Identification and cultivation of hydrogenotrophic methanogens from palm oil mill effluent for high methane production

By means of biorefinery, biogas production through anaerobic digestion is one of the most common treatments of wastewater in the palm oil industry. After biogas production, the treated palm oil mill effluent (POME) is generally discharged into the environment. However, certain level of hazardous compounds still exists in the treated wastewater, which can lead to the pollution of water bodies. In this study, we have investigated the dynamics of volatile organic acids dwelling in consecutive POME treatment lagoons as well as identified, and categorized, microbial species responsible for the treatment process. Bacteria and methanogens, both hydrogenotrophic and acetoclastic, related to methane production were identified using mcrA and 16S rRNA genes specific primers. Two hydrogenotrophic methanogens, Methanoculleus marisnigri and Methanoculleus chikugoensis, were found abundant in accordance with high formate concentration throughout the process of anaerobic digestion. This study has also isolated eight consortia of microbes that yielded different methane productions by utilizing formate as the substrate in the synthetic medium. The consortia of a group, containing M. marisnigri, M. chikugoensis, uncultured bacteria, Aminobacterium sp., and Ruminobacillus xylanolyticum, produced the highest methane yield of 259 mL/g COD after 25 days of incubation in the laboratory. The findings from this study are contributing to optimize and increase biogas production in POME, which will allow higher efficiency in palm oil mill wastewater treatment.     

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.     

Functional properties of protein isolate obtained from physic nut (<Emphasis Type="Italic">Jatropha curcas</Emphasis> L.) seed cake

Physic nut (Jatropha curcas L.) protein isolate was successfully achieved from physic nut seed cake by an alkaline extraction and followed by an isoelectric precipitation. The protein isolate had small amounts of phorbol esters, phytic acid, and saponin without any lectin. Its minimum and maximum solubility were at pH 4.0 and 12.0, respectively. Its water and oil binding capacities were 3.22 g water/g protein and 1.86mL oil/g protein, respectively. Its foaming capacity and emulsion activity showed high values in a range of basic pHs. Its foaming and emulsion stability values decreased with increasing time and exhibited high levels under basic pH conditions. Physic nut protein isolate had unique functional properties in water binding capacity, emulsion activity, and emulsion stability indicating an important role in food systems. It may be applied to salad dressing, mayonnaise, sausage, and meat products. Therefore, physic nut seed cake has a potential to be exploited as a novel source of functional protein for food or feed applications.     

Magnetic field effects as a result of the radical pair mechanism are unlikely in redox enzymes

Environmental exposure to electromagnetic fields is potentially carcinogenic. The radical pair mechanism is considered the most feasible mechanism of interaction between weak magnetic fields encountered in our environment and biochemical systems. Radicals are abundant in biology, both as free radicals and reaction intermediates in enzyme mechanisms. The catalytic cycles of some flavin-dependent enzymes are either known or potentially involve radical pairs. Here, we have investigated the magnetic field sensitivity of a number of flavoenzymes with important cellular roles. We also investigated the magnetic field sensitivity of a model system involving stepwise reduction of a flavin analogue by a nicotinamide analogue—a reaction known to proceed via a radical pair. Under the experimental conditions used, magnetic field sensitivity was not observed in the reaction kinetics from stopped-flow measurements in any of the systems studied. Although widely implicated in radical pair chemistry, we conclude that thermally driven, flavoenzyme-catalysed reactions are unlikely to be influenced by exposure to external magnetic fields.     

Identification of a catalytic base for sugar oxidation in the pyranose 2-oxidase reaction

Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK(a) >10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK(a) value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K(d) for D-glucose binding was 19 times greater.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering

We have employed computational approaches—FireProt and FRESCO—to predict thermostable variants of the reductase component (C₁) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C₁ variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6–5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C₁ variants remain active and generate reduced flavin mononucleotide (FMNH⁻) for reactions catalyzed by bacterial luciferase and by the monooxygenase C₂ more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300–500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C₁ enzyme can lead to broad-spectrum uses of C₁ as a redox biocatalyst for future industrial applications.     

Identification of Ellagic Acid in Blackberry Juice Sediment

ABSTRACT: The sediment in a commercial reconstituted 'Evergreen' blackberry ( Rubus laciniatus L.) juice concentrate was found to be composed of ellagic acid, protein, and other unidentified compounds. The qualitative tannin and protein-tannin haze test indicated that the sediment was predominantly tannin or protein-tannin complexes. Nitrogen determination showed the sediment to be 6.69%± 2.21% protein on a dry-weight basis. Almost all of the extractable material was identified as ellagic acid by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The ellagic acid content of the wet sediment was 0.05 g/100 g, whereas it was 7.41 g/100 g in freeze-dried sediment. Tannase enzyme did not significantly decrease the concentration of ellagitannins in Marion blackberry ( Rubus spp. Hyb.) juice in this study.