An extremely alkaline mannanase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS428 hydrolyzes galactomannan producing series of mannooligosaccharides

An alkaline-thermostable mannanase from Streptomyces sp. CS428 was produced, purified, and biochemically characterized. The extracellular mannanase (Mn428) was purified to homogeneity with 12.4 fold, specific activity of 2406.7 U/mg, and final recovery of 37.6 %. The purified β-mannanase was found to be a monomeric protein with a molecular mass of approximately 35 kDa as analyzed by SDS-PAGE and zymography. The first N-terminal amino acid sequences of mannanase enzyme were HIRNGNHQLPTG. The optimal temperature and pH for enzyme were 60 °C and 12.5, respectively. The mannanase activities were significantly affected by the presence of metal ions, modulators, and detergents. K_m and V_max values of Mn428 were 1.01 ± 3.4 mg/mL and 5029 ± 85 µmol/min mg, respectively when different concentrations (0.6–10 mg/mL) of locust bean gum galactomannan were used as substrate. The substrate specificity of enzyme showed its highest specificity towards galactomannan which was further hydrolyzed to produce mannose, mannobiose, mannotriose, and a series of mannooligosaccharides. Mannooligosaccharides can be further converted to ethanol production, thus the purified β-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.     

Studies on seasonal dynamics of soil-higher fungal communities in Mongolian oak-dominant Gwangneung forest in Korea

We surveyed macrofungi biweekly at defined plots from April to December in 2014, in the Mongolian oak-dominant forest, Gwangneung Forest, Pochen-si, Korea, and analyzed a soilhigher fungal diversity during four seasons (represented by April, August, October, and December). Based on morphological observation of collected specimens, the collected macrofungi were classified into 2 phyla 3 classes 7 orders, 14 families, 21 genera, and 33 species (36 specimens). DNA-based community analyses indicated that soil-higher fungi were classified into 2 phyla, 18 classes, 49 orders, 101 families, and 155 genera (83,360 sequence reads), defined herein as 155 genus-level operational taxonomic units (GOTUs). In the present study, we evaluated and discussed the fungal diversity in seasonal dynamics and soil layers based on collected macrofungi and pyrosequencing data while considering environmental parameters (pH, exchangeable K, T-P, NH ₄ ⁺ , NO ₃ ^- , OM, WR, TOC, and T-N). Moreover, principal components analysis (PCA) showed distinct clusters of the GOTU assemblage associated with the seasons.     

Crystal structure and modeling of the tetrahedral intermediate state of methylmalonate-semialdehyde dehydrogenase (MMSDH) from <Emphasis Type="Italic">Oceanimonas doudoroffii</Emphasis>

The gene product of dddC (Uniprot code G5CZI2), from the Gram-negative marine bacterium Oceanimonas doudoroffii, is a methylmalonate-semialdehyde dehydrogenase (OdoMMSDH) enzyme. MMSDH is a member of the aldehyde dehydrogenase superfamily, and it catalyzes the NADdependent decarboxylation of methylmalonate semialdehyde to propionyl-CoA. We determined the crystal structure of OdoMMSDH at 2.9 Å resolution. Among the twelve molecules in the asymmetric unit, six subunits complexed with NAD, which was carried along the protein purification steps. OdoMMSDH exists as a stable homodimer in solution; each subunit consists of three distinct domains: an NAD-binding domain, a catalytic domain, and an oligomerization domain. Computational modeling studies of the OdoMMSDH structure revealed key residues important for substrate recognition and tetrahedral intermediate stabilization. Two basic residues (Arg103 and Arg279) and six hydrophobic residues (Phe150, Met153, Val154, Trp157, Met281, and Phe449) were found to be important for tetrahedral intermediate binding. Modeling data also suggested that the backbone amide of Cys280 and the side chain amine of Asn149 function as the oxyanion hole during the enzymatic reaction. Our results provide useful insights into the substrate recognition site residues and catalytic mechanism of OdoMMSDH.     

Antioxidative effect of phycoerythrin derived from <Emphasis Type="Italic">Grateloupia filicina</Emphasis> on rat primary astrocytes

Astrocytes, which support neuronal tissue and activity in the brain, are receiving attention as a possible target for treating neurological damage. Phycoerythrin extract, a pigment protein of red algae, is known to have anti-inflammatory, anti-cancer, and anti-viral effects. In this study, Phycoerythrin extract from Grateloupia filicina (GfPE) was used to treat astrocytes and then assessed for its ability to protect against physiological changes under oxidative stress via H₂O₂. GfPE had a good effect on viability and proliferation of astrocytes that were downregulated under oxidative stress. Accordingly, GfPE alleviated the increasing effect of H₂O₂ on ROS of astrocytes.     

Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors

Glucose oxidase (GOx) was immobilized onto graphene oxide (GRO) via three different preparation methods: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC), and enzyme adsorption, precipitation and crosslinking (EAPC). EAPC formulations, prepared via enzyme precipitation with 60% ammonium sulfate, showed 1,980 and 1,630 times higher activity per weight of GRO than those of EA and EAC formulations, respectively. After 59 days at room temperature, EAPC maintained 88% of initial activity, while EA and EAC retained 42 and 45% of their initial activities, respectively. These results indicate that the steps of precipitation and crosslinking in the EAPC formulation are critical to achieve high enzyme loading and stability of EAPC. EA, EAC and EAPC were used to prepare enzyme electrodes for use as glucose biosensors. Optimized EAPC electrode showed 93- and 25-fold higher sensitivity than EA and EAC, respectively. To further increase the sensitivity of EAPC electrode, multi-walled carbon nanotubes (MWCNTs) were mixed with EAPC for the preparation of enzyme electrode. Surprisingly, the EAPC electrode with additional 99.5 wt% MWCNTs showed 7,800-fold higher sensitivity than the EAPC electrode without MWCNT addition. Immobilization and stabilization of enzymes on GRO via the EAPC approach can be used for the development of highly sensitive biosensors as well as to achieve high enzyme loading and stability.     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.