Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO₂). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO₂, a major greenhouse gas, making this a promising alternative for chemical CO₂ mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO₂ sequestration by mineral carbonation, a process with the potential to store large quantities of CO₂. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO₂ compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO₃) formation and exerted a striking impact on the maximal amount of CaCO₃ produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO₂ sequestration.     

Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering

Zinc oxide (ZnO) nanostructures have been commonly studied for electronic purposes due to their unique piezoelectric and catalytic properties; however, recently, they have been also exploited for biomedical applications. The purpose of this study was to fabricate ZnO-doped poly(urethane) (PU) nanocomposite via one-step electrospinning technique. The utilized nanocomposite was prepared by using colloidal gel composed of ZnO and PU, and the obtained mats were vacuum dried at 60 °C overnight. The physicochemical characterization of as-spun composite nanofibers was carried out by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, and transmission electron microscopy, whereas the thermal behavior was analyzed by thermogravimetric analysis. The viability, attachment, and proliferation of NIH 3T3 mouse fibroblast cells on the ZnO/PU composite nanofibers were analyzed by in vitro cell compatibility test. The morphological features of the cells attached on nanofibers were examined by Bio-SEM. We conclude that the electrospun nanofibrous scaffolds with unique spider nets had good biocompatibility. Cytotoxicity experiments indicated that the mouse fibroblasts could attach to the nanocomposite after being cultured. Thus, the current work demonstrates that the as-synthesized ZnO/PU hybrid nanofibers represent a promising biomaterial to be exploited for various tissue engineering applications.     

A record of N2O and CH4 emissions and underlying soil processes of Korean rice paddies as affected by different water management practices

Rice is staple food of half of mankind and paddy soils account for the largest anthropogenic wetlands on earth. Ample of research is being done to find cultivation methods under which the integrative greenhouse effect caused by emitted CH₄ and N₂O would be mitigated. Whereas most of the research focuses on quantifying such emissions, there is a lack of studies on the biogeochemistry of paddy soils. In order to deepen our mechanistic understanding of N₂O and CH₄ fluxes in rice paddies, we also determined NO₃ ^? and N₂O concentrations as well as N₂O isotope abundances and presence of O₂ along soil profiles of paddies which underwent three different water managements during the rice growing season(s) in (2010 and) 2011 in Korea. Largest amounts of N₂O (2 mmol m^?2) and CH₄ (14.5 mol m^?2) degassed from the continuously flooded paddy, while paddies with less flooding showed 30–60 % less CH₄ emissions and very low to negative N₂O balances. In accordance, the global warming potential (GWP) was lowest for the Intermittent Irrigation paddy and highest for the Traditional Irrigation paddy. The N₂O emissions could the best be explained (*P < 0.05) with the δ¹⁵N values and N₂O concentrations in 40–50 cm soil depth, implying that major N₂O production/consumption occurs there. No significant effect of NO₃ ^? on N₂O production has been found. Our study gives insight into the soil of a rice paddy and reveals areas along the soil profile where N₂O is being produced. Thereby it contributes to our understanding of subsoil processes of paddy soils.     

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-l-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-l-fucose resulted in a maximum GDP-l-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer–Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.     

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.     

Candidate target genes for the Saccharomyces cerevisiae transcription factor, Yap2

In Saccharomyces cerevisiae, the Yap family of basic leucine zipper (bZip) proteins contains eight members. The Yap family proteins are implicated in a variety of stress responses; among these proteins, Yap1 acts as a major regulator of oxidative stress responses. However, the functional roles of the remaining Yap family members are poorly understood. To elucidate the function of Yap2, we mined candidate target genes of Yap2 by proteomic analysis. Among the identified genes, FRM2 was previously identified as a target gene of Yap2, which confirmed the validity of our screening method. YNL134C and YDL124W were also identified as candidate Yap2 target genes. These genes were upregulated in strains overexpressing Yap2 and possess Yap2 target sequences in their promoter regions. Furthermore, chromatin immunoprecipitation assays showed that YNL134C and YDL124W have Yap2 binding motif. These data will help to elucidate the functional role of Yap2.     

Non hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydrophobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial peptides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic stability makes these molecules promising candidates for novel antimicrobial therapeutics.     

Pyrrole and furan oligoglycosides from the starfish Asterina batheri and their inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide-stimulated bone marrow-derived dendritic cells

Three new pyrrole oligoglycosides, astebatheriosides A–C (1–3), and a new furan oligoglycoside, astebatherioside D (4), were isolated from the starfish Asterina batheri by various chromatographic methods. Their structures were elucidated by spectroscopic and chemical methods. Compounds 2, 3, and 4 moderately inhibited IL-12 p40 production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs) with IC₅₀ values of 36.4, 31.6, and 22.8 μM, respectively.     

Acid-Catalyzed Stereoselective Cyclization of Germacrene D

A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The K_m for iodide and SAM were 12 mM and 12 μM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I^-,Br^-, and Cl^- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.