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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 9, Issue 6 - Dec 1999
Volume 9, Issue 5 - Oct 1999
Volume 9, Issue 4 - Aug 1999
Volume 9, Issue 3 - Jun 1999
Volume 9, Issue 2 - Apr 1999
Volume 9, Issue 1 - Feb 1999
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Roles of the Conserved Carboxylic Residues in the Active-Site of 5'-3' Exonuclease of Taq DNA Polymerase
Kim, Young-Soo ; Shin, Joong-Chul ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 381~385
Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7
radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.
Large-Scale Culture of Hepatitis A Virus in Human Diploid MRC-5 Cells and Partial Purification of the Viral Antigen for Use as a Vaccine
Kim, Hyun-Seok ; Chung, Yong-Ju ; Jeon, Yeong-Joong ; Lee, Sung-Hee ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 386~392
A large-scale culture of hepatitis A virus in human diploid MRC-5 cells was conducted. In a roller bottle culture, the virus was grown to a maximum titer in 3 weeks after infection. Over 95％ of the cell-associated virus was excreted after culturing the infected cells in suspension media without fetal bovine serum for 3 days. The cultured virus was inactivated with formalin, concentrated by ultrafiltration, and partially purified by ultracentrifugation in a non-ionic gradient medium of Renocal. Two separate peak fractions showing high anti-HAY ELISA titer were pooled and about 40％ of HAV antigen was recovered by this purification procedure. Of the partially purified vaccine, the protein pattern in SDS-PAGE and immunogenicity in mice were compared with a commercial HAV vaccine. In SDS-PAGE, the purified vaccine in this study and the commercial vaccine showed almost the same protein pattern. The seroconversion rate of the purified vaccine in mice was not different from that of the commercial vaccine. Therefore, we could prepare a good grade of HAV vaccine by a simple purification procedure although the purification itself was not completed.
Cloning and Expression of the UDP-Galactose-4-Epimerase Gene (galE) Constituting the gal/lac Operon of Lactococcus lactis ssp. lactis ATCC7962
Lee, Jung-Min, Choi, Jae-Yeon ; Lee, Jong-Hoon ; Chang, Hae-Choon ; Chung, Dae-Kyun ; Kim, Jeong-Hwan ; Lee, Hyong-Joo ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 393~397
The gene (galE) encoding UDP-galactose-4-epimerase, operative in the galactose metabolic pathway, was cloned together with the
-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis ATCC7962 (L. lactis 7962). galE was found to have a length of 981 bps and encoded a protein with a molecular mass of 36,209 Da. The deduced amino acid sequence showed a homology with GalE proteins from several other microorganisms. A Northern analysis demonstrated that galE was constitutively expressed by its own promoter. When galactose or lactose was added into medium, the galE transcription was induced by several upstream promoters. The structure of the gal/lac operon of L. lactis 7962 was partially characterized and the gene order around galE was galT-lacA-lacZ-galE-orfX.
Examination of Cytopathic Effect and Apoptosis in Listeria monocytogenes-Infected Hybridoma B-Lymphocyte (Ped-2E9) Line In Vitro
Bhunia, Arun-Kumar ; Feng, Xiang ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 398~403
In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua (
) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua (
) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).
Random Sequence Analysis of the Genomic DNA of Methanopyrus kandleri and Molecular Cloning of the Gene Encoding a Homologue of the Catalytic Subunit of Carbon Monoxide Dehydrogenase
Shin, Hyun-Seock ; Ryu, Jae-Ryeon ; Han, Ye-Sun ; Choi, Yong-Jin ; Yu, Yeon-Gyu ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 404~413
Methanopyrus kandleri is a hyperthermophilic methanogen that represents one of the most heat-resistant organisms: the maximum growth temperature of M. kandleri is
. A random sequence analysis of the genomic DNA of M. kandleri has been performed to obtain genomic information. More than 200 unique sequence tags were obtained and compared with the sequences in the GenBank and PIR databases. About 30% of the analyzed tags showed strong sequence similarity to previously identified genes involved in various cellular processes such as biosynthesis, transport, methanogenesis, or metabolism. When statistics relating to the frequency of codons were examined, the sequenced open reading frames showed highly biased codon usage and a high content of charged amino acids. Among the identified genes, a homologue of the catalytic subunit of carbon monoxide dehydrogenase (CODH) that reduces
to CO was cloned and sequenced in order to examine its detailed gene structure. The cloned gene includes consensus promoters. The amino acid sequence of the cloned gene shows a strong homology with the CODH genes from methanogenic Archaea, especially in the presumed binding sites for Fe-S centers.
Synergistic Inhibition of Membrane ATPase and Cell Growth of Helicobacter pylori by ATPase Inhibitors
Ki, Mi-Ran ; Yun, Soon-Kyu ; Lim, Wang-Jin ; Hong, Bum-Shik ; Hwang, Se-Young ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 414~421
Helicobacter pylori were found to be resistant to azide but sensitive to vanadate, suggesting that defect in the P-type ATPase activity rather than F-type ATPase would be lethal to cell survival or growth. To elucidate the relationship between this enzyme inhibition and H. pylori death, we determined the effect of omeprazole (OMP) plus vanadate on enzyme activity and cell growth. The minimum inhibitory concentration (MIC; ca. 0.8
mol/disk) of vanadate for H. pylori growth was lowered over l0-fold with the aid of OMP, whereby its inhibitory potential toward the P-type ATPase activity was diametrically increased. Alternatively, we found that this enzyme activity was essential for active transport in H. pylori. From these observations, we strongly suggest that the immediate cause of the growth inhibition of H. pylori cells with OMP and/or vanadate might be defective in the cell's active transport due to the lack of P-type ATPase activity. From the spectral data with circular dichroism (CD) spectroscopy, we found that activated OMP (OAS) at concentration below MIC did not disrupt helical structures of membrane proteins. Separately, we determined the cytopathic effect of OAS by SDS-PAGE, indicating the change in the production of cytoplasmic protein but not cell membrane.
Responses of Pseudomonas sp. DJ-12 to Pollutant Stresses of Benzoate and 4-Chlorobenzoate
Ko, Yeon-Ja ; Park, Sang-Ho ; Park, Yong-Keun ; Kim, Chi-Kyung ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 422~428
Aromatic hydrocarbons can be utilized as carbon and energy sources by some microorganisms at lower concentrations. However, they can also act as stresses to these organisms at higher concentrations. Pseudomonas sp. DJ-12 is capable of degrading 0.5 mM concentration of benzoate and 4-chlorobenzoate (4CBA). In this study, the exposure of Pseudomonas sp. DJ-12 to the pollutant stresses of benzoate and 4CBA at various concentrations was comparatively studied for its cellular responses, including survival tolerance, degradability of the aromatics, and morphological changes. Pseudomonas sp. DJ-12 utilized 0.5 to 1.0mM benzoate and 4CBA as carbon and energy sources for growth. However, the organism could not degrade the aromatics at concentrations of 3 mM or higher, resulting in reduced cell viability due to the destruction of the cell envelopes. Pseudomonas sp. DJ-12 cells produced stress-shock proteins such as DnaK and GroEL when treated with benzoate and 4CBA at concentrations of 0.5mM, or higher as sublethal dosage; Yet, there were differing responses between the cells treated with either benzoate or 4CBA. 4CBA affected the degradability of the cells more critically than benzoate. The DnaK and GroEL stress-shock proteins were produced either by 1mM benzoate with 5 min treatment or by higher concentrations after 10min. The proteins were also induced by 0.5mM 4CBA, however, it needed at least 20min treatment or longer. These results indicate that the chlorination of benzoate increased the recalcitrance of the pollutant aromatics and changed the conditions to lower concentrations and longer treatment times for the production of stress-shock proteins. of stress-shock proteins produced by the aromatics at sublethal concentrations functioned interactively between the aromatics for survival tolerance to lethal concentrations.
Isolation, Purification, and Partial Characterization of an AMP Deaminase from Saccharomyces cerevisiae D
Kim, Myung-Hee ; Lee, Jung-Kee ; Kim, Hyung-Kwoun ; Oh, Tae-Kwang ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 429~435
An adenosine 5'-monophosphate deaminase (AMP aminohydrolase, EC 22.214.171.124) was purified to homogeneity from the cell-free extract of Saccharomyces cerevisiae DKCTC7248. The molecular mass of subunit was estimated to be 80 kDa on SDS-PAGE, and that of the holoenzyme was shown to be 240 kDa by gel filtration. The isoelectric point of the enzyme (AMP deaminase D) was determined to be 6.2. The AMP deaminase D was specific towards AMP with an apparent
value of 4.1 mM and a Hill coefficient,
, of 2.2. Both ATP and ADP were positive allosteric effectors of the AMP deaminase D: The apparent
was decreased to 1.6 mM and 3.3 mM in the presence of 0.1 mM ATP and ADP, respectively, lowering
to 1.0. Univalent cations like
activated the enzyme but some divalent cations such as
showed strong inhibitory effects. This enzyme displayed optimum activity at
and pH 7.0. In addition, it was stable up to
and over a wide pH range(pH 5.5-9.0). Amino acid sequences of its N-terminal region were analyzed to be ADYKMQMFADDA.
Symmetry Region at Beginning of Transcript Inhibits Expression of Escherichia coli aeg-46.5 Operon
Lee, Seung-Hwa ; Lee, Sang-Ho ; Sung, Ha-Chin ; Kim, Joon ; Choe, Mu-Hyeon ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 436~442
The aeg-46.5 operon of Escherichia coli is induced by nitrate and anaerobic conditions. Positive regulators Fnr and NarP, and a negative regulator NarL control the expression of the aeg-46.5. It has two symmetry regions , one of which is located between +37 and +56 bp from the 5'end of the anaerobic transcription initiation site. In this study, mutagenized symmetry regions were transferred from plasmid to chromosome by homologous recombination to evaluate the mutation as a single copy in the fnr, narL, narP, and narL-narP double mutant background. The expressions of the aeg-46.5 operon with these mutations indicated that the control was not through the possible stem-loop structure. Whether there is a protein that mediates this control remains to be seen. The results from the narL-narP double mutant indicated that the anaerobic Fill induction was independent of NarL repression.
Production of a Fibrinolytic Enzyme in Bioreactor Culture by Bacillus subtilis BK-17
Lee, Jin-Wook ; Park, Sung-Yurb ; Choi, Won-A ; Lee, Kyung-Hee ; Jeong, Yong-Kee ; Kong, In-Soo ; Park, Sung-Hoon ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 443~449
Bacillus subtilis BK-17 which produces a novel protease with fibrinolytic activity was isolated from soybean paste. Bioreactor production of the enzyme was studied in order to optimize fermentation conditions such as medium concentration, pH, agitation speed, and temperature. Under most cultural conditions, enzyme production initially began when the cell growth stopped. The onset of the enzyme production was indicated by rapid increase in both dissolved oxygen (DO) and pH. Two- to three-times more concentrated medium than the flask optimum medium yielded higher enzyme production in the bioreactor fermentation. When the medium pH was controlled constant, pH 6.5 exhibited the highest activity in the range of 6.0 to 7.5, but the activity was similar to the case when the pH was initially adjusted to 7.5 and subsequently maintained within a relatively wide range of 6.4 to 7.8. Agitation speed did not affect the enzyme production with an exception of DO reaching zero. Fermentation time was reduced when temperature increased within the range of
. However, the highest activity, along with the slow decrease of the enzymatic activity after reaching the maximum value, was observed at
. By shifting the temperature from
immediately after DO reached the minimum level, the high enzyme production of 1,100 U/ml along with the short fermentation period of 13 h could be obtained.
Anti-Complementary Properties of Polysaccharides Isolated from Fruit Bodies of Mushroom Pleurotus ostreatus
Kweon, Mee-Hyang ; Jang, Hyo ; Lim, Wang-Jin ; Chang, Hyo-Ihl ; Kim, Chan-Wha ; Yang, Han-Chul ; Hwang, Han-Joon ; Sung, Ha-Chin ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 450~456
A high molecular-weight water-soluble fraction(PO) obtained by the ethanol precipitation of 0.1 N NaOH extracts of the mushroom Pleurotus ostreatus showed 82% anti-complementary activity for complement consumption hemolysis. The PO consisted of 42% carbohydrate (w/w), 50% protein (w/w), and 3% uronic acid (w/w). Fifty-eight percent of the anti-complementary activity decreased by periodate oxidation and 22% by protease digestion, suggesting that the sugar and protein moieties are essential for this activity. Two polysaccharide fractions, PO-IIIa-1 and PO-IIIa-2, with anti-complementary activity were isolated from the PO using DEAE-Sepharose FF followed by Sephadex G-75 and Sepharose CL-6B gel permeation chromatographies. The PO-IIIa-2 was found by HPLC to be nearly homogeneous, with the molecular mass of 531 kDa, and showed 96%
(inhibition against the total complement hemolysis of deionized water as the control) at a concentration of 1 mg/ml. This fraction contained galactose, mannose, fucose, and glucose with molar ratios of 1.75:1:0.65 and 0.59, respectively. The majority of galactose and mannose units in the PO-IIIa-2 were composed of TGalp1 ->, ->6Galp1->, ->2,6Galp1->, and ->Manp1->. The PO-IIIa-1 (molecular mass of 2000 kDa), exhibiting higher activity than the PO-IIIa-2, was further purified into two fractions, unbound proteoglycan (PO-IIIa-1A) and bound glucomannan (PO-IIIa-lB), by affinity chromatography using ConA-Sepharose CL-4B. The anti-complementary activity of each affinity purified fraction decreased as compared to that of the native PO-IIIa-1 fraction, indicating that the formation of complex between both polysaccharide fractions was necessary for full anti-complementary activity.
Circular Permutation of the DNA Genome of Temperate Bacteriophage
from Enterococcus faecalis KBL 703
Kim, Young-Woo ; Jang, Se-Hwan ; Hong, Bum-Shik ; Lim, Wang-Jin ; Kim, Chan-Wha ; Sung, Ha-Chin ; Chang, Hyo-Ihl ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 457~463
The physical map of bacteriophage
DNA was constructed with the restriction endonucleases SalI, BamHI, EcoRI, XbaI, and AvaI. The 40.5-kb DNA restriction map is shown to be circularly permuted representing the headful packaging mechanism of the phage. The DNA restriction fragments containing the packaging initiation site(pac) was localized on the restriction map and the nucleotide sequences of the region were analyzed. Four open reading frames (ORFs), following one another with the same orientation, were found at the region. The 2nd ORF (ORF-ts) has significant amino acid sequence homologies to the previously known terminase small subunits of other bacteriophages. The putative terminase small subunit gene has a presumptive NTP-hydrolysis motif and a helix-turn-helix motif. The cleavage site for the first round of packaging was found to be located at the coding sequence of the putative terminase small subunit gene. The fourth ORF, even if partially sequenced, has a good amino acid sequence homology to the portal vertex proteins of other bacteriophages representing the evolutionarily conserved arrangements of genes near the pac site of this bacteriophage,
Effects of Temperature and Compost Conditions on the Biodegradation of Degradable Polymers
Jung, Eun-Joo ; Shin, Pyong-Kyun ; Bae, Hee-Kyung ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 464~468
The effectiveness of current biodegradation test methods for degradable polymers under controlled composting conditions was studied in regards to the test temperature and compost condition. When biodegradability tests for the natural (starch, cellulose, PHB/HV) and synthetic (PCL, SG, PLA) polymers were conducted at temperature levels of 35 and
with compost cured at ambient temperature, the degradations of cellulose and starch were higher at
because of the priming effect. On the other hand, degradations of other polymers were higher at
. In the biodegradation test at
, compost harvested right after the thermophilic degradation stage showed higher biodegradation activities than the cured compost for both the synthetic aliphatic polyester (SG) and a natural polymer, cellulose. These results suggest that the biodegradation test conducted at
with the compost, harvested right after the thermophilic degradation stage during composting, showed the highest biodegradation activity under controlled composting conditions.
Minor Thermostable Alkaline Protease Produced by Thermoactinomyces sp. E79
Kim, Young-Ok ; Lee, Jung-Kee ; Sunitha, Kandula ; Kim, Hyung-Kwoun ; Oh, Tae-Kwang ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 469~474
Thermoactinomyces sp. E79 produced two types of thermostable alkaline proteases extracellularly. A minor protease was separated from a major protease by using DEAE-column chromatography. This enzyme was purified to homogeneity by ammonium sulfate and DEAE-Sepharose ion-exchange chromatography. The purified minor protease showed different biochemical properties compared to the major protease. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 36 kDa. Its optimum temperature and pH for proteolytic activity against Hammarsten casein were
and 9.0, respectively. The enzyme was stable up to
and in an alkaline pH range of 9.0-11.0. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF) and
, indicating that the enzyme may be a cysteine-dependent serine protease. In addition, the enzyme cleaved the endoproteinase substrate, succinyl-Ala-Ala-Pro-Phe-p- nitroanilide, and the
value for the substrate was 1.2 mM.
Molecular Characterization of an Apple cDNA Encoding Cinnamyl Alcohol Dehydrogenase
Kim, Sung-Hyun ; Lee, Jae-Rin ; Shin, Yong-Uk ; An, Gyn-Heung ; Kim, Seong-Ryong ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 475~481
The study of lignin, a major component of secondary cell wall, has been partly focused on its removal from the woody part in the kraft pulping industry. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.l95) catalyzes the synthesis of cinnamyl alcohols from corresponding cinnamaldehydes. A cDNA clone, MdCADl, encoding putative CAD from apples (Malus domestica Borkh. cv Fuji) was characterized in this study. The clone contains an open reading frame of 325 amino acid residues, which shows a greater than 80％ identity with Eucalyptus CADl. MdCADl mRNA was detectable in vegetative tissues and was strongly expressed in the fruit. The expression pattern of MdCADl mRNA in the fruit peel after light exposure was also examined. The mRNA was rapidly increased until 1 day after light exposure and remained stable thereafter, suggesting that MdCADl is light inducible. The inducibility of the MdCADl gene was examined using several environmental stresses. Mechanical wounding of leaves increased the MdCADl mRNA level and the induction was further increased by salicylic acid. Southern blot hybridization showed that there is either one or a few copies of CAD genes in apples. To our knowledge, it is believed that MdCADl is the first CAD clone expressed predominantly in fruit.
Production of Salicylic Acid from Naphthalene by Immobilized Pseudomonas sp. Strain NGK1
Shinde, Manohar ; Kim, Chi-Kyung ; Karegoudar, Timmanagouda-Baramanagouda ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 482~487
The Pseudomonas sp. strain NGK1 (NCIM 5120) was immobilized in calcium alginate, agar, and polyacrylamide gel matrices. The salicylic acid-producing capacity of freely suspended cells was compared with immobilized cells in batches with a shake culture and continuous culture system in a packed bed reactor. Freely suspended cells (
) produced 12 mM of salicylic acid, whereas cells immobilized in calcium alginate (
cfu/g beads), agar (
cfu/g beads), and polyacrylamide (
cfu/g beads) produced 15, 11, and 16mM of salicylic acid, respectively, from naphthalene at an initial concentration of 25 mM. The continuous production of salicylic acid from naphthalene was investigated in a continuous packed bed reactor with two different cell populations. The longevity of the salicylic acid-producing activity of the immobilized cells from naphthalene was also studied in semi continuous fermentations. The immobilized cells could be reused 18, 13, and more than 20 times without losing salicylic acid-producing activity in calcium alginate-,agar-, and polyacrylamide-entrapped cells, respectively. The study reveals a more efficient utilization of naphthalene and salicylic acid production by the immobilized Pseudomonas sp. strain NGK1 as compared to the free cells.
Heterologous Expression of Streptomyces albus Genes Linked to an Integrating Element and Activation of Antibiotic Production
Kwon, Hyung-Jin ; Lee, Soon-Youl ; Hong, Soon-Kwang ; Park, Uhn-Mee ; Suh, Joo-Won ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 488~497
Probing Streptomyces albus ATCC 21838 chromosomal DNA with a proline tRNA sequence resulted in an isolation of a putative integrating element in the 6.4-kb EcoRI fragment. It was found that Streptomyces lividans TK-24 transformed with a cloned DNA fragment on a multicopy plasmid, produced a higher level of spore pigment and mycelial red pigment on a regeneration agar. Furthermore, the transformant S. lividans TK-24 produced a markedly increased level of undecylprodigiosin in a broth culture. A nucleotide sequence analysis of the cloned region revealed several open reading frames homologous to the integrases of integrating plasmids or temperate bacteriophages, signal-transducing regulatory proteins with a conserved ATP-binding domain, oxidoreductases (
-ketoacyl reductase), and an AraC-like transcriptional regulator. To examine the effect on antibiotic production, each coding region was overexpressed separately from the other genes in the region in S. lividans TK-24 with; pJHS3044 for the expression of the signal-transducing regulatory protein homologue, pJHS3045 for the homologue of oxidoreductase, and pJHS3051 for the homologue of the AraC-like transcriptional regulator. Phenotypic studies of S. lividans TK-24 strains harboring plasmids for the overexpression of individual genes suggested the following effects of the genes on antibiotic production: The oxidoreductase homologue stimulated the production of actinorhodin and undecylprodigiosin, which was influenced by the culture conditions; the homologue of the AraC-like transcriptional regulator was the most effective factor in antibiotic production within all the culture conditions tested; the signal-transducing regulatory protein homologue repressed the effect due to the homologue of the AraC-like transcriptional regulator, however, the antibiotic production was derepressed upon entering the stationary phase.
Large-Scale Purification of Protease Produced by Bacillus sp. from Meju by Consecutive Polyethylene Glycol/Potassium Phosphate Buffer Aqueous Two-Phase System
Cho, Seong-Jun ; Kim, Chan-Hwa ; Yim, Moo-Hyun ; Lee, Cherl-Ho ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 498~503
Protease produced from Bacillus sp. FSE-68 was isolated from Meju, a Korean fermented soybean starter, and was purified by a two-consecutive aqueous two-phase system. The change of partition coefficient (K) in the polyethylene glycol (PEG)/potassium phosphate buffer (PPB) aqueous two-phase system was measured at different pHs (6.0- 9.2), PPB concentrations (8-12%), and temperatures (4 and
). As the PPB concentration in the aqueous two-phase system increased, the protease concentration in the top phase (PEG-rich phase) increased, thereby enhancing the partition coefficient. The minimum partition coefficient of the protease was achieved at pH 7.0, whereas that of the total protein was at pH 6.0. The biggest difference in partition coefficients of total protein and protease occurred at pH 6.0. It was interesting to note that the partition coefficient of protease decreased as the temperature increased. The optimum condition of the primary aqueous two-phase extraction of Bacillus sp. FSE-68 was pH 6.0, 14% (w/w) PPB, and 16% (w/w) PEG at
, and the crude enzyme concentration in this system was 50% (w/w). The protease, which was concentrated in the top phase, was further mixed with 15% (w/w) PPB (pH 7.0) in the ratio of 1:1 at
to elute the bottom phase (PPB-rich phase). Using these steps, the purification fold achieved was 9.2 with a 44.7% yield.
Selective Cytotoxic Effects of Doenjang (Korean Soybean Paste) Fermented with Bacillus Strains on Human Liver Cell Lines
Choi, Myeong-Rak ; Lim, Hyun-Soo ; Chung, Yoon-Ju ; Yoo, Eun-Jeong ; Kim, Jong-Kyu ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 504~508
This report compares the selective cytotoxic effects of Doenjang fermented by various Bacillus strains (Bacillus sp. SS9, SSA3, and PM3) on human liver cell lines with that of conventional Doenjang (DTY, DTG, and DTK) and commercial Doenjang (DCM, DCD, and DCS). To investigate selective cytotoxic effects of Doenjang extracts, the cell density of HepG2 (Hepatocellular carcinoma) and CCL-13 (cells derived from human normal liver) was estimated after addition of the extracts by using a viable cell counting method. The maximum selectivity ratio (
value against CCL-13/
value aganist HepG2) was observed by PM3 (extracts of Doenjang fermented with Bacillus sp. PM3). As for morphological changes shown by the addition of PM3 into HepG2 and CCL-13 cultures, HepG2 was significantly disrupted, however, CCL-13 was not affected. Also, the growth rate of HepG2 was decreased significantly by the addition of PM3. Consequently, PM3 showed a more detrimental effect on HepG2 than that on CCL-13.
Molecular Characterization and Bitter Taste Formation of Tryptic Hydrolysis of 11S Glycinin
Kim, Mi-Ryung ; Choi, Sang-Yun ; Lee, Cherl-Ho ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 509~513
The molecular size reduction and the formation of bitterness during a tryptic hydrolysis of soybean 11S glycinin were determined by using quantitative analysis and organoleptic evaluation. The 11S glycinin of 90% purity was prepared by cryoprecipitation and Con A Sepharose 4B affinity chromatography, and hydrolyzed with trypsin in a pH-stat reactor for 4 h. Bitterness was formed within 1 h of hydrolysis, and then slowly increased up to
M quinine-HCl equivalent. The extent of hydrolysis (DH) was 7% at 1 h and increased up to 12% by the end of the reaction. The -amino nitrogen content increased from an initial 0.7 mM to 7 mM at the end of the period. The SDS-PAGE analysis showed that the acidic subunit of 11S glycinin was mostly hydrolyzed. The GP-HPLC analysis indicated that the bitterness was mainly contributed by the peptide fractions of molecular weights of 360-2,100 Da.
Hydrolysis of Empty Fruit Bunch of Oil Palm Using Cellulolytic Enzymes from Aspergillus terreus IMI 28243
Kader, Jalil ; Krishnasamy, Getha ; Mohtar, Wan ; Omar, Othman ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 514~517
Hydrolysis of EFB (empty fruit bunch) derived from oil palm was studied using crude enzyme from Aspergillus terreus IMI 282743 along with commercial enzymes from Trichoderma reesei and Aspergillus niger. Hydrolysis at
-cellulose or EFB gave significantly lower yield when commercial enzymes of T. reesei and A. niger were used and the hydrolysis time extended beyond 10 h. After 24 h of hydrolysis at
, the filter paper activity (Fpase) from A. terreus retained as much activity as A. niger and it was significantly higher than T. reesei. Glucose concentration of 0.25% and 0.5% caused significant inhibition in the crude enzyme, but in regards to the commercial enzymes it only showed a slight effect. Crude enzymes from A. terreus could produce the highest reducing sugars when compared to commercial enzymes from T. reesei or A. niger. Nevertheless, low yield of sugar was observed for EFB for all treatments.
Hyper-CMCase-Producing Mutants of Bacillus sp. 79-23 Induced by Gamma- Radiation
Yoon, Ki-Hong ; Shin, In-Kyung ; Jung, Kyung-Hwa ; Park, Seung-Hwan ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 518~521
Bacillus sp. 79-23 spores were irradiated with
gamma-rays at doses ranging from 0.5 to 5 kGy. Following gamma-irradiation, seven mutant strains were isolated by scoring the halo sizes formed around the colonies grown on LB agar plates containing 4% carboxymethylcellulose (CMC) and trypan blue. The mutant strains showed a 1.5 to 2-fold increase in carboxymethylcellulase (CMCase) activity over the parent strain. Wheat bran acted as an effective inducer for CMCase production in the parent and mutant strains. Mutant strains 68 and 70 were identified as exhibiting higher CMCase activities than those of other mutants in LB media both with and without 3% wheat bran. In addition, these strains seem to produce substantially lower amounts of capsular materials, whereas the parent strain produced large amounts of them in both liquid and solid LB media. In flask cultures, the CMCase production by mutants 68 and 70 reached maximum levels of 17.5 unit/ml and 15.7 unit/ml, respectively, in an LB medium containing 3% wheat bran.
A Method for the Separation of Mouse Pancreatic Islets Using Discontinuous Percoll Gradient Centrifugation
Cho, Yu-Ree ; Kim, Sang-Duk ; Chang, Hyo-Ihl ; Sung, Ha-Chin ; Lee, Cherl-Ho ; Kim, Chan-Wha ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 522~524
A discontinuous Percoll gradient was used to separate islets from the collagenase-treated mouse pancreas easily and rapidly. Since the osmolality of Percoll is very low, adjustment of its osmolality to 340 mOs/kg
O was essential for securing the optimal separation. A discontinuous gradient layering with Percoll solution of 1.09 g, 1.07g, and1.05g/m, respectively, when centrifuged at 800
g for 10 min, resulted in an optimal condition for separation and yielded a banding pattern with an even distribution of islet cells. No significant difference was observed in the morphological features between the Percoll-isolated and the manually-isolated islets. In conclusion, the discontinuous Percoll gradient can be effectively used to isolate the pancreatic islets from mice with four-fold higher efficiency compared to the handpicking method.
Antimutagenic Potential of Phellinus igniarius
Shon, Yun-Hee ; Lee, Jae-Sung ; Lee, Hang-Woo ; Nam, Kyung-Soo ;
Journal of Microbiology and Biotechnology, volume 9, issue 4, 1999, Pages 525~528
Mutagenic activities of extracts from the filtrate of the cultured broth (PI-I), mycelia (pI-II), and the fruiting bodies (PI-III) of Phellinus igniarius were examined by Ames/Salmonella tests. No mutagenic activity was found in Salmonella typhimurium strains TA98 and TA100, either with or without S9 activation. In contrast, PI-I, PI-II, and PI-III showed inhibitory effects on the mutagenic activities by the directly-acting mutagens, 4-nitro-ο-phenylenediamine(NPD) and sodium azide ($NaN_3$), and also by the indirectly-acting mutagens, 2-aminofluorene (2-AF) and benzo[a]pyrene (B[a]P). These results suggest that P. igniarius possesses some antimutagenic activity and may contain some chemopreventive agents.