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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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sanN Encoding a Dehydrogenase is Essential for Nikkomycin Biosynthesis in Streptomyces ansochromogenes
Ling, Hong-Bo ; Wang, Guo-Jun ; Li, Jin-E ; Tan, Hua-Rong ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 397~403
Nikkomycins are a group of peptidyl nucleoside antibiotics with potent fungicidal, insecticidal, and acaricidal activities. sanN was cloned from the partial genomic library of Streptomyces ansochromogenes 7100. Gene disruption and complementation analysis demonstrated that sanN is essential for nikkomycin biosynthesis in S. ansochromogenes. Primer extension assay indicated that sanN is transcribed from two promoters (sanN-P1 and sanN-P2), and sanN-P2 plays a more important role in nikkomycin biosynthesis. Purified recombinant SanN acts as a dehydrogenase to convert benzoate-CoA to benzaldehyde in a random-order mechanism in vitro, with respective
toward benzoate-CoA and NADH, suggesting that SanN catalyzes the formation of picolinaldehyde during biosynthesis of nikkomycin X and Z components in the wild-type stain. These data would facilitate us to understand the biosynthetic pathway of nikkomycins and to consider the combinatorial synthesis of novel antibiotic derivatives.
Functional Analysis of a Gene Encoding Endoglucanase that Belongs to Glycosyl Hydrolase Family 12 from the Brown-Rot Basidiomycete Fomitopsis palustris
Song, Byeong-Cheol ; Kim, Ki-Yeon ; Yoon, Jeong-Jun ; Sim, Se-Hoon ; Lee, Kang-Seok ; Kim, Yeong-Suk ; Kim, Young-Kyoon ; Cha, Chang-Jun ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 404~409
The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and
-glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.
Cloning, Expression, and Characterization of Protease-resistant Xylanase from Streptomyces fradiae var. k11
Li, Ning ; Yang, Peilong ; Wang, Yaru ; Luo, Huiying ; Meng, Kun ; Wu, Nigfeng ; Fan, Yunliu ; Yao, Bin ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 410~416
The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-
-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and
, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by
and strongly inhibited by
and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.
Functional Effects of Increased Copy Number of the Gene Encoding Proclavaminate Amidino Hydrolase on Clavulanic Acid Production in Streptomyces clavuligerus ATCC 27064
Song, Ju-Yeon ; Kim, Eun-Sook ; Kim, Dae-Wi ; Jesen, Susan E. ; Lee, Kye-Joon ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 417~426
The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by increasing dosoge of a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.
Expression and Characterization of Polyketide Synthase Module Involved in the Late Step of Cephabacin Biosynthesis from Lysobacter lactamgenus
Lee, Ji-Seon ; Vladimirova, Miglena G. ; Demirev, Atanas V. ; Kim, Bo-Geum ; Lim, Si-Kyu ; Nam, Doo-Hyun ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 427~433
The cephabacins produced by Lysobacter lactamgenus are
-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains,
-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of
-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-
-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with
-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.
Enhanced Enzyme Activities of Inclusion Bodies of Recombinant
-Galactosidase via the Addition of Inducer Analog after L-Arabinose Induction in the araBAD Promoter System of Escherichia coli
Jung, Kyung-Hwan ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 434~442
We observed that an inclusion body (IB) of recombinant
-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the
-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific
-galactosidase production, although
-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of
-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of
-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.
-Glycosidase-CBD Fusion Protein for Biochemical Analysis of Cotton Scouring Efficiency
Ha, Jae-Seok ; Lee, Young-Mi ; Choi, Su-Lim ; Song, Jae-Jun ; Shin, Chul-Soo ; Kim, Ju-Hea ; Lee, Seung-Goo ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 443~448
Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable
-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric
-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.
Molecular Cloning and Characterization of a Bile Salt Hydrolase from Lactobacillus acidophilus PF01
Oh, Hae-Keun ; Lee, Ji-Yoon ; Lim, Soo-Jin ; Kim, Min-Jeong ; Kim, Geun-Bae ; Kim, Jung-Hoan ; Hong, Soon-Kwang ; Kang, Dae-Kyung ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 449~456
Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a
-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately
. The enzyme maintained approximately 70% of its maximum activity even at
, whereas its activity rapidly decreased at below
. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.
Purification and Characterization of Branching Specificity of a Novel Extracellular Amylolytic Enzyme from Marine Hyperthermophilic Rhodothermus marinus
Yoon, Seong-Ae ; Ryu, Soo-In ; Lee, Soo-Bok ; Moon, Tae-Wha ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 457~464
An extracellular enzyme (RMEBE) possessing
-transferring activity was purified to homogeneity from Rhodothermus marin us by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex-200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at
. Its half-life was determined to be 73.7 and 16.7 min at 80 and
, respectively. The enzyme was also halophilic and highly halotolerant up to about 2M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial
-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited
-transferase activity for small maltooligosaccharides, producing disproportionated
-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.
Development of Magnetically Separable Immobilized Lipase by Using Cellulose Derivatives and Their Application in Enantioselective Esterification of Ibuprofen
Lee, Go-Woun ; Joo, Hong-Il ; Kim, Jung-Bae ; Lee, Jung-Heon ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 465~471
Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.
Analysis of the Dimerization of Human CD99 Using Bimolecular Fluorescence Complementation Technique
Lee, Mi-Kyung ; Kim, Hyun-Soo ; Kim, Seung-Seok ; Cho, Myung-Hwan ; Lee, Im-Soon ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 472~476
Two isoforms of human CD99 have been identified, but only heterotypic interaction between the isomers was recently demonstrated. In this study, we performed bimolecular fluorescence complementation analysis to further characterize the interaction in vivo. Upon transiently transfecting plasmids expressing either of the two isoforms fused with yellow fluorescent protein (YFP) fragments, all the YFP-tagged CD99 molecules were properly localized on cell surfaces, and formed fluorescent dimers. Interestingly, however, unlike the previous report, the homodimers formed as efficiently as the heterodimer via their extracellular domains, implying its distinct regulatory role through modulating the complex profile.
Comparative Biodegradation of HDPE and LDPE Using an Indigenously Developed Microbial Consortium
Satlewal, Alok ; Soni, Ravindra ; Zaidi, Mgh ; Shouche, Yogesh ; Goel, Reeta ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 477~482
A variety of bacterial strains were isolated from waste disposal sites of Uttaranchal, India, and some from artificially developed soil beds containing maleic anhydride, glucose, and small pieces of polyethylene. Primary screening of isolates was done based on their ability to utilize high- and low-density polyethylenes (HDPE/LDPE) as a primary carbon source. Thereafter, a consortium was developed using potential strains. Furthermore, a biodegradation assay was carried out in 500-ml flasks containing minimal broth (250ml) and HDPE/LDPE at 5mg/ml concentration. After incubation for two weeks, degraded samples were recovered through filtration and subsequent evaporation. Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric-differential thermogravimetry-differential thermal analysis (TG-DTG-DTA) were used to analyze these samples. Results showed that consortium-treated HDPE (considered to be more inert relative to LDPE) was degraded to a greater extent (22.41% weight loss) in comparison with LDPE (21.70% weight loss), whereas, in the case of untreated samples, weight loss was more for LDPE than HDPE (4.5% and 2.5%, respectively) at
. Therefore, this study suggests that polyethylene could be degraded by utilizing microbial consortia in an eco-friendly manner.
Selection of Newly Isolated Mushroom Strains for Tolerance and Biosorption of Zinc In Vitro
Gonen Tasdemir, F. ; Yamac, M. ; Cabuk, A. ; Yildiz, Z. ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 483~489
Nine newly isolated mushroom strains were tested to assess both their zinc tolerance and potential for zinc removal from an aqueous solution. Four strains of ectomycorrhizal fungi, namely Clavariadelphus truncatus (T 192), Rhizopogon roseolus (T 21), Lepista nuda (T 373), and Tricholoma equestre (T 174), along with five strains of white rot fungi, Lenzites betulina (S 2), Trametes hirsuta (T 587), Ganoderma spp. (T 99), Polyporus arcularius (T 438), and Ganoderma carnosum (M 88), were investigated using zinc-amended solid and liquid media. Their biosorption properties were also determined. The colony diameter and dry weight were used as tolerance indices for fungal growth. C. truncatus and T. equestre were not strongly inhibited at the highest concentrations of (225 mg/l) zinc in solid media. The most tolerant four strains with solid media, C. truncatus, G carnosum, T. hirsuta, and T. equestre, were then chosen for tolerance tests in liquid media. An ectomycorrhizal strain, C. truncatus, was also detected as the most tolerant strain in liquid media. However, the metal-tolerant strains demonstrated weak activity in the biosorption studies. In contrast, the highest biosorption activity was presented by a more sensitive strain, G. carnosum. In addition, seven different biosorbent types from G. carnosum (M 88) were compared for their Zn (II) biosorption in batch experiments.
Characterization of Growth-supporting Factors Produced by Geobacillus toebii for the Commensal Thermophile Symbiobacterium toebii
Kim, Joong-Jae ; Masui, Ryoji ; Kuramitsu, Seiki ; Seo, Jin-Ho ; Kim, Kwang ; Sung, Moon-Hee ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 490~496
Symbiobacterium toebii is a commensal symbiotic thermophile that cannot grow without support from a partner bacterium. We investigated the properties of Symbiobacterium growth-supporting factors (SGSFs) produced by the partner bacterium Geobacillus toebii. SGSFs occurred in both the cell-free extract (CFE) and culture supernatant of G. toebii and might comprise multifarious materials because of their different biological properties. The heavy SGSF contained in the cytosolic component exhibited heat- and proteinase-sensitive proteinaceous properties and had a molecular mass of >50 kDa. In contrast, the light SGSF contained in the extracellular component exhibited heat-stable, proteinase-resistant, nonprotein properties and had a molecular mass of <10 kDa. Under morphological examination using light microscopy, S. toebii cultured with the culture supernatant of G. toebii was filamentous, whereas S. toebii cultured with the CFE of G. toebii was rod-shaped. These results strongly suggest that the SGSFs produced by G. toebii comprise two or more types that differ in their growth-supporting mechanisms, although all support the growth of S. toebii. Upon the examination of the distribution of SGSFs in other bacteria, both cytosolic and extracellular components of Geobacillus kaustophilus, Escherichia coli, and Bacillus subtilis had detectable growth-supporting effects for S. toebii, indicating that common SGSF materials are widely present in various bacterial strains.
Chemical Composition and Antimicrobial Activity of Essential Oil from Cones of Pinus koraiensis
Lee, Jeong-Ho ; Yang, Hye-Young ; Lee, Hong-Sub ; Hong, Soon-Kwang ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 497~502
The essential oil from the cones of Pinus koraiensis was prepared after removing the seeds, and its chemical composition analyzed using gas chromatography-mass spectrometry (GC-MS). Hydrodistillation of the P. koraiensis cones yielded 1.07% (v/w) of essential oil, which was almost three times the amount of essential oil extracted from the needles of the same plant. Moreover, the antimicrobial activities of the oil against the growth of Gram-positive bacteria, Gram-negative bacteria, and fungi were evaluated using the agar disc diffusion method and broth microdilution method. Eighty-seven components, comprising about 96.8% of the total oil, were identified. The most abundant oil components were limonene (27.90%),
-pinene (12.02%), 3-carene(4.95%),
-myrcene (4.53%), isolongifolene (3.35%), (-)-bornyl acetate (2.02%), caryophyllene (1.71%), and camphene (1.54%). The essential oil was confirmed to have significant antimicrobial activities, especially against pathogenic fungal strains such as Candida glabrata YFCC 062 and Cryptococcus neoformans B 42419. Therefore, the present results indicate that the essential oil from the cones of Pinus koraiensis can be used in various ways as a nontoxic and environmentally friendly disinfectant.
Evaluation of Biomolecular Interactions of Sulfated Polysaccharide Isolated from Grateloupia filicina on Blood Coagulation Factors
Athukorala, Yasantha ; Jung, Won-Kyo ; Park, Pyo-Jam ; Lee, Young-Jae ; Kim, Se-Kwon ; Vasanthan, Thava ; No, Hong-Kyoon ; Jeon, You-Jin ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 503~511
An edible marine red alga, Grateloupia filicina, collected from Jeju Island of Korea was hydrolyzed by cheap food-grade carbohydrases (Viscozyme, Celuclast, AMC, Termamyl, and Ultraflo) to investigate its anticoagulant activity. Among the tested enzymatic extracts of G. filicina, a Termamyl extract showed the highest anticoagulant activity. Anion-exchange chromatography on DEAE-cellulose and gel-permeation chromatography on Sepharose-4B were used to purify the active polysaccharide from the crude polysaccharide fraction of G. filicina. The purified sulfated polysaccharide (0.42 sulfate/total sugar) showed
molecular mass and was comprised mainly of galactose(98%) and 1-2% of glucose. The sample showed potential anticoagulant activity on activated partial thromboplastin time (APTT) thrombin time (TT) assays. The purified G. filicina anticoagulant (GFA) inhibited the coagulation factor X (92%), factor II (82%), and factor VII (68%) of the coagulation cascade, and the molecular interaction (protein-polysaccharide) was highly enhanced in the presence of ATIII (antithrombin III). The dissociation constant of polysaccharide towards serine proteins decreased in the order of FXa (58.9 nM) >FIIa (74.6 nM) >FVII (109.3 nM). The low/less cytotoxicity of the polysaccharide benefits its use in the pharmaceutical industry; however, further studies that would help us to elucidate the mechanism of its activity are needed.
Apoptosis of Human Hepatocarcinoma (HepG2) and Neuroblastoma (SK-N-SH) Cells Induced by Polysaccharides-Peptide Complexes Produced by Submerged Mycelial Culture of an Entomopathogenic Fungus Cordyceps sphecocephala
Oh, Jung-Young ; Baek, Yu-Mi ; Kim, Sang-Woo ; Hwang, Hye-Jin ; Hwang, Hee-Sun ; Lee, Sung-Hak ; Yun, Jong-Won ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 512~519
Three different polysaccharide-peptide complexes (PPC, named as Fr-I, Fr-II, and Fr-III) were produced by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala, and their anticancer activities were investigated in human hepatocarcinoma (HepG2) and neuroblastoma (SK-N-SH) cells. The highest inhibitory effects of PPC on both HepG2 and SK-N-SH cells were achieved with Fr-I, whereas Fr-III with low molecular mass showed lower inhibition effects. Interestingly, the inhibitory effects of the three fractions were increased after protease digestion, suggesting that the inhibitory effects resulted mainly from the carbohydrate moiety, at least in the case of Fr-II and Fr-III, of PPC. The results of DNA fragmentation in PPC-induced apoptotic cells were confirmed by both DNA ladder assay and comet assay. Our investigation also showed that PPC-induced apoptosis of both cancer cells was associated with intracellular events including DNA fragmentation, activation of caspase-3, and modulation of Bcl-2 and Bax. We conclude that PPC has potential as a novel therapeutic agent for the treatment of both HepG2 and SK-N-SH cancer cells without any cytotoxicity against normal cells.
Soraphinol C, a New Free-Radical Scavenger from Sorangium cellulosum
Li, Xuemei ; Yu, Tae-Kyung ; Kwak, Jong-Hwan ; Son, Byoung-Yil ; Seo, Young-Wan ; Zee, Ok-Pyo ; Ahn, Jong-Woong ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 520~522
A new compound named soraphinol C (1) was isolated from myxobacteria Sorangium cellulosum KM1001 a soil isolate, together with a structurally related known compound, 4-hydroxysattabacin (2). These compounds were isolated by silica gel column chromatography and recycling preparative HPLC, consecutively. The structures of the compounds were determined on the basis of combined spectroscopic analyses. Compounds 1 and 2 exhibited antioxidant activity as a radical scavenger in the experiment using a hydrophilic free-radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride with ORAC values of 0.956 and 0.617, respectively.
Sensitization of the Apoptotic Effect of
-Irradiation in Genistein-pretreated CaSki Cervical Cancer Cells
Shin, Jang-In ; Shim, Jung-Hyun ; Kim, Ki-Hong ; Choi, Hee-Sook ; Kim, Jae-Wha ; Lee, Hee-Gu ; Kim, Bo-Yeon ; Park, Sue-Nie ; Park, Ok-Jin ; Yoon, Do-Young ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 523~531
Radiotherapy is currently applied in the treatment of human cancers. We studied whether genistein would enhance the radiosensitivity and explored its precise molecular mechanism in cervical cancer cells. After co-treatment with genistein and irradiation, the viability, cell cycle analysis, and apoptosis signaling cascades were elucidated in CaSki cells. The viability was decreased by co-treatment with genistein and irradiation compared with irradiation treatment alone. Treatment with only
-irradiation led to cell cycle arrest at the
phase. On the other hand, co-treatment with genistein and
-irradiation caused a decrease in the
phase and a concomitant increase up to 56% in the number of
phase. In addition, co-treatment increased the expression of p53 and p21, and Cdc2-tyr-15-p, supporting the occurrence of
arrest. In general, apoptosis signaling cascades were activated by the following events: release of cytochrome c, upregulation of Bax, down regulation of Bcl-2, and activation of caspase-3 and -8 in the treatment of genistein and irradiation. Apparently, co-treatment downregulated the transcripts of E6*I, E6*II, and E7. Genistein also stimulated irradiation-induced intracellular reactive oxygene, species (ROS) production, and co-treatment-induced apoptosis was inhibited by the antioxidant N-acetylcysteine, suggesting that apoptosis has occurred through the increase in ROS by genistein and
-irradiation in cervical cancer cells. Gamma-irradiation increased cyclooxygenase-1 (COX-2) expression, whereas the combination with genistein and
-irradiation almost completely prevented irradiation-induced COX-2 expression and
production. Co-treatment with genistein and
-irradiation inhibited proliferation through
arrest and induced apoptosis via ROS modulation in the CaSki cancer cells.
Carbon and Energy Balances of Glucose Fermentation with Hydrogen-producing Bacterium Citrobacter amalonaticus Y19
Oh, You-Kwan ; Park, Sung-Hoon ; Seol, Eun-Hee ; Kim, Seo-Hyoung ; Kim, Mi-Sun ; Hwang, Jae-Woong ; Ryu, Dewey D.Y. ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 532~538
For the newly isolated
-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5-9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (
, ethanol, acetate, lactate,
, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal Hz production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.
Enhanced Biomass and
-Linolenic Acid Production of Mutant Strain Arthrospira platensis
Choi, Gang-Guk ; Bae, Myong-Sook ; Ahn, Chi-Yong ; Oh, Hee-Mock ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 539~544
A mutant of Arthrospira platensis PCC 9108, strain M9108, obtained by mutagenesis with UV treatment, was able to mixotrophically grow in an SOT medium containing 40 g of glucose/l. The biomass and specific growth rate of strain M9108 (4.10 g/l and 0.70/d) were 1.9-fold and 1.4-fold higher, respectively, than those of the wild type (2.21 g/l and 0.58/d) under mixotrophic culture condition. In addition, when compared with the wild type, the content of
-linolenic acid (GLA) in the mutant was increased when glucose concentration was increased. Compared with the wild type, the GLA content of the mutant was 2-fold higher in autotrophic culture and about 3-fold higher in mixotrophic culture. Thus, the mutant appears to possess more efficient facility to assimilate and metabolize glucose and to produce more GLA than its wild-type strain.
Production of Ethanol Directly from Potato Starch by Mixed Culture of Saccharomyces cerevisiae and Aspergillus niger Using Electrochemical Bioreactor
Jeon, Bo-Young ; Kim, Dae-Hee ; Na, Byung-Kwan ; Ahn, Dae-Hee ; Park, Doo-Hyun ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 545~551
When cultivated aerobically, Aspergillus niger hyphae produced extracellular glucoamylase, which catalyzes the saccharification of unliquified potato starch into glucose, but not when grown under anaerobic conditions. The
of the extracellular glucoamylase were 652.3 mg/l of starch and 253.3 mg/l/min of glucose, respectively. In mixed culture of A. niger and Saccharomyces cerevisiae, oxygen had a negative influence on the alcohol fermentation of yeast, but activated fungal growth. Therefore, oxygen is a critical factor for ethanol production in the mixed culture, and its generation through electrolysis of water in an electrochemical bioreactor needs to be optimized for ethanol production from starch by coculture of fungal hyphae and yeast cells. By applying pulsed electric fields (PEF) into the electrochemical bioreactor, ethanol production from starch improved significantly: Ethanol produced from 50 g/l potato starch by a mixed culture of A. niger and S. cerevisiae was about 5 g/l in a conventional bioreactor, but was 9 g/l in 5 volts of PEF and about 19 g/l in 4 volts of PEF for 5 days.
R-Stereoselective Amidase from Rhodococcus erythropolis No. 7 Acting on 4-Chloro-3-Hydroxybutyramide
Park, Ha-Ju ; Uhm, Ki-Nam ; Kim, Hyung-Kwoun ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 552~559
Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolisstrain (No.7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at
. The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with mid-chain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 22.214.171.124). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.
Factors Influencing the Production of Water-soluble Endopolysaccharides and Exopolysaccharides from Lentinus lepideus and their Effects on Immune Cytokine Production
Lee, Wi-Young ; Ahn, Jin-Kwon ; Kim, Dong-Hyun ; Ka, Kang-Hyeon ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 560~567
An efficient method to produce water-soluble polysaccharides from Lentinus lepideus is described. The productivity of both endopolysaccharides (PPS) and exopolysaccharides (EPS) was compared under various culture conditions. The effect of treating their own PPS and EPS on immune cytokine production was also studied in relation to culture factors. High yield production of EPS required a moderate culture temperature
as well as long culture period (16-20 days). In contrast, PPS production required a high culture temperature
and short culture period (8 days). Most of the carbon sources did not affect polysaccharides and mycelial production except for sucrose. Immune cytokine levels in the EPS treatment varied among carbon sources or culture periods. PPS did not appear to affect much on the production of cytokines, regardless of the culturing factors, except for the culture period. These results suggest that the optimal culture conditions for L. lepideus vary according to culture purposes, and different culture conditions should be used for different targets including mycelial biomass, EPS, and PPS. Whereas the immunomodulating activitiy of EPS appeared to be affected by culture conditions in L. lepideus, that of PPS did not.
Antimicrobial (BN/PE) Film Combined with Modified Atmosphere Packaging Extends the Shelf Life of Minimally Processed Fresh-Cut Iceberg Lettuce
Kang, Sun-Chul ; Kim, Min-Jeong ; Park, In-Sik ; Choi, Ung-Kyu ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 568~572
This study was conducted to investigate the effect of modified atmosphere packaging (MAP) in combination with BN/PE film on the shelf life and quality of fresh-cut iceberg lettuce during cold storage. The total mesophilic population in the sample packed in BN/PE film under MAP conditions was dramatically reduced in comparison with that of PE film, PE film under MAP conditions, and BN/PE film. The
concentration in the BN/PE film under MAP conditions decreased slightly as the storage period progressed. The coloration of the iceberg lettuce progressed the slowest when it was packaged in BN/PE film under MAP conditions, followed by BN/PE film, PE film, and PE film under MAP conditions. The shelf life of fresh-cut iceberg lettuce packaged in the BN/PE film under MAP conditions was extended by more than 2 days at
as compared with that of the BN/PE film in which the extension effect was more than 2 days longer than that of PE, PET, and OPP films.
Hydrolysis of Isoflavone Glucosides in Soymilk Fermented with Single or Mixed Cultures of Lactobacillus paraplantarum KM, Weissella sp. 33, and Enterococcus faecium 35 Isolated from Humans
Chun, Ji-Yeon ; Jeong, Woo-Ju ; Kim, Jong-Sang ; Lim, Jin-Kyu ; Park, Cheon-Seok ; Kwon, Dae-Young ; Choi, In-Duck ; Kim, Jeong-Hwan ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 573~578
Lactobacillus paraplantarum KM (Lp), Weissella sp. 33 (Ws), and Enterococcus faecium 35 (Ef) were used in single (Lp, Ws, Ef) or mixed cultures (Lp+Ws, Lp+Ef, Ws+Ef) for soy milk fermentation (
, 12 h). After 12 h, the cell numbers, pH, and TA of soymilk were
, 3.8-4.5, and 0.59-0.70%, respectively. Changes in the contents of glycitin and genistin in soymilk fermented with Ef were not significant. The contents of isoflavone glucosides in soymilk fermented with the other cultures decreased significantly with an increase of aglycone contents (p<0.05). It corresponded well with a sharp increase in
-glucosidase activity during fermentation. About 92-100% of the daidzin and 98-100% of the genistin in soymilk were converted to corresponding aglycones by Lp, Ws, or Lp+Ef within 12 h.
Evaluation of a Chromogenic Medium Supplemented with Glucose for Detecting Enterobacter sakazakii
Song, Kwang-Young ; Hyeon, Ji-Yeon ; Shin, Ho-Chul ; Park, Chan-Kyu ; Choi, In-Soo ; Seo, Kun-Ho ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 579~584
A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakit) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces
-glucosidase. The enzyme
-glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3-indolyl-
, producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at
. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.
Analysis of Natural Recombination in Porcine Endogenous Retrovirus Envelope Genes
Lee, Dong-Hee ; Lee, Jung-Eun ; Park, Nu-Ri ; Oh, Yu-Kyung ; Kwon, Moo-Sik ; Kim, Young-Bong ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 585~590
Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.
Protective Immunity Induced by Systemic and Mucosal Delivery of DNA Vaccine Expressing Glycoprotein B of Pseudorabies Virus
Yoon, Hyun-A ; Han, Young-Woo ; Aleyas, Abi George ; George, June Abi ; Kim, Seon-Ju ; Kim, Hye-Kyung ; Song, Hee-Jong ; Cho, Jeong-Gon ; Eo, Seong-Kug ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 591~599
A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.
Role of CAGE, a Novel Cancer/Testis Antigen, in Various Cellular Processes, Including Tumorigenesis, Cytolytic T Lymphocyte Induction, and Cell Motility
Kim, Young-Mi ; Jeoung, Doo-Il ;
Journal of Microbiology and Biotechnology, volume 18, issue 3, 2008, Pages 600~610
A cancer-associated antigen gene (CAGE) was identified by serological analysis of a recombinant cDNA expression library (SEREX). The gene was identified by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera from patients with gastric cancer. CAGE was found to contain a D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. The CAGE gene is widely expressed in various cancer tissues and cancer cell lines. Demethylation plays a role in the activation of CAGE in certain cancer cell lines where the gene is not expressed. The functional roles of CAGE in tumorigenesis, the molecular mechanisms of CAGE expression, and cell motility are also discussed.