies have similarities with regard to the content disclosed in the Annual Sustainability Reports. Such similarities occur in spite of the fact that companies operate in different industrial segments (petrochemical, food, wood, pulp and paper products and personal use products) and probably come from a homogeneous perception of the segments interested in the information, which would be in reduced number. Although the surveyed companies operate in different segments, they are subject to similar pressures by stakeholders and that imposes an apparent homogeneity to their reports.