Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

Jo, B.H.,Kim, J.Y.H.,Seo, J.H.,Cha, H.J. Pergamon Press ; Elsevier Science Ltd 2014 International journal of hydrogen energy Vol.39 No.20

H<SUB>2</SUB> production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H<SUB>2</SUB> production by fermentative bacteria by maximizing the number of electrons that are available for H<SUB>2</SUB>. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O<SUB>2</SUB>)-binding protein on oxic H<SUB>2</SUB> production in a closed batch system that was supplemented with glucose. The H<SUB>2</SUB> yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O<SUB>2</SUB> tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H<SUB>2</SUB> yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H<SUB>2</SUB> production and not by working as an O<SUB>2</SUB>-scavenger during H<SUB>2</SUB> production. This new approach allowed more substrate to remain to be further utilized for the production of more H<SUB>2</SUB> from limited resources. We expect that VHb can be successfully engineered in potential aerobic H<SUB>2</SUB>-producing microbial systems to enhance the overall H<SUB>2</SUB> production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H<SUB>2</SUB>.

Expression of Suppressors of Cytokine Signaling-3 in Helicobacter pylori-Infected Rat Gastric Mucosal RGM-1Cells

CHA, B.,KIM, K. H.,MATSUI, H.,KIM, H. Wiley (Blackwell Publishing) 2007 Annals of the New York Academy of Sciences Vol.1096 No.1

<P>Our previous studies show that Helicobacter pylori (H. pylori) induces oxidative stress and the expression of proinflammatory cytokines in gastric epithelial cells. H. pylori induces the expression of molecular chaperones and proteins involved in protein-folding machinery as a defense mechanism against cellular stress. The suppressors of cytokine signaling (SOCS) are known as negative regulators of major immune signal pathways. The purpose of this article is to determine whether H. pylori in a Korean isolate (HP99) induces the expression of SOCS in rat gastric mucosal RGM-1 cells as a defense mechanism. As a result, HP99 induced SOCS-3 expression time-dependently in RGM-1 cells. SOCS-1 was not expressed while SOCS-2 expression was not changed by HP99 infection in RGM-1 cells. SOCS-3 might have a defensive role in H. pylori-infected gastric mucosal cells. Further study by manipulating SOCS-3 gene should be performed to investigate the physiological meaning of SOCS-3 induced by H. pylori in gastric mucosal cells.</P>

Draft genome sequence of Hydrogenovibrio marinus MH-110, a model organism for aerobic H₂ metabolism

Jo, B.H.,Hwang, B.H.,Cha, H.J. Elsevier Science Publishers 2014 Journal of biotechnology Vol.185 No.-

Hydrogenovibrio marinus, an obligate chemolithoautotroph isolated from oceanic surface water, is a Knallgas bacterium that conserves energy by oxidizing H<SUB>2</SUB> in the presence of O<SUB>2</SUB>. The strain possesses a periplasmic membrane-bound respiratory [NiFe]-hydrogenase with high O<SUB>2</SUB> tolerance, hence is of great biotechnological importance in the development of H<SUB>2</SUB>-based technologies for a promising alternative energy. Here, we report the draft genome of H. marinus MH-110, providing genomic information on the biosynthesis of the hydrogenase, aerobic H<SUB>2</SUB> metabolism, and autotrophic carbon assimilation.

Escherichia coli-based expression of functional novel DNA-binding histone H1 from Carassius auratus

Wei, Q.,Jung, H.J.,Hwang, D.S.,Hwang, B.H.,Gim, Y.,Cha, H.J. IPC Science and Technology Press ; Elsevier Scienc 2007 Enzyme and microbial technology Vol.40 No.6

Histones are DNA-binding proteins that assist in DNA packaging and protection. Here, we, for the first time, cloned a novel histone H1 cDNA (638bp) from the goldfish, Carassius auratus. Sequencing revealed that this histone H1 shared 68.1% amino acid identity and 73.9% similarity with that from the rainbow trout, Salmo gairdneri. We successfully expressed a full-length recombinant version (∼20kDa) and a truncated C-terminal fragment (∼6kDa; 61 amino acids) of this histone H1 as a partially soluble form using a maltose binding protein (MBP) fusion strategy in an Escherichia coli expression system. Our results revealed that both these recombinant histone H1 versions had DNA binding and protection functions, and MBP fusion system was an effective way to produce biological functional recombinant histone proteins in E. coli. This novel recombinant histone H1 protein and/or its C-terminal peptide could be used as potential mediators for efficacious gene delivery.

Efficacy of HVT-IBD vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a Korean very virulent IBDV in commercial broiler chickens

Roh, J.-H.,Kang, M.,Wei, B.,Yoon, R.-H.,Seo, H.-S.,Bahng, J.-Y.,Kwon, J.-T.,Cha, S.-Y.,Jang, H.-K. Elsevier 2016 Poultry science Vol.95 No.5

<P>The production performance, efficacy, and safety of two types of vaccines for infectious bursal disease virus (IBDV) were compared with in-ovo vaccination of Cobb 500 broiler chickens for gross and microscopic examination of the bursa of Fabricius, bursa/body weight (b/B) ratio, flow cytometry, and serologic response to Newcastle disease virus (NDV) vaccination. One vaccine was a recombinant HVT-IBD vector vaccine (HVT as for herpesvirus of turkeys) and the other was an intermediate plus live IBDV vaccine. A significant difference was detected at 21 d. Eight of 10 chickens that received the IBDV live vaccine had severe bursal lesions and a relatively low b/B ratio of 0.95, and an inhibited NDV vaccine response. On the other hand, the HVT-IBD vector vaccine resulted in mild bursal lesions and a b/B ratio of 1.89. Therefore, the live vaccine had lower safety than that of the HVT-IBD vector vaccine. To determine the protective efficacy, chickens were intraocularly challenged at 24 d. Eight of 10 chickens in the IBDV live vaccination group showed gross and histological lesions characterized by hemorrhage, cyst formation, lymphocytic depletion, and a decreased b/B ratio. In contrast, the HVT-IBD vector vaccinated chickens showed mild gross and histological lesions in three of 10 chickens with a b/B ratio of 1.36, which was similar to that of the unchallenged controls. Vaccinated chickens showed a significant increase in IBDV antibody titers, regardless of the type of vaccine used. In addition, significantly better broiler flock performance was observed with the HVT-IBD vector vaccine compared to that of the live vaccine. Our results revealed that the HVT-IBD vector vaccine could be used as an alternative vaccine to increase efficacy, and to have an improved safety profile compared with the IBDV live vaccine using in-ovo vaccination against the Korean very virulent IBDV in commercial broiler chickens.</P>

혈액검체로부터 분리된 황색포도구균의 독소유전자형 및 PFGE 분석

이영선,차정옥,정영희,최연화,양숙자,김홍빈,유재일,김봉수 국립보건연구원 2000 국립보건원보 Vol.37 No.-

Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli

Kim, J.Y.H.,Jo, B.H.,Cha, H.J. Elsevier Science Publishers 2011 Journal of biotechnology Vol.155 No.3

Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8nmol H<SUB>2</SUB>/(minmg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.

Ethanol extract of Prunus mume fruit attenuates hydrogen peroxide-induced oxidative stress and apoptosis involving Nrf2/HO-1 activation in C2C12 myoblasts

Kang, J.S.,Kim, D.J.,Kim, G.Y.,Cha, H.J.,Kim, S.,Kim, H.S.,Park, C.,Hwang, H.J.,Kim, B.W.,Kim, C.M.,Choi, Y.H. Sociedade Brasileira de Farmacognosia 2016 Revista brasileira de farmacognosia Vol.26 No.2

<P>The fruit of the Prunus mume (Siebold) Siebold & Zucc., Rosaceae (Korean name: Maesil) has long been used as a health food or valuable medicinal material in traditional herb medicine in Southeast Asian countries. In this study, we determined the potential therapeutic efficacy of the ethanol extract of P. mume fruits (EEPM) against H2O2-induced oxidative stress and apoptosis in the murine skeletal muscle myoblast cell line C2C12, and sought to understand the associated molecular mechanisms. The results indicated that exposure of C2C12 cells to H2O2 caused a reduction in cell viability by increasing the generation of intracellular reactive oxygen species and by disrupting mitochondrial membrane permeability, leading to DNA damage and apoptosis. However, pretreatment of the cells with EEPM before H2O2 exposure effectively attenuated these changes, suggesting that EEPM prevented H2O2-induced mitochondria-dependent apoptosis. Furthermore, the increased ex-pression and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and up-regulation of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme, were detected in EEPM-treated C2C12 cells. We also found that zinc protoporphyrin IX, an HO-1 inhibitor, attenuated the protective effects of EEPM against H2O2-induced reactive oxygen species accumulation and cytotoxicity. Therefore, these results indicate that the activation of the Nrf2/HO-1 pathway might be involved in the protection of EEPM against H2O2-induced cellular oxidative damage. In conclusion, these results show that EEPM contributes to the prevention of oxidative damage and could be used as a nutritional agent for oxidative stress-related diseases. (C) 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.</P>

<i>MICB</i> polymorphisms and haplotypes with <i>MICA</i> and HLA alleles in Koreans

Cha, C.‐,H.,Sohn, Y.‐,H.,Oh, H.‐,B.,Ko, S.‐,Y.,Cho, M.‐,C.,Kwon, O.‐,J. Blackwell Publishing Ltd 2011 Tissue antigens Vol.78 No.1

<P>Major histocompatibility complex (MHC) class I chain‐related gene B (<I>MICB</I>) is located within the human MHC class I region. The location of <I>MICB</I> in the MHC region may imply the presence of linkage disequilibrium with polymorphic <I>MICA</I> and human leukocyte antigen (HLA) loci. <I>MICB</I> is also polymorphic; however, <I>MICB</I> polymorphisms have not been investigated in Koreans. Using sequence‐based typing (SBT), we estimated the allelic frequencies of <I>MICB</I> and haplotypes with <I>MICA</I>, <I>HLA‐B</I>, and <I>HLA‐DRB1</I> at high resolution in a population of 139 unrelated Korean individuals. Eight <I>MICB</I> alleles were identified. The most frequent allele was <I>MICB*005:02/*010</I> (57.2%), followed by <I>*002</I> (11.5%), <I>*004</I> (8.3%), <I>*005:03</I> (8.3%), and <I>*008</I> (6.8%). The most common two‐locus haplotypes were <I>MICB*005:02/*010‐MICA*010</I> (19.4%), <I>MICB*005:02/*010‐DRB1*15:01</I> (6.5%), and <I>MICB*005:02/*010‐B*15:01</I> (10.4%); the most common three‐locus haplotypes were <I>B*15:01‐MICA*010‐MICB*005:02/*010</I> (5.8%) and <I>MICA*010‐MICB*005:02/*010‐DRB1*04:06</I> (10.4%); and the most common four‐locus haplotype was <I>B*15:01‐MICA*010‐MICB*005:02/*010‐DRB1*04:06</I> (5.8%). This is the first study to provide information about <I>MICB</I> allele frequencies and haplotypes with HLA in Koreans. These study results should help understand mechanisms of disease association between the <I>MICB</I> locus and neighboring loci in Koreans.</P>

Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase

Cha, H.J.,Jang, D.S.,Jeong, J.H.,Hong, B.H.,Yun, Y.S.,Shin, E.J.,Choi, K.Y. Elsevier Science 2016 Biochimica et biophysica acta. Proteins and proteo Vol.1864 No.10

<P>Ketosteroid isomerase (3-oxosteroid Delta(5)-Delta(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Delta(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the k(cat) by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased K-D values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (T-m) by 6.4 degrees C and 2.5 degrees C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased k(cat) by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing T-m by 4.1 degrees C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core. (C) 2016 Published by Elsevier B.V.</P>