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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 18, Issue 12 - Dec 2008
Volume 18, Issue 11 - Nov 2008
Volume 18, Issue 10 - Oct 2008
Volume 18, Issue 9 - Sep 2008
Volume 18, Issue 8 - Aug 2008
Volume 18, Issue 7 - Jul 2008
Volume 18, Issue 6 - Jun 2008
Volume 18, Issue 5 - May 2008
Volume 18, Issue 4 - Apr 2008
Volume 18, Issue 3 - Mar 2008
Volume 18, Issue 2 - Feb 2008
Volume 18, Issue 1 - Jan 2008
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Bacterial and Fungal Communities in Bulk Soil and Rhizospheres of Aluminum-Tolerant and Aluminum-Sensitive Maize (Zea mays L.) Lines Cultivated in Unlimed and Limed Cerrado Soil
Mota, Da ; Faria, Fabio ; Gomes, Eliane Aparecida ; Marriel, Ivanildo Evodio ; Paiva, Edilson ; Seldin, Lucy ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 805~814
Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.
Application of rDNA-PCR Amplification and DGGE Fingerprinting for Detection of Microbial Diversity in a Malaysian Crude Oil
Liew, Pauline Woan Ying ; Jong, Bor Chyan ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 815~820
Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.
Identification and Characterization of a New Strain of the Unicellular Green Alga Dunaliella salina (Teod.) from Korea
Polle, Jurgen E.W. ; Struwe, Lena ; Jin, Eon-Seon ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 821~827
The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately
wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of
-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of
-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate
-carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.
Positive Regulation of Pyoluteorin Biosynthesis in Pseudomonas sp. M18 by Quorum-Sensing Regulator VqsR
Huang, Xianqing ; Zhang, Xuehong ; Xu, Yuquan ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 828~836
The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain MI8. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of N-acylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.
Deletion of xylR Gene Enhances Expression of Xylose Isomerase in Streptomyces lividans TK24
Heo, Gun-Youn ; Kim, Won-Chan ; Joo, Gil-Jae ; Kwak, Yun-Young ; Shin, Jae-Ho ; Roh, Dong-Hyun ; Park, Heui-Dong ; Rhee, In-Koo ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 837~844
Glucose (xylose) isomerases from Streptomyces sp. have been used for the production of high fructose corn syrup for industrial purposes. An 11-kb DNA fragment containing the xyl gene cluster was isolated from Streptomyces lividans TK24 and its nucleotide sequences were analyzed. It was found that the xyl gene cluster contained a putative transcriptional repressor (xylR), xylulokinase (xylB), and xylose isomerase (xylA) genes. The transcriptional directions of the xylB and xylA genes were divergent, which is consistent to those found in other streptomycetes. A gene encoding XylR was located downstream of the xylB gene in the same direction, and its mutant strain produced xylose isomerase regardless of xylose in the media. The enzyme expression level in the mutant was 4.6 times higher than that in the parent strain under xylose-induced condition. Even in the absence of xylose, the mutant strain produce over 60% of enzyme compared with the xylose-induced condition. Gel mobility shift assay showed that XylR was able to bind to the putative xyl promoter, and its binding was inhibited by the addition of xylose in vitro. This result suggested that XylR acts as a repressor in the S. lividans xylose operon.
Expression and Biochemical Characterization of the Periplasmic Domain of Bacterial Outer Membrane Porin TdeA
Kim, Seul-Ki ; Yum, Soo-Hwan ; Jo, Wol-Soon ; Lee, Bok-Luel ; Jeong, Min-Ho ; Ha, Nam-Chul ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 845~851
TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic
-helical barrel domain and a membrane-embedded
-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.
Purification and Cloning of an Extracellular Serine Protease from the Nematode-Trapping Fungus Monacrosporium cystosporium
Yang, Jin-Kui ; Ye, Feng-Ping ; Mi, Qi-Li ; Tang, Song-Qing ; Li, Juan ; Zhang, Ke-Qin ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 852~858
An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and
(over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (
for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were
per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might playa role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mcl showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mcl possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.
Biochemical Characterization of a Glycosyltransferase Homolog from an Oral Pathogen Fusobacterium nucleatum as a Human Glycan-Modifying Enzyme
Kim, Seong-Hun ; Oh, Doo-Byoung ; Kwon, Oh-Suk ; Jung, Jae-Kap ; Lee, Yun-Mi ; Ko, Ki-Sung ; Ko, Jeong-Heon ; Kang, Hyun-Ah ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 859~865
Bacterial glycosyltransferases have drawn growing attention as economical enzymes for oligosaccharide synthesis, with their easy expression and relatively broad substrate specificity. Here, we characterized a glycosyltransferase homolog (Fnu_GT) from a human oral pathogen, Fusobacterium nucleatum. Bioinformatic analysis showed that Fnu_GT belongs to the glycosyltransferases family II. The recombinant Fnu_GT (rFnu_GT) expressed in Escherichia coli displayed the highest glycosylation activity when UDP-galactose (Gal) was used as a donor nucleotide-sugar with heptose or N-acetylglucosamine (GlcNAc) as an acceptor sugar. Interestingly, rFnu_GT transferred the galactose moiety of UDP-Gal to a nonreducing terminal GlcNAc attached to the trimannosyl core glycan, indicating its potential as an enzyme for human-type N-glycan synthesis.
Heterologous Production and Detection of Recombinant Directing 2-Deoxystreptamine (DOS) in the Non-Aminoglycoside-Producing Host Streptomyces venezuelae YJ003
Kurumbang, Nagendra Prasad ; Oh, Tae-Jin ; Liou, Kwangkyoung ; Sohng, Jae-Kyung ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 866~873
2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESI-MS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.
Inhibitory Effect of Dalbergioidin Isolated from the Trunk of Lespedeza cyrtobotrya on Melanin Biosynthesis
Baek, Seung-Hwa ; Kim, Jin-Hee ; Kim, Dong-Hyun ; Lee, Chan-Yong ; Kim, Ji-Young ; Chung, Dae-Kyun ; Lee, Choong-Hwan ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 874~879
Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be reduced by treatment with depigmenting agents. The methanol extract of Lespedeza cyrtobotrya
showed inhibitory activity against mushroom tyrosinase. The active compound was purified from the methanol extract of L. cyrtobotrya, followed by several chromatographic methods, and identified as dalbergioidin (DBG) by spectroscopic methods. The results showed that DBG exhibited tyrosinase inhibitory activity with an
. The kinetic analysis of tyrosinase inhibition revealed that DBG acted as a noncompetitive inhibitor. In addition, DBG showed a melanin biosynthesis inhibition zone in the culture plate of Streptomyces bikiniensis that has commonly been used as an indicator organism. Furthermore,
DBG decreased more than 50% of melanin contents on the pigmentation using the immortalized mouse melanocyte, melan-a cell.
Antifungal Activity of Valinomycin, a Peptide Antibiotic Produced by Streptomyces sp. Strain M10 Antagonistic to Botrytis cinerea
Park, Cheol-Nam ; Lee, Jung-Min ; Lee, Dong-Ho ; Kim, Beom-Seok ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 880~884
A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.
Continuous Cell-Free Protein Synthesis Using Glycolytic Intermediates as Energy Sources
Kim, Ho-Cheol ; Kim, Tae-Wan ; Park, Chang-Gil ; Oh, In-Seok ; Park, Kyung-Moon ; Kim, Dong-Myung ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 885~888
In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from a CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis a more realistic alternative for rapid protein production.
Modeling of Esterase Production from Saccharomyces cerevisiae
Thilakavathi, Thilakavathi ; Basak, Tanmay ; Panda, Tapobrata ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 889~896
A suitable simple model tested by experiments is required to address complex biological reactions like esterase synthesis by Saccharomyces cerevisiae. Such an approach might be the answer to a proper bioprocessing strategy. In this regard, a logistic model for esterase production from Saccharomyces cerevisiae has been developed, which predicts well the cell mass, the carbon source (glucose) consumption, and the esterase activity. The accuracy of the model has been statistically examined by using the Student's t-test. The parameter sensitivity analysis showed that all five parameters (
) have significant influence on the predicted values of esterase activity.
Enhancement of Geldanamycin Production by pH Shock in Batch Culture of Streptomyces hygroscopicus subsp. duamyceticus
Song, Jae-Yang ; Kim, Yoon-Jung ; Hong, Young-Soo ; Chang, Yong-Keun ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 897~900
Various sequences of pH change were applied in a batch bioreactor to investigate pH shock effects on geldanamycin production by Streptomyces hygroscopicus subsp. duamyceticus JCM4427. In the control culture where the pH was not controlled, the maximum geldanamycin concentration was 414 mg/l. With the pHS1 mode of pH shock, that is, an abrupt pH change from pH 6.5 to pH 5.0 and then being maintained at around pH 5.0 afterward, 768 mg/l of geldanamycin was produced. With pHS2, in which the pH was changed sequentially from pH 6.7 to pH 5.0 and then back to pH 6.0, 429 mg/l of geldanamycin was produced. With pHS3 having a sequential pH change from pH 6.0 to pH 4.0 and then back to pH 6.5 followed by the third pH shock to pH 5.5, no geldanamycin production was observed. Considering that the productivity with pHS1 was about two-fold of that of the control culture with no pH control, we concluded that a more sophisticated manipulation of pH would further promote geldanamycin production.
Modulation of the Regioselectivity of a Thermotoga neapolitana
-Glucosidase by Site-Directed Mutagenesis
Choi, Ki-Won ; Park, Kyung-Min ; Jun, So-Young ; Park, Cheon-Seok ; Park, Kwan-Hwa ; Cha, Jae-Ho ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 901~907
-glucosidase (BglA) was subjected to site-directed mutagenesis in an effort to increase its ability to synthesize arbutin derivatives by transglycosylation. The transglycosylation reaction of the wild-type enzyme displays major
regioselectivity. The three mutants, N291T, F412S, and N291T/F412S, increased the ratio of transglycosylation/hydrolysis compared with the wild-type enzyme when pNPG and arbutin were used as a substrate and an acceptor, respectively. N291T and N219T/F412S had transglycosylation/hydrolysis ratios about 3- and 8-fold higher, respectively, than that of the wild-type enzyme. This is due to the decreased hydrolytic activity of the mutant rather than increased transglycosylation activity. Interestingly, N291T showed altered regioselectivity, as well as increased transglycosylation products. TLC analysis of the transglycosylation products indicated that N291T retained its
regioselectivity, but lost its
regioselectivity. The altered regioselectivity of N291T using two other acceptors, esculin and salicin, was also confirmed by TLC. The major transglycosylation products of the wild type and N291T mutant were clearly different. This result suggests that Asn-291 is highly involved in the catalytic mechanism by controlling the transglycosylation reaction.
Succinic Acid Production by Continuous Fermentation Process Using Mannheimia succiniciproducens LPK7
Oh, In-Jae ; Lee, Hye-Won ; Park, Chul-Hwan ; Lee, Sang-Yup ; Lee, Jin-Won ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 908~912
To achieve a higher succinic acid productivity and evaluate the industrial applicability, this study used Mannheimia succiniciproducens LPK7 (knock-out: ldhA, pflB, pta-ackA), which was recently designed to enhance the productivity of succinic acid and reduce by-product secretion. Anaerobic continuous fermentation of Mannheimia succiniciproducens LPK7 was carried out at different glucose feed concentrations and dilution rates. After extensive fermentation experiments, a succinic acid yield and productivity of 0.38 mol/mol and 1.77 g/l/h, respectively, were achieved with a glucose feed concentration of 18.0 g/l and
dilution rate. A similar amount of succinic acid production was also produced in batch culture experiments. Therefore, these optimal conditions can be industrially applied for the continuous production of succinic acid. To examine the quantitative balance of the metabolism, a flux distribution analysis was also performed using the metabolic network model of glycolysis and the pentose phosphate pathway.
Spore Inoculum Optimization to Maximize Cyclosporin A Production in Tolypocladium niveum
Lee, Mi-Jin ; Lee, Han-Na ; Han, Kyu-Boem ; Kim, Eung-Soo ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 913~917
The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs. It is generated nonribosomally from a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized. Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.
High Cell Density Culture of Anabaena variabilis with Controlled Light Intensity and Nutrient Supply
Yoon, Jong-Hyun ; Shin, Jong-Hwan ; Ahn, Eun-Kyung ; Park, Tai-Hyun ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 918~925
Controlling the light energy and major nutrients is important for high cell density culture of cyanobacterial cells. The growth phase of Anabaena variabilis can be divided into an exponential growth phase and a deceleration phase. In this study, the cell growth in the deceleration phase showed a linear growth pattern. Both the period of the exponential growth phase and the average cell growth rate in the deceleration phase increased by controlling the light intensity. To control the light intensity, the specific irradiation rate was maintained above
dry cell by increasing the incident light intensity stepwise. The final cell density increased by controlling the nutrient supply. For the control of the nutrient supply, nitrate, phosphate, and sulfate were intermittently added based on the growth yield, along with the combined control of light intensity and nutrient concentration. Under these control conditions, both final cell concentration and cell productivity increased, to 8.2 g/l and 1.9 g/l/day, respectively.
Improved Coexpression and Multiassembly Properties of Recombinant Human Ferritin Subunits in Escherichia coli
Lee, Jung-Lim ; Levin, Robert E. ; Kim, Hae-Yeong ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 926~932
Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. H-ferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LH-ferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.
Purification and Characterization of an Extracellular
-Glucosidase from Monascus purpureus
Daroit, Daniel J. ; Simonetti, Aline ; Hertz, Plinho F. ; Brandelli, Adriano ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 933~941
-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A
central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at
and pH 5.5. The
-glucosidase showed moderate thermostability, was inhibited by
, whereas other reagents including
-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-
-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-
-D-glucopyranoside, and maltose indicates that the
-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-
-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.
Safety Assessment of Commercial Enterococcus Probiotics in Korea
Lee, Ki-Eun ; Lee, Min-Young ; Lee, Yeon-Hee ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 942~945
There have been concerns about possible pathogenicity and antimicrobial resistance in Enterococcus, which constitute more than 50% of probiotics in the worldwide market. In this study, Enterococcus in sixteen products manufactured by ten different companies was tested for the presence of six virulence genes and two vancomycin resistance genes. Results in this study showed the safety of Enterococcus on the Korean market and the importance of screening vanA, vanE, agg, cylA, esp, and gelE. Pulse-field gel electrophoresis showed that the sixteen isolates tested in this study are originated from three strains.
Biodegradation of Organophosphate Pesticide Using Recombinant Cyanobacteria with Surface- and Intracellular-Expressed Organophosphorus Hydrolase
Chungjatupornchai, Wipa ; Fa-Aroonsawat, Sirirat ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 946~951
The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency (
) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.
Isolation and Characterization of Comprehensive Polychlorinated Biphenyl-Degrading Bacterium, Enterobacter sp. LY402
Jia, Ling-Yun ; Zheng, Ai-Ping ; Xu, Li ; Huang, Xiao-Dong ; Zhang, Qing ; Yang, Feng-Lin ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 952~957
A Gram-negative bacterium, named LY402, was isolated from contaminated soil. 16S rDNA sequencing and measurement of the physiological and biochemical characteristics identified it as belonging to the genus Enterohacter. Degradation experiments showed that LY402 had the ability to aerobically transform 79 of the 91 major congeners of Aroclor 1242, 1254, and 1260. However, more interestingly, the strain readily degraded certain highly chlorinated and recalcitrant polychlorinated biphenyls (PCBs). Almost all the tri- and tetra-chlorobiphenyls (CBs), except for 3,4,3',4'-CB, were degraded in 3 days, whereas 73% of 3,4,3',4'-, 92% of the penta-, 76% of the hexa-, and 37% of the hepta-CBs were transformed after 6 days. In addition, among 12 octa-CBs, 2,2',3,3',5,5',6,6-CB was obviously degraded, and 2,2',3,3',4,5,6,6'- and 2,2',3,3',4,5,5',6'-CB were slightly transformed. In a metabolite analysis, mono- and dichlorobenzoic acids (CBAs) were identified, and parts of them were also transformed by strain LY402. Analysis of PCB degradation indicated that strain LY402 could effectively degrade PCB congeners with chlorine substitutions in both ortho- and para-positions. Consequently, this is the first report of an Enterobacteria that can efficiently degrade both low and highly chlorinated PCBs under aerobic conditions.
Effectiveness of Rhizobacteria Containing ACC Deaminase for Growth Promotion of Peas (Pisum sativum) Under Drought Conditions
Zahir, Z.A. ; Munir, A. ; Asghar, H.N. ; Shaharoona, B. ; Arshad, M. ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 958~963
A series of experiments were conducted to assess the effectiveness of rhizobacteria containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase for growth promotion of peas under drought conditions. Ten rhizobacteria isolated from the rhizosphere of different crops (peas, wheat, and maize) were screened for their growth promoting ability in peas under axenic condition. Three rhizobacterial isolates, Pseudomonas fluorescens biotype G (ACC-5), P. fluorescens (ACC-14), and P. putida biotype A (Q-7), were selected for pot trial on the basis of their source, ACC deaminase activity, root colonization, and growth promoting activity under axenic conditions. Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (4 seeds/pot) at different soil moisture levels (25, 50, 75, and 100% of field capacity). Results revealed that decreasing the soil moisture levels from 100 to 25% of field capacity significantly decreased the growth of peas. However, inoculation of peas with rhizobacteria containing ACC deaminase significantly decreased the "drought stress imposed effects" on growth of peas, although with variable efficacy at different moisture levels. At the lowest soil moisture level (25% field capacity), rhizobacterial isolate Pseudomonas fluorescens biotype G (ACC-5) was found to be more promising compared with the other isolates, as it caused maximum increases in fresh weight, dry weight, root length, shoot length, number of leaves per plant, and water use efficiency on fresh and dry weight basis (45, 150, 92, 45, 140, 46, and 147%, respectively) compared with respective uninoculated controls. It is highly likely that rhizobacteria containing ACC deaminase might have decreased the drought-stress induced ethylene in inoculated plants, which resulted in better growth of plants even at low moisture levels. Therefore, inoculation with rhizobacteria containing ACC deaminase could be helpful in eliminating the inhibitory effects of drought stress on the growth of peas.
Vaccination of Shrimp (Penaeus chinensis) against White Spot Syndrome Virus (WSSV)
Ha, Yu-Mi ; Gong, Soo-Jung ; Nguyen, Thi-Hoai ; Ra, Chae-Hun ; Kim, Ki-Hong ; Nam, Yoon-Kwon ; Kim, Sung-Koo ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 964~967
Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.
Proteomic Identification and Characterization of Vibrio vulnificus Proteins Induced upon Exposure to INT-407 Intestinal Epithelial Cells
Oh, Man-Hwan ; Jeong, Hee-Gon ; Choi, Sang-Ho ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 968~974
Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as amino acid transport/metabolism, nucleotide transport/metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsuA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.
Enhancement of In Vivo Bone Regeneration Efficacy of Human Mesenchymal Stem Cells
Kang, Sun-Woong ; Lee, Jae-Sun ; Park, Min Sun ; Park, Jung-Ho ; Kim, Byung-Soo ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 975~982
We investigated whether transplantation of osteogenically differentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and the use of an hydroxyapatite (HAp) scaffold can enhance the in vivo bone formation efficacy of human BMMSCs. Three months after implantation to the subcutaneous dorsum of athymic mice, transplantation of osteogenically differentiated human BMMSCs increased the bone formation area and calcium deposition to 7.1- and 6.2-folds, respectively, of those of transplantation of undifferentiated BMMSCs. The use of the HAp scaffold increased the bone formation area and calcium deposition to 3.7- and 3.5-folds, respectively, of those of a polymer scaffold. Moreover, a combination of transplantation of osteogenically differentiated BMMSCs and HAp scaffold further increased the bone formation area and calcium deposition to 10.6- and 9.3-folds, respectively, of those of transplantation of undifferentiated BMMSCs seeded onto polymer scaffolds. The factorial experimental analysis showed that osteogenic differentiation of BMMSCs prior to transplantation has a stronger positive effect than the HAp scaffold on in vivo bone formation.
-Mercaptoethanol and Hydrogen Peroxide on Enzymatic Conversion of Human Proinsulin to Insulin
Son, Young-Jin ; Kim, Chang-Kyu ; Choi, Byoung-Taek ; Park, Yong-Cheol ; Seo, Jin-Ho ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 983~989
Human insulin is a hormone well-known to regulate the blood glucose level. Recombinant preproinsulin, a precursor of authentic insulin, is typically produced in E. coli as an inactive inclusion body, the solubilization of which needs the addition of reducing agents such as
-mercaptoethanol. To make authentic insulin, recombinant preproinsulin is modified enzymatically by trypsin and carboxypeptidase B. The effects of
-mercaptoethanol on the formation of human insulin derivatives were investigated in the enzymatic modification by using commercially available human proinsulin as a substrate. Addition of 1 mM
-mercaptoethanol induced the formation of various insulin derivatives. Among them, the second major one, impurity 3, was found to be identical to the insulin B chain fragment from
. Minimization of the formation of insulin derivatives and concomitant improvement of the production yield of human insulin were achieved by the addition of hydrogen peroxide. Hydrogen peroxide bound with
-mercaptoethanol and thereby reduced the negative effects of
-mercaptoethanol considerably. Elimination of the impurity 3 and other derivatives by the addition of over 10 mM hydrogen peroxide in the presence of
-mercaptoethanolled to a 1.3-fold increase in the recovery efficiency of insulin, compared with those for the case without hydrogen peroxide. The positive effects of hydrogen peroxide were also confirmed with recombinant human preproinsulin expressed in recombinant E. coli as an inclusion body.
Effective Antibacterial Action of Tat (47-58) by Increased Uptake into Bacterial Cells in the Presence of Trypsin
Jung, Hyun-Jun ; Jeong, Kyu-Shik ; Lee, Dong-Gun ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 990~996
In a previous study, we found an antifungal effect on human pathogenic fungi by the cell-penetrating peptide Tat (47-58) derived from HIV-1. Tat (47-58) immediately entered into the fungal nucleus and affected some physiological changes on the intracellular condition. In this study, Tat (47-58) showed a broad spectrum of antibacterial activity against pathogenic bacteria including bacterial clinical isolates. To improve resistance against proteases for use in vivo, we synthesized an analog of Tat (47-58) by substituting the L-amino acid for the D-amino acid. The D-enantiomer of Tat (47-58) also exhibited a broad spectrum of antibacterial activity at almost the same level of L-Tat (47-58) concentration. Unlike L-Tat (47-58), D-Tat (47-58) showed a significant proteolytic resistance against all proteases tested and antimicrobial activities in the presence of trypsin. Moreover, D-Tat (47-58) inhibited MRSA infection in human HeLa cells whereas L-Tat (47-58) partially allowed MRSA infection, and the results were due to the proteolytic resistance of D-Tat (47-58).
Dry-Heat Treatment Process for Enhancing Viral Safety of an Antihemophilic Factor VIII Concentrate Prepared from Human Plasma
Kim, In-Seop ; Choi, Yong-Woon ; Kang, Yong ; Sung, Hark-Mo ; Shin, Jeong-Sup ;
Journal of Microbiology and Biotechnology, volume 18, issue 5, 2008, Pages 997~1003
Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (
for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at
. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were
for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.