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Characterization of NAD-Dependent Formate Dehydrogenase from Trametes versicolor Using a Cell-Free Protein Expression System

  • LEE, Su-Yeon;JANG, Seokyoon;LEE, Soo-Min
    • Journal of the Korean Wood Science and Technology
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    • v.50 no.3
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    • pp.159-166
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    • 2022
  • CO2 emissions are the primary reason for global warming; hence, biological and chemical technologies for converting CO2 into useful compounds are being actively studied. Biological methods using enzymes can convert CO2 under mild conditions. Formate dehydrogenase (FDH) is a representative CO2 conversion enzyme. Its function was revealed after isolation from bacteria, yeast, and plants. In this study, we evaluated the CO2 conversion potential of FDH isolated from wood-rotting fungi. After isolating the FDH gene (TvFDH) from Trametes versicolor, we cloned the full-length FDH from T. versicolor and expressed it in a cell-free expression system. The gene encoding TvFDH was identified as 1,200 bp open reading frame (ORF) and the expected molecular weight of the protein was approximately 42 kDa. Overexpression of the recombinant crude protein including TvFDH was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme activities and metabolite analyses confirmed the efficiency of TvFDH for CO2 reduction.

Studies on improvement of manufacturing method of enzymic source for Maggerley(Korea wine) brewing(I) (막걸리의 제조를 위한 효소제의 개발연구 1)

  • 이성범;최경환;임동순;김덕치
    • Korean Journal of Microbiology
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    • v.7 no.4
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    • pp.159-166
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    • 1969
  • It is necessry to develop and strengthen the activity of enzymic source which in low applied for maggerley brewing as an amylolytic and proteolytic starter, recently in this country the active and strong enzymic starter is required for the better brewing and to substitute another starch material for the present wheat flour. In this study, manufacturing method the strong enzymic source have been developed and established with use of raw wheat bran plus fungal strains of Rhizopus sp. and Aspergillus usamii the culture of starter. The results on experimental the activities of enzymic sources (stater) are as following ; 1. Method of making the enzymic source (starter) is to cultivate the strains of Asperguillus orzyae, Asp. kawachii, Asp usamii and Rhizopus sp. in the acid treated raw or heatboiled wheat bran. 2. The saccharogenic pwoer (S.P.) of enzymic source which consisted of raw bran plus fungi and cultured in it is generally stronger than those of heat-boiled bran plus fungi, the strongest power was shown in the culture of Rhizopus plus raw bran, and the next other is in mixture of Asp.usamii and Rhizopus on raw wheat bran. 3. The most strong alpha amylase activity was expressed in the plot of Asp.oryzae on heat-boiled wheat bran, the next was in the culture of Rhizopus nad Aspergillus usamii on raw wheatbran. 4. The most vigourous acidic proteinase activity was expressed in the micture of raw bran plus Asp. usamii and Rhizopus those were independentlu cu;tured before mixing for neutral proteinase activity, it was shown in the mixed culture of Asp. usamii and Rhizopus on raw wheat bran, the msot active alkaline proteinase activity of enzymic source was found in the plot of raw bran material. 5. For poly-preptidase activity in pH 6.5 it is found that the culture of Rhizopus and Asp.usamii on raw bran was most active among them of enzymic sources. 6. Generally, it is concluded that culture of fungi on acid treated raw wheat bran is stronger in its activity than those of heat boiled wheat bran, especially the culture of Rhizopus nad Asp.usamii on raw bran exhibited the most vigorous and non-polarized activity for all aspects, so it is considered to be most desirable enzymic stater in Korean Maggerley brewing and this would be able to substitute brewing material for the present wheat flour because of its strong and wide hand activity of amylolytic and proteolytic action.

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Isolation and Characterization of a Formate Dehydrogenase cDNA in Poplar (Populus alba ${\times}$ P. glandulosa) (현사시나무에서 Formate Dehydrogenase cDNA의 분리와 특성 구명)

  • Bae, Eun-Kyung;Lee, Hyoshin;Lee, Jae-Soon;Choi, Young-Im;Yoon, Seo-Kyung;Eo, Soo Hyung
    • Journal of Korean Society of Forest Science
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    • v.102 no.3
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    • pp.331-337
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    • 2013
  • Formate dehydrogenase (FDH), catalyzing the oxidation of the formate ion to carbon dioxide, is known as the stress protein in response to drought, low temperature and pathogen infection. To study the functions of FDH in poplar (Populus alba ${\times}$ P. glandulosa), we isolated a FDH cDNA (PagFDH1) and examined its expressional characteristics. The PagFDH1 is 1,499 base pairs long and encodes a putative 388 amino acid protein with an expected molecular mass of 42.5 kDa. The PagFDH1 protein has N-terminal mitochondria signal peptide and $NAD^+$ binding domain. Southern blot analysis indicated that a single copy of the PagFDH1 is present in the poplar genome. PagFDH1 is expressed highly in the suspension cells (especially in the lag and early exponential phases) and moderately in roots, flowers and leaves. ABA-mediated enhanced expression of PagFDH1 in response to drought and salt stress treatments indicates that the gene product could play an important role in the development of stress resistant trees.

Molecular Identification of Taenia hydatigena from Sheep in Khartoum, Sudan

  • Muku, Rosline James;Yan, Hong-Bin;Ohiolei, John Asekhaen;Saaid, Abubakar Ahmed;Ahmed, Sara;Jia, Wan-Zhong;Fu, Bao-Quan
    • Parasites, Hosts and Diseases
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    • v.58 no.1
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    • pp.93-97
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    • 2020
  • The cestode Taenia hydatigena uses canids, primarily dogs, as definitive hosts, while the metacestode larval stage cysticercus infects a range of intermediate hosts, including domestic animals such as goats, sheep, and pigs. Cysticercosis due to T. hydatigena has large veterinary and economic drawbacks. Like other taeniids, e.g., Echinococcus, intraspecific variation is found among the members of the genus Taenia. In Africa, few studies are available on the epidemiology and distribution of T. hydatigena, and even fewer studies are available on its genetic variation. In this study, we molecularly identified 11 cysticerci from sheep in Sudan and demonstrated the genetic variation based on the NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) mitochondrial genes. The isolates were correctly identified as T. hydatigena with more than 99% similarity to those in the GenBank database. Low diversity indices and insignificant neutrality indices were observed, with 3 and 2 haplotypes for the nad1 and cox1 genes, respectively. The results suggest the presence of unique T. hydatigena haplotypes in Sudan, as haplotypes with 100% similarity were not found in the GenBank database. With few available studies on the genetic variation of T. hydatigena in Africa, this report represents the first insights into the genetic variation of T. hydatigena in Sudan and constitutes useful data.

Isolation and characterization of a monodehydroascorbate reductase gene in poplar (Populus alba × P. glandulosa) (현사시나무 monodehydroascorbate reductase (MDHAR) 유전자의 분리 및 발현특성)

  • Yoon, Seo-Kyung;Park, Eung-Jun;Bae, Eun-Kyung;Choi, Young-Im;Kim, Joon-Hyeok;Lee, Hyoshin
    • Journal of Plant Biotechnology
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    • v.41 no.4
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    • pp.194-200
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    • 2014
  • Monodehydroascorbate reductase (MDHAR) is an important enzyme that plays a role in the detoxification of reactive oxygen species (ROS) by maintaining reduced pool of ascorbate through recycling the oxidized form of ascorbate. In this study, we isolated a PagMDHAR1 gene from Populus alba ${\times}$ P. glandulosa, and investigated its expression characteristics. The PagMDHAR1 cDNA encodes a putative 434 amino acids containing FAD- and NAD(P)H-binding domains. Southern blot analysis indicated that a single nuclear gene encodes this enzyme. Northern hybridization analysis revealed that PagMDHAR1 is highly expressed in both suspension cells and flower tissues, while its expression levels were enhanced by drought, salt, cold, wounding and ABA. Therefore, PagMDHAR1 might be expressed in response to abiotic stress through the ABA-mediated signaling pathway in this poplar species, suggesting that the PagMDHAR1 plays an important role in the defense mechanisms against oxidative stress.

Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose

  • Lu, Xinyao;Fu, Xiaomeng;Zong, Hong;Zhuge, Bin
    • Journal of Microbiology and Biotechnology
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    • v.26 no.7
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    • pp.1252-1258
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    • 2016
  • 1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD+ ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae.

Purification and Properties of Thermostable L-Lactate Dehydrogenase Produced by Escherichia Coli (대장균으로 부터 생산된 L-lactate Dehydrogenase의 정제 및 특성)

  • Song, Jae-Young;Kim, Kyoug-Sook
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.23 no.6
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    • pp.964-972
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    • 1994
  • The 4.3-kb gene coding for L-lactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment , DEAE-Sephadex, and NAD++ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200 . The molecular weight of the purfied enzyme was estimated to be about 35, 000 and 140, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. indicating that the enzyme is composed of four identical subunits. THe enzyme for pyruvate reduction and lactate oxdiation was stable at 60 and 75$^{\circ}C$ for 30 min, and the optimal temperatures for both reactions were 60 and 7$0^{\circ}C$, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme of pyruvate reduction was table between pH 5 and 7. more than 90% of enzyme activity was lost at 1mM FeSO4 and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1, 6-bisphosphate.

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Two- Dimensional Electrophoresis Analysis of Proteins; Bacillus subtilis LTD and Its Antifungal Activity Deficient Mutant

  • Lee, Young-Keun;Dinh, Le Thi;Jang, Yu-Sin;Chung, Hye-Young;Chang, Hwa-Hyoung
    • Korean Journal of Environmental Biology
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    • v.22 no.4
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    • pp.487-493
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    • 2004
  • To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.

Metabolic Flux Shift of Weissella kimchii sk10 Grown Under Aerobic Conditions

  • Park, Sun-Mi;Kang, Hye-Sun;Park, Doo-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.919-923
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    • 2004
  • The sk10 isolated from kimchi was identified as W. kimchii on the basis of l6s-rDNA sequencing. Studies were made to analyze the metabolic flux shift of the sk10 on glucose under aerobic growth conditions. The sk10 produced 38.2 mM acetate, 16.3 mM ethanol, and 33.2 mM lactate under aerobic conditions, but 2.4 mM acetate, 48.0 mM ethanol, and 44.1 mM lactate under anaerobic conditions. The NADH peroxidase (NADH-dependent hydrogen peroxidase) activity of sk10 grown under aerobic conditions was 11 times higher than that under anaerobic conditions. Under the low ratio of $NADH/NAD^+$, the metabolic flux toward lactate and ethanol was shifted to the flux through acetate kinase without NADH oxidation. The kinds of enzymes and metabolites of sk10 were close to those in the pathway of Leuconostoc sp., but the metabolites produced under aerobic growth conditions were different from those of Leuconostoc sp. The stoichiometric balance calculated using the concentrations of metabolites and substrate was about 97%, coincident with the theoretical values under both aerobic and anaerobic conditions. From these results, it was concluded that the metabolic flux of W. kimchii sk10 was partially shifted from lactate and ethanol to acetate under aerobic conditions only.

Effects of Hanghaedan on the Cytokine Secretory Function of Monocytes of Atopic Dermatitis Patients (항해단(沆瀣丹)이 아토피 피부염 환자 단핵세포의 Cytokine 분비능에 미치는 영향)

  • Lee, Jin-Yong;Kim, Woo-Yeoun
    • The Journal of Pediatrics of Korean Medicine
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    • v.21 no.2
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    • pp.169-184
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    • 2007
  • Objetives The late research tendency on atopic dermatitis is mainly focused on the experiment of unbalance of cytokine released from T cell subtype. Merhods As there's no experiment with herbal medicine using Hanghaedan(HHD), we isolated and cultured monocytes of atopic dermatitis pateints (AD) peripheral blood, for knowing the influence of HHD on the secretory function of IL-4, IL-5, $IFN-{\gamma}$. Results 1. In AD group, HHD exhibits statistically significant inhibitory effect on IL-4(p<0.05). 2. In AD group, HHD exhibits statistically significant inhibitory effect on IL-5(p<0.05). 3. In AD group, HHD exhibits statistically significant inhibitory effect on $IFN-{\gamma}$ (p<0.01). 4. Sample group comparing with the cytokine concentration rate value of AD patients and that of NAD patients IL-4 of AD patients exhibits statistically significant decrease(p<0.05). 5. In AD group, there is no significant relation between serum IgE and cytokines. 6. In AD group, there is no significant relation between total eosinophil and IL-5. Conclusions Effects of Hanghaedan on the cytokine(IL-4, IL-5, $IFN-{\gamma}$) secretory function of monocytes of atopic dermatitis patients is recognized.

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