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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 19, Issue 12 - Dec 2009
Volume 19, Issue 11 - Nov 2009
Volume 19, Issue 10 - Oct 2009
Volume 19, Issue 9 - Sep 2009
Volume 19, Issue 8 - Aug 2009
Volume 19, Issue 7 - Jul 2009
Volume 19, Issue 6 - Jun 2009
Volume 19, Issue 5 - May 2009
Volume 19, Issue 4 - Apr 2009
Volume 19, Issue 3 - Mar 2009
Volume 19, Issue 2 - Feb 2009
Volume 19, Issue 1 - Jan 2009
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A Novel pH-Stable, Bifunctional Xylanase Isolated from a Deep-Sea Microorganism, Demequina sp. JK4
Meng, Xin ; Shao, Zongze ; Hong, Yuzhi ; Lin, Ling ; Li, Chanjuan ; Liu, Ziduo ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1077~1084
DOI : 10.4014/jmb.0901.017
A genomic library was constructed to clone a xylanase gene (Mxyn10) from Demequina sp. JK4 isolated from a deep sea. Mxyn10 encoded a 471 residue protein with a calculated molecular mass of 49 kDa. This protein showed the highest sequence identity (70%) with the xylanase from Streptomyces lividans. Mxyn10 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10) and a carbohydrate-binding module (CBM) belonging to family 2. The optimum pH and temperature for enzymatic activity were pH 5.5 and
, respectively. Mxyn10 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 3.5 to 10.0. The protein was not significantly affected by a variety of chemical reagents, including some compounds that usually inhibit the activity of other related enzymes. In addition, Mxyn10 showed activity on cellulose. These properties mark Mxyn10 as a potential enzyme for industrial application and saccharification processes essential for bioethanol production.
Novel Low-Temperature-Active Phytase from Erwinia carotovora var. carotovota ACCC 10276
Huang, Huoqing ; Luo, Huiying ; Wang, Yaru ; Fu, Dawei ; Shao, Na ; Yang, Peilong ; Meng, Kun ; Yao, Bin ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1085~1091
DOI : 10.4014/jmb.0901.039
A phytase with high activity at low temperatures has great potential for feed applications, especially in aquaculture. Therefore, this study used a degenerate PCR and TAIL PCR to clone a phytase gene from Erwinia carotovora var. carotovota, the cause of soft rot of vegetables in the ground or during cold storage. The full-length 2.5-kb fragment included an open reading frame of 1,302 bp and encoded a putative phytase of 45.3 kDa with a 50% amino acid identity to the Klebsiella pneumoniae phytase. The phytase contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. The enzyme was expressed in Escherichia coli, purified, and displayed the following characteristics: a high catalytic activity at low temperatures (retaining over 24% activity at
) and remarkably thermal lability (losing >96% activity after incubation at
for 2 min). The optimal phytase activity occurred at pH 5.5 and
, and the enzyme activity rapidly decreased above
. When compared with mesophilic counterparts, the phytase not only exhibited a high activity at a low temperature, but also had a low
. These temperature characteristics and kinetic parameters are consistent with low-temperature-active enzymes. To our knowledge, this would appear to be the first report of a low-temperature-active phytase and its heterogeneous expression.
Biochemical Characteristics and Function of a Fucosyltransferase Encoded by ste7 in Ebosin Biosynthesis of Streptomyces sp. 139
Chang, Ming ; Bai, Li-Ping ; Shan, Jung-Jie ; Jiang, Rong ; Zhang, Yang ; Guo, Lian-Hong ; Zhang, Ren ; Li, Yuan ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1092~1097
DOI : 10.4014/jmb.0903.03021
A novel exopolysaccharide named Ebosin was produced by Streptomyces sp. 139, with medicinal activity. Its biosynthesis gene cluster (ste) has been previously identified. For the functional study of the ste7 gene in Ebosin biosynthesis, it was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS-7m produced by the mutant strain Streptomyces sp. 139 (
) was found altered from that of Ebosin, with fucose decreasing remarkably. For biochemical characterization of Ste7, the ste7 gene was cloned and expressed in Escherichia coli BL21. With a continuous coupled spectrophotometric assay, Ste7 was demonstrated to have the ability of catalyzing the transfer of fucose specifically from GDP-
-L-fucose to a fucose acceptor, the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 (
). Therefore, the ste7 gene has been identified to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugars units during Ebosin biosynthesis.
Production of Gamma-Linolenic Acid in Pichia pastoris by Expression of a Delta-6 Desaturase Gene from Cunninghamella echinulata
Wan, Xia ; Zhang, Yinbo ; Wang, Ping ; Huang, Fenghong ; Chen, Hong ; Jiang, Mulan ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1098~1102
DOI : 10.4014/jmb.0902.071
Gamma-linolenic acid (GLA, C18:3
) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2
) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.
Pathway Analysis in HEK 293T Cells Overexpressing HIV-1 Tat and Nucleocapsid
Lee, Min-Joo ; Park, Jong-Hoon ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1103~1108
DOI : 10.4014/jmb.0903.03005
The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.
Proteomic Analysis in ob/ob Mice Before and After Hypoglycemic Polysaccharide Treatments
Kim, Sang-Woo ; Hwang, Hye-Jin ; Baek, Yu-Mi ; Hwang, Hee-Sun ; Yun, Jong-Won ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1109~1121
DOI : 10.4014/jmb.0901.056
In an attempt to discover novel biomarker proteins in type 2 diabetes prognosis, we investigated the influence of hypoglycemic extracellular polysaccharides (EPS) obtained from the macrofungus Tremella fuciformis on the differential levels of plasma proteins in ob/ob mice using two-dimensional gel electrophoresis (2-DE). The 2-DE analysis demonstrated that 92 spots from about 900 visualized spots were differentially regulated, of which 40 spots were identified as principal diabetes-associated proteins. By comparing control with EPS-fed mice, we found that at least six proteins were significantly altered in ob/ob mice, including Apo A-I, IV, C-III, E, retinol-binding protein 4, and transferrin, and their levels were interestingly normalized after EPS treatment. Western blot analysis revealed that the altered levels of the two regulatory molecules highlighted in diabetes and obesity (e.g., resistin and adiponectin) were also normalized in response to EPS. The Mouse Diabetes PCR Array profiles showed that the expression of 84 genes related to the onset, development, and progression of diabetes were significantly downregulated in liver, adipocyte, and muscle of ob/ob mice. EPS might act as a potent regulator of gene expression for a wide variety of genes in ob/ob mice, particularly in obesity, insulin resistance, and complications from diabetes mellitus.
Cadaverine is Transported into Vibrio vulnificus Through its CadB in Alkaline Environment
Kang, In-Hye ; Kim, Eui-Jin ; Lee, Jeong-K. ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1122~1126
DOI : 10.4014/jmb.0903.03012
The exogenously added cadaverine is effective in protecting Vibrio vulnificus from methyl viologen (MV)-induced superoxide stress at pH 8.5. Such a protective effect by cadaverine was not observed at pH 7.5. Consistently, the accumulated level of intracellular cadaverine at pH 8.5 is approximately four times as much as that of the control cell at pH 7.5. Cadaverine accumulation is not affected by MV. The protection of V. vulnificus by cadaverine from superoxide stress was abolished when cadB coding for the lysine-cadaverine antiporter was interrupted. However, the cadaverine-mediated protection was complemented with cadB DNA. Therefore, CadB of V. vulnificus not only acts as a lysine-cadaverine antiporter at acid pH to neutralize the external medium, but also mediates cadaverine uptake at alkaline pH to result in cell protection from superoxide stress.
Acetate Consumption Activity Directly Determines the Level of Acetate Accumulation During Escherichia coli W3110 Growth
Shin, Soo-An ; Chang, Dong-Eun ; Pan, Jae-Gu ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1127~1134
DOI : 10.4014/jmb.0902.097
Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.
Antitumor Components from Naematoloma fasciculare
Ding, Yan ; Bao, Hai Ying ; Bau, Tolgor ; Li, Yu ; Kim, Young-Ho ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1135~1138
DOI : 10.4014/jmb.0901.022
The bioassay-guided fractionation of MeOH extract from Naematoloma fasciculare afforded a petroleum ether fraction (NFPF) and four known compounds, which showed good antitumor activities to inhibit MCF-7 cell line proliferation in vitro and tumor growth in
implanted mice in vivo. In addition, a number of unsaturated aliphatic acids were identified in NFPF by GC analysis. These results showed that NFPF inhibits tumor growth through the activity of unsaturated aliphatic acids together with two active compounds, ergosterol peroxide (1: 62.17 mg/g in NFPF) and ergosterol (2: 3.13 mg/g in NFPF), and indicate the potential utility of NFPF as an antitumor drug.
Clitocybin D, a Novel Human Neutrophil Elastase Inhibitor from the Culture Broth of Clitocybe aurantiaca
Kim, Young-Hee ; Ryoo, In-Ja ; Choo, Soo-Jin ; Xu, Guang-Hua ; Lee, Sang-Ku ; Seok, Soon-Ja ; Bae, Ki-Hwan ; Yoo, Ick-Dong ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1139~1141
DOI : 10.4014/jmb.0903.03033
Clitocybin D, a novel human neutrophil elastase inhibitor, was isolated from the culture broth of Clitocybe aurantiaca. This compound was purified by solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. The compound was determined to be 4-(4,6-dihydroxy-3-methoxy-3H-isoindol-1-yl)-benzoic acid on the basis of 1D and 2D NMRs and MS spectroscopic analysis. Analysis of the human neutrophil elastase (HNE) inhibitory activity of the isolated compound revealed that it showed significant HNE inhibitory activity with an
Effects of Nutrients on Quorum Signals and Secondary Metabolite Productions of Burkholderia sp. O33
Keum, Young-Soo ; Lee, Young-Ju ; Lee, Youn-Hyung ; Kim, Jeong-Han ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1142~1149
DOI : 10.4014/jmb.0801.465
Several bioactive metabolites, including pyrrolnitrin, N-acylhomoserine lactones, and polyhydroxyalkanoates were isolated from Burkholderia sp. O33. Effects of various nutrients, including sugars, gluconolactone, glycerol, tryptophan, chloride, and zinc were investigated in relation to the production of these metabolites. Logarithmic increase of pyrrolnitrin was observed between 2-5 days and reached a maximum at 7-10 days. Tryptophan concentration reached the maximum at 3 days, whereas 7-chlorotryptophan was gradually increased throughout the studies. Among various carbon sources, gluconolactone, trehalose, and glycerol enhanced pyrrolnitrin production, whereas strong inhibitory effects were found with glucose. Relative concentrations of pyrrolnitrin and its precursors were in the order of pyrrolnitrin
dechloroaminopyrrolnitrin or aminopyrrolnitrin throughout the experiments. Among three N-acylhomoserine lactones, the N-octanoyl analog was the most abundant quorum sensing signal, of which the concentrations reached the maximum in 2-3 days, followed by a rapid dissipation to trace level. No significant changes in pyrrolnitrin biosynthesis were observed by external addition of N-acylhomoserine lactones. Polyhydroxyalkanoates accumulated up to 3-4 days and decreased slowly thereafter. According to the kinetic analyses, no strong correlations were found between the levels of pyrrolnitrin, N-acylhomoserine lactones, and polyhydroxyalkanoates.
Microbial Transformation of a Monoterpene, Geraniol, by the Marine-derived Fungus Hypocrea sp.
Leutou, Alain S. ; Yang, Guohua ; Nenkep, Viviane N. ; Siwe, Xavier N. ; Feng, Zhile ; Khong, Thang T. ; Choi, Hong-Dae ; Kang, Jung-Sook ; Son, Byeng-Wha ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1150~1152
DOI : 10.4014/jmb.0904.04013
Geraniol (1) is the biogenetic precursor of a number of monoterpenes. We tested various marine-derived microorganisms to determine their ability to biotransform 1. Only Hypocrea sp. was capable of transforming 1 into its oxidized derivative, 1,7-dihydroxy-3,7-dimethyl-(E)-oct-2-ene (2). The structure of the metabolite obtained was assigned on the basis of detailed spectroscopic data analyses.
Production of Lactosucrose from Sucrose and Lactose by a Levansucrase from Zymomonas mobilis
Han, Woo-Cheul ; Byun, Sun-Ho ; Kim, Mi-Hyun ; Sohn, Eun-Hwa ; Lim, Jung-Dae ; Um, Byung-Hun ; Kim, Chul-Ho ; Kang, Soon-Ah ; Jang, Ki-Hyo ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1153~1160
DOI : 10.4014/jmb.0901.045
-D-galactosylsucrose) is an oligosaccharide consisting of galactose, glucose, and fructose. In this study, we prepared lactosucrose from lactose and sucrose using a levansucrase derived from Zymomonas mobilis. Optimum conditions for lactosucrose formation were
, pH 7.0, 18.0% (w/v) lactose monohydrate, and 18% (w/v) sucrose as substrates, and 1 unit of enzyme/ml of reaction mixture. Under these conditions, the lactosucrose conversion efficiency was 28.5%. The product was purified and confirmed to be O-
-D-fructofuranoside, or lactosucrose. A mixed-enzyme system containing a levansucrase and a glucose oxidase was applied in order to increase the efficiency of lactose and sucrose conversion to lactosucrose, which rose to 43.2% as a result.
Ethanol Production from Rice Winery Waste - Rice Wine Cake by Simultaneous Saccharification and Fermentation Without Cooking
Vu, Van Hanh ; Kim, Keun ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1161~1168
DOI : 10.4014/jmb.0907.07001
Ethanol production by the simultaneous saccharification and fermentation (SSF) of low-value rice wine cake (RWC) without cooking was investigated. RWC is the filtered solid waste of fermented rice wine mash and contains 53% raw starch. For the SSF, the RWC slurry was mixed with the raw-starch-digesting enzyme of Rhizopus sp. and yeast, where the yeast strain was selected from 300 strains and identified as Saccharomyces cerevisiae KV25. The highest efficiency (94%) of ethanol production was achieved when the uncooked RWC slurry contained 23.03% starch. The optimal SSF conditions were determined as 1.125 units of the raw-starch-digesting enzyme per gram of RWC, a fermentation temperature of
, slurry pH of 4.5, 36-h-old seeding culture, initial yeast cell number of
per ml of slurry, 17 mM of urea as the nitrogen additive, 0.25 mM of
as the metal ion additive, and a fermentation time of 90 h. Under these optimal conditions, the ethanol production resulting from the SSF of the uncooked RWC slurry was improved to 16.8% (v/v) from 15.1% (v/v) of pre-optimization.
Analysis of the Involvement of Chitin-Binding Domain of ChiCW in Antifungal Activity, and Engineering a Novel Chimeric Chitinase with High Enzyme and Antifungal Activities
Huang, Chien-Jui ; Guo, Shu-Huei ; Chung, Shu-Chun ; Lin, Yu-Ju ; Chen, Chao-Ying ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1169~1175
DOI : 10.4014/jmb.0811.624
An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated,
(lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since
(lacking the chitin-binding domain) could not be expressed in Escherichia coli as
did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW (
) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with
. When the chitin-binding domain of ChiA1 was replaced with
, the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that
may play an important role in the antifungal activity of ChiCW.
Characterization of Surface Layer Proteins in Lactobacillus crispatus Isolate ZJ001
Chen, Xueyan ; Chen, Yang ; Li, Xiaoliang ; Chen, Ning ; Fang, Weihuan ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1176~1183
DOI : 10.4014/jmb.0901.010
Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-S-layer layer genes were sequenced and expressed in E. coli to characterize the function of proteins with this particular strain. L. crispatus ZJ001 harbored two S-layer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disc-like structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and His-slpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.
Purification and Characterization of Endo-
-1,4 Mannanase from Aspergillus niger gr for Application in Food Processing Industry
Naganagouda, K. ; Salimath, P.V. ; Mulimani, V.H. ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1184~1190
DOI : 10.4014/jmb.0901.029
A thermostable extracellular
-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The
-mannanase exhibited optimum catalytic activity at pH 5.5 and
. It was thermostable at
, and retained 50% activity after 6 h at
. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions
inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with
of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.
Medium Optimization and Application of Affinity Column Chromatography for Trypsin Production from Recombinant Streptomyces griseus
Chi, Won-Jae ; Song, Ju-Hyun ; Oh, Eun-A. ; Park, Seong-Whan ; Chang, Yong-Keun ; Kim, Eung-Soo ; Hong, Soon-Kwang ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1191~1196
DOI : 10.4014/jmb.0901.001
The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and
, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to
. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.
Purification and Characterization of Neoagarotetraose from Hydrolyzed Agar
Jang, Min-Kyung ; Lee, Dong-Guen ; Kim, Nam-Young ; Yu, Ki-Hwan ; Jang, Hye-Ji ; Lee, Seung-Woo ; Jang, Hyo-Jung ; Lee, Ye-Ji ; Lee, Sang-Hyeon ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1197~1200
DOI : 10.4014/jmb.0906.06045
The whitening effect, tyrosinase inhibition, and cytotoxicity of neoagarotetraose were measured after its purification from hydrolyzed agar by gel filtration chromatography. In melanoma B16F10 cells, the melanin content of neoagarotetraose-treated cells was the same as that treated by kojic acid or arbutin. In addition, tyrosinase of melanoma cells was strongly inhibited by neoagarotetraose at a concentration of
and similarly inhibited at 10 and
compared with those by arbutin or kojic acid. The activity of mushroom tyrosinase showed a 38% inhibition by neoagarotetraose at
, and this inhibitory effect was more efficient than that by kojic acid. Neoagarotetraose revealed a similar
(50% inhibition concentration) value for mushroom tyrosinase as that by kojic acid. These data suggest that the neoagarotetraose generated from agar by recombinant
-agarase might be a good candidate as a cosmetic additive for the whitening effect.
Effect of Amylose Content on Corn Starch Modification by Thermus aquaticus 4-
Cho, Kyoung-Hee ; Auh, Joong-Hyuck ; Kim, Jung-Hwan ; Ryu, Je-Hoon ; Park, Kwan-Hwa ; Park, Cheon-Seok ; Yoo, Sang-Ho ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1201~1205
DOI : 10.4014/jmb.0905.05048
Corn starches with different amylose contents were enzymatically modified using Thermus aquaticus 4-
). Upon the enzyme treatment, the chain-length distributions of isoamylolytically debranched products became broader [degree of polymerization (DP): 3-40] than those of untreated corn starches. In addition, a variety of cycloamyloses (CAs) with different sizes were formed by the glucanotransfer activity of
. CAs with DP 5-40 were detectable in all of the
-treated corn starches. From the results of high-performance anion-exchange chromatography and high-performance size-exclusion chromatography analyses, it was suggested that the amount of CAs produced by the enzyme treatment increased as the amylose content of the starches increased. Thus, we concluded that the extent of modification of starch molecules was enhanced in proportion to amylose content by the transfer activity of
. This finding could be useful for developing an efficient process of CA production using this enzyme.
Use of FT-IR to Identify Enhanced Biomass Production and Biochemical Pool Shifts in the Marine Microalgae, Chlorella ovalis, Cultured in Media Composed of Different Ratios of Deep Seawater and Fermented Animal Wastewater
Kim, Mi-Kyung ; Jeune, Kyung-Hee ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1206~1212
DOI : 10.4014/jmb.0901.041
Growth rates, photosystem II photosynthesis, and the levels of chlorophyll
and secondary metabolites of Chlorella ovalis were estimated to determine if they were enhanced by the addition of swine urine (BM) or cow compost water (EP) that had been fermented by soil bacteria to deep seawater (DSW) in an attempt to develop media that enabled batch mass culture at lower costs. Growth of C. ovalis in f/2, f/2-EDTA+BM60%, DSW+BM30%, and DSW+EP60% was enhanced and maintained in the log phase of growth for 16 days. The cell densities of C. ovalis in DSW+EP60% (
Cells/ml) were higher than those of f/2 (
Cells/ml), f/2-E+BM60% (
Cells/ml), and DSW+BM30% (
Cells/ml). The growth rate was also more favorable for C. ovalis cultured in DSW+EP60% (
) than that of C. ovalis cultured in the control medium (f/2) (
). Furthermore, the chlorophyll a concentration of C. ovalis cultured in DSW+EP60% (4.56 mg/l) was more than 2-fold greater than that of C. ovalis cultured in f/2 (2.35 mg/l). Moreover, the maximal quantum yields of photo system II at 470 nm (Fv/Fm) were significantly higher in organisms cultured at f/2-E+BM60% (0.53) and DSW+EP60% (0.52) than in the other treatment groups. Finally, Fourier transformation infrared (FT-IR) spectroscopy revealed that C. ovalis grown in DSW+EP60% had more typical peaks and various biochemical pool shifts than those grown in other types of media. Taken together, the results of this study indicate that the use of DSW+EP60% to culture C. ovalis can reduce maintenance expenses and promote higher yields.
Characterization of Plant Growth-Promoting Traits of Free-Living Diazotrophic Bacteria and Their Inoculation Effects on Growth and Nitrogen Uptake of Crop Plants
Islam, Md. Rashedu ; Madhaiyan, M. ; Boruah, Hari P.Deka ; Yim, Woo-Jong ; Lee, Gill-Seung ; Saravanan, V.S. ; Fu, Qingling ; Hu, Hongqing ; Sa, Tongmin ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1213~1222
DOI : 10.4014/jmb.0903.03028
The search for diverse plant growth-promoting (PGP) diazotrophic bacteria is gaining momentum as efforts are made to exploit them as biofertilizers for various economically important crops. In the present study, 17 diazotrophic strains belonging to eight different genera isolated from rice paddy fields were screened for multiple PGP traits and evaluated for their inoculation effects on canola and rice plants. All of the strains tested positive for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and production of indole 3-acetic acid (IAA) and ammonia (
). Additionally, four of the strains were able to solubilize phosphorus (P), five tested positive for zinc (Zn) solubilization and sulfur (S) oxidation, and eight strains produced siderophores. Based on the presence of multiple PGP traits, 10 strains were selected for inoculation studies. Treatment with Herbaspirillum sp. RFNB26 resulted in maximum root length (54.3%), seedling vigor, and dry biomass in canola, whereas Paenibacillus sp. RFNB4 exhibited the lowest activity under gnotobiotic conditions. However, under pot culture conditions, Paenibacillus sp. RFNB4 significantly increased plant height and dry biomass production by 42.3% and 29.5%, respectively. Canola plants and rhizosphere soils inoculated with Bacillus sp. RFNB6 exhibited significantly higher nitrogenase activity. In greenhouse experiments, Serratia sp. RFNB18 increased rice plant height by 35.1%, Xanthomonas sp. RFNB24 enhanced biomass production by 84.6%, and rice rhizosphere soils inoculated with Herbaspirillum sp. RFNB26 exhibited the highest nitrogenase activity. Our findings indicate that most of the selected strains possess multiple PGP properties that significantly improve the growth parameters of the two plants when tested under controlled conditions.
Construction of a Recombinant Bacillus velezensis Strain as an Integrated Control Agent Against Plant Diseases and Insect Pests
Roh, Jong-Yul ; Liu, Qin ; Choi, Jae-Young ; Wang, Yong ; Shim, Hee-Jin ; Xu, Hong Guang ; Choi, Gyung-Ja ; Kim, Jin-Cheol ; Je, Yeon-Ho ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1223~1229
DOI : 10.4014/jmb.0902.065
To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.
Selective Plugging Strategy Based Microbial Enhanced Oil Recovery Using Bacillus licheniformis TT33
Suthar, Harish ; Hingurao, Krushi ; Desai, Anjana ; Nerurkar, Anuradha ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1230~1237
DOI : 10.4014/jmb.0904.04043
The selective plugging strategy of Microbial Enhanced Oil Recovery (MEOR) involves the use of microbes that grow and produce exopolymeric substances, which block the high permeability zones of an oil reservoir, thus allowing the water to flow through the low permeability zones leading to increase in oil recovery. Bacillus licheniformis TT33, a hot water spring isolate, is facultatively anaerobic, halotolerant, and thermotolerant. It produces EPS as well as biosurfactant and has a biofilm-forming ability. The viscosity of its cell-free supernatant is
. Its purified EPS contained 26% carbohydrate and 3% protein. Its biosurfactant reduced the surface tension of water from 72 to 34 mN/m. This strain gave
oil recovery in a sand pack column. Environmental scanning electron microscopy analysis showed bacterial growth and biofilm formation in the sand pack. Biochemical tests and Amplified Ribosomal DNA Restriction Analysis confirmed that the oil recovery obtained in the sand pack column was due to Bacillus licheniformis TT33.
Antimicrobials Effective for Inhibition of Enterohemorrhagic Escherichia coli Strains O26, O111, and O157 and Their Effects on Shiga Toxin Releases
Lee, John-Hwa ; Stein, Barry D. ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1238~1243
DOI : 10.4014/jmb.0903.03006
The susceptibilities of major enterohemorrhagic Escherichia coli (EHEC) strains to antimicrobial agents and the cytotoxicity of these agents were examined using a total of 38 strains of E. coli O26, O111, and O157, which are the major serogroups of EHEC. Among the 38 strains, 35, 36, and 36 were susceptible to amikacin, imipenem, and norfloxacin, respectively. These antimicrobial agents were further examined to determine their cytotoxicity on Vero cells as well as their effect on the release of Shiga toxins along with trimethoprim/sulfamethoxazole. Each of the E. coli O26, O111, and O157 strains containing both the stx1 and stx2 genes were grown in the absence or presence of these agents at 1/4 minimal inhibitory concentration for 6 h, 12 h, and 18 h. At the concentrations used in this study, none of the agents significantly altered cell count compared with the control group. The level of cytotoxicity in the imipenem group was lower at 12 hand 18 h than their respective controls. In contrast, the level of cytotoxicity in cultures treated with trimethoprim/sulfamethoxazole, norfloxacin, and amikacin was increased. The strains were also examined for the release of Shiga toxins 1 and 2 following treatment with the agents, which were measured by the reversed passive latex agglutination (RPLA) method. The RPLA assay showed a suppression of release of Shiga toxin 2 in the strain cultures containing imipenem. These results indicate that imipenem may be a safe and effective agent for inhibition of these bacteria, which has clinical implications for the treatment of EHEC infections.
Phoma herbarum as a New Gibberellin-Producing and Plant Growth-Promoting Fungus
Hamayun, Muhammad ; Khan, Sumera Afzal ; Khan, Abdul Latif ; Rehman, Gauhar ; Sohn, Eun-Young ; Shah, Aamer Ali ; Kim, Sang-Kuk ; Joo, Gil-Jae ; Lee, In-Jung ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1244~1249
DOI : 10.4014/jmb.0901.030
Endophytic fungi are known for the production of valuable metabolites, but information on the gibberellin production capacity of this group is limited. We isolated 9 endophytic fungi from the roots of salt-stressed soybean plants and screened them on waito-c rice, in order to identify plant growth promoting fungal strains. The fungal isolate TK-2-4 gave maximum plant length (20.35 cm) promotion in comparison with wild-type Gibberella fujikuroi (19.5 cm). In a separate experiment, bioassay of TK-2-4 promoted plant length and biomass of soybean cultivar Taegwangkong. The TK-2-4 culture filtrate was analyzed for the presence of gibberellins, and it was found that all physiologically active gibberellins, especially
, were present in higher amounts (
, 0.11 ng/ml;
, 2.91 ng/ml;
, 3.21 ng/ml; and
, 1.4 ng/ml) in conjunction with physiologically inactive
(0.23 ng/ ml),
(0.53 ng/ml), and
(0.06 ng/ml). The fungal isolate TK-2-4 was later identified as a new strain of Phoma herbarum, through the phylogenetic analysis of 28S rDNA sequence.
Molecular Mechanism of Plant Growth Promotion and Induced Systemic Resistance to Tobacco Mosaic Virus by Bacillus spp.
Wang, Shuai ; Wu, Huijun ; Qiao, Junqing ; Ma, Lingli ; Liu, Jun ; Xia, Yanfei ; Gao, Xuewen ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1250~1258
DOI : 10.4014/jmb.0901.008
Bacillus spp., as a type of plant growth-promoting rhizobacteria (PGPR), were studied with regards promoting plant growth and inducing plant systemic resistance. The results of greenhouse experiments with tobacco plants demonstrated that treatment with the Bacillus spp. significantly enhanced the plant height and fresh weight, while clearly lowering the disease severity rating of the tobacco mosaic virus (TMV) at 28 days post-inoculation (dpi). The TMV accumulation in the young non-inoculated leaves was remarkably lower for all the plants treated with the Bacillus spp. An RT-PCR analysis of the signaling regulatory genes Coil and NPR1, and defense genes PR-1a and PR-1b, in the tobacco treated with the Bacillus spp. revealed an association with enhancing the systemic resistance of tobacco to TMV. A further analysis of two expansin genes that regulate plant cell growth, NtEXP2 and NtEXP6, also verified a concomitant growth promotion in the roots and leaves of the tobacco responding to the Bacillus spp.
Comparative Evaluation of Three Culture Methods for the Isolation of Mycobacteria from Clinical Samples
Sorlozano, Antonio ; Soria, Isabel ; Roman, Juan ; Huertas, Pilar ; Soto, Maria Jose ; Piedrola, Gonzalo ; Gutierrez, Jose ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1259~1264
DOI : 10.4014/jmb.0901.059
We assessed the capacity of two liquid-medium culture methods with automated incubation and reading systems (MB/BacT ALERT 3D System and BACTEC MGIT 960 System) and one solid-medium culture method (
-Jensen) to detect mycobacteria in different types of clinical samples. Out of 1,770 cultured clinical samples (1,519 of respiratory origin and 251 of non respiratory origin), mycobacteria were isolated in 156 samples (135 M. tuberculosis complex, 8 M. chelonae, 6 M. kansasii, 4 M. fortuitum, 2 M. gordonae, and 1 M. marinum) by at least one of the methods used. The BACTEC MGIT 960 System proved to be the most sensitive method (86.5%), especially in the detection of M. tuberculosis complex (89.1%). However,
-Jensen culture was the most sensitive (76.2%) to detect nontuberculous mycobacteria. The BACTEC MGIT 960 System showed the lowest mean detection time for mycobacterial growth (15.3 days), significantly shorter than the other two methods. Highest sensitivity (95.5%) and specificity (99.6%) values were obtained using the BACTEC MGIT 960 System with the
-Jensen culture method, which was also the only combination capable of detecting 100% of the nontuberculous mycobacteria.
Antibiograms and Molecular Subtypes of Methicillin-Resistant Staphylococcus aureus in Local Teaching Hospital, Malaysia
Thong, Kwai Lin ; Junnie, June ; Liew, Fong Yin ; Yusof, Mohd Yasim ; Hanifah, Yasmin A. ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1265~1270
DOI : 10.4014/jmb.0809.504
The objectives of this study were to determine the antibiotypes, SCCmec subtypes, PVL carriage, and genetic diversity of MRSA strains from a tertiary hospital. Sixty-six MRSA strains were selected randomly (2003, 2004, and 2007) and tested for the Panton-Valentine leukocidin gene, mecA gene, and SCCmec type via a PCR. The antibiograms were determined using a standard disc diffusion method, and the genetic diversity of the isolates was determined by PFGE. Thirty-four antibiograms were obtained, with 55% of the 66 strains exhibiting resistance to more than 4 antimicrobials. All the isolates remained susceptible to vancomycin, and low resistance rates were noted for fusidic acid (11%), rifampicin (11%), and clindamycin acid (19%). The MRSA isolates that were multisensitive (n=12) were SCCmec type IV, whereas the rest (multiresistant) were SCCmec type III. Only two isolates (SCCmec type IV) tested positive for PVL, whereas all the isolates were mecA-positive. The PFGE was very discriminative and subtyped the 66 isolates into 55 pulsotypes (F=0.31-1.0). The multisensitive isolates were distinctly different from the multidrug-resistant MRSA. In conclusion, no vancomycin-resistant isolate was observed. The Malaysian MDR MRSA isolates were mostly SCCmec type III and negative for PVL. These strains were genetically distinct from the SCCmec type IV strains, which were sensitive to SXT, tetracycline, and erythromycin. Only two strains were SCCmec IV and PVL-positive. The infections in the hospital concerned were probably caused by multiple subtypes of MRSA.
Multi-Immunogenic Outer Membrane Vesicles Derived from a MsbB-Deficient Salmonella enterica Serovar Typhimurium Mutant
Lee, Sang-Rae ; Kim, Sang-Hyun ; Jeong, Kang-Jin ; Kim, Keun-Su ; Kim, Young-Hyun ; Kim, Sung-Jin ; Kim, E-Kyune ; Kim, Jung-Woo ; Chang, Kyu-Tae ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1271~1279
DOI : 10.4014/jmb.0901.505
To develop low endotoxic and multi-immunogenic outer membrane vesicles (OMVs), a deletion mutant of the msbB gene in Salmonella enterica serovar Typhimurium (S. Typhimurium) was used as a source of low endotoxic OMV, and an expression vector of the canine parvovirus (CPV) VP2 epitope fused to the bacterial OmpA protein was constructed and transformed into the Salmonella
mutant. In a lethality test, BALB/c mice injected intraperitoneally with the Salmonella
mutant survived for 7 days, whereas mice injected intraperitoneally with the wild type survived for 3 days. Moreover, all mice inoculated orally with the
mutant survived for 30 days, but 80% of mice inoculated orally with the wild type survived. The OmpA::CPV VP2 epitope fusion protein was expressed successfully and associated with the outer membrane and OMV fractions from the mutant S. Typhimurium transformed with the fusion protein-expressing vector. In immunogenicity tests, sera obtained from the mice immunized with either the Salmonella msbB mutant or its OMVs containing the OmpA::CPV VP2 epitope showed bactericidal activities against wild-type S. Typhimurium and contained specific antibodies to the CPV VP2 epitope. In the hemagglutination inhibition (HI) assay as a measurement of CPV-neutralizing activity in the immune sera, there was an 8-fold increase of HI titer in the OMV-immunized group compared with the control. These results suggested that the CPV-neutralizing antibody response was raised by immunization with OMV containing the OmpA::CPV VP2 epitope, as well as the protective immune response against S. Typhimurium in BALB/c mice.
Characterization of a 27 kDa Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang[Author's correction]
Kim, Gyoung-Min ; Lee, Ae-Ran ; Lee, Kang-Wook ; Park, Ae-Yong ; Chun, Ji-Yeon ; Cha, Jae-Ho ; Song, Young-Sun ; Kim, Jeong-Hwan ;
Journal of Microbiology and Biotechnology, volume 19, issue 10, 2009, Pages 1280~1280