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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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New Gene Cluster from Thermophile Bacillus fordii MH602 for Conversion of DL-5-Substituted Hydantoins to L-Amino Acids
Mei, Yan-Zhen ; Wan, Yong-Min ; He, Bing-Fang ; Ying, Han-Jie ; Ouyang, Ping-Kai ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1497~1505
DOI : 10.4014/jmb.0904.04048
The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-
-amino acids. Since the reaction occurs at higher temperature, the advantages for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile is firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2-kb DNA fragment by restriction site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein gene hyuP, hyperprotein gene hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-
-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 compared respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.
Rational Introduction of Disulfide Bond to Enhance Optimal Temperature of Lipomyces starkeyi
-Dextranase Expressed in Pichia pastoris
Chen, Lin ; Yu, Chao ; Zhou, Xiangshan ; Zhang, Yuanxing ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1506~1513
DOI : 10.4014/jmb.0902.0096
-Dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable
-dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi
-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi
-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wild-type dextranase, the mutant DexM2 (D279C/S289C) showed a more than
improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest
Cloning of the Bacillus subtilis AMX-4 Xylanase Gene and Characterization of the Gene Product
Yoon, Ki-Hong ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1514~1519
DOI : 10.4014/jmb.0907.07004
A gene encoding the xylanase of Bacillus subtilis AMX-4 isolated from soil was cloned into Escherichia coli and the gene product was purified from the cell-free extract of the recombinant strain. The gene, designated xylA, consisted of 639 nucleotides encoding a polypeptide of 213 residues. The deduced amino acid sequence was highly homologous to those of xylanases belonging to glycosyl hydrolase family 11. The molecular mass of the purified xylanase was 23 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum of 6.0-7.0 and a temperature optimum of
. Xylanase activity was significantly inhibited by 5 mM
and 5 mM
, and noticeably enhanced by 5 mM
. The enzyme was active on xylans including arabinoxylan, birchwood xylan, and oat spelt xylan, but it did not exhibit activity toward carboxymethylcellulose or p-nitrophenyl-
-xylopyranoside. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylobiose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylotriose.
IscR Modulates Catalase A (KatA) Activity, Peroxide Resistance, and Full Virulence of Pseudomonas aeruginosa PA14
Kim, Seol-Hee ; Lee, Bo-Young ; Lau, Gee W. ; Cho, You-Hee ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1520~1526
DOI : 10.4014/jmb.0906.06028
We have identified the iscR (PA3815) gene encoding an iron-sulfur cluster assembly regulator homolog as one of the genes required for peroxide resistance in Pseudomonas aeruginosa PA14. Here, we present the phenotypic characterization of an iscR deletion mutant in terms of KatA expression, stress responses, and virulence. The iscR null mutant exhibited reduced KatA activity at the posttranslational level, hypersensitivity to hydrogen peroxide, and virulence-attenuation in Drosophila melanogaster and mouse peritonitis models. These phenotypes were fully restored by multicopy-based expression of katA. These results suggest that the requirement of IscR in P. aeruginosa is related to the proper activity of KatA, which is crucial for peroxide resistance and full virulence of this bacterium.
Polyphosphate Kinase Affects Oxidative Stress Response by Modulating cAMP Receptor Protein and rpoS Expression in Salmonella Typhimurium
Cheng, Yuanyuan ; Sun, Baolin ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1527~1535
DOI : 10.4014/jmb.0903.03030
Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered
sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.
Thermostability of Chimeric Cytidine Deaminase Variants Produced by DNA Shuffling
Park, Yu-Mi ; Phi, Quyet Tien ; Song, Bang-Ho ; Ghim, Sa-Youl ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1536~1541
DOI : 10.4014/jmb.0903.03017
The DNA shuffling technique has been used to generate libraries of evolved enzymes in thermostability. We have shuffled two thermostable cytidine deaminases (CDAs) from Bacillus caldolyticus DSM405 (T53) and B. stearothermophilus IFO12550 (T101). The shuffled CDA library (SH1067 and SH1077 from the first round and SH2426 and SH2429 from the second round) showed various patterns in thermostability. The CDAs of SH1067 and SH1077 were more thermostable than that of T53. SH2426 showed 150% increased halftime than that of T53 at
. The CDA of SH2429 showed about 200% decreased thermostability than that of T53 at
. A single amino acid residue replacement that presented between SH1077 and SH2429 contributed to dramatic changes in specific activity and thermostability. On SDS-PAGE, the purified CDA of SH1077 tetramerized, whereas that of SH2429 denatured and became almost monomeric at
. A simulated three-dimensional structure for the mutant CDA was used to interpret the mutational effect.
Cloning and Characterization of the Zeaxanthin Glucosyltransferase Gene (crtX) from the Astaxanthin-Producing Marine Bacterium, Paracoccus haeundaensis
Seo, Yong-Bae ; Choi, Seong-Seok ; Nam, Soo-Wan ; Lee, Jae-Hyung ; Kim, Young-Tae ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1542~1546
DOI : 10.4014/jmb.0904.04033
Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa. Biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin
Recombinant Expression and Characterization of Thermoanaerobacter tengcongensis Thermostable
-Glucosidase with Regioselectivity for High-Yield Isomaltooligosaccharides Synthesis
Zhou, Cheng ; Xue, Yanfen ; Zhang, Yueling ; Zeng, Yan ; Ma, Yanhe ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1547~1556
DOI : 10.4014/jmb.0905.05006
A novel thermostable
-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2,253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, p-nitrophenyl-
-D-glucopyranide, and dextrin with an exotype cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at
and pH 5.5. The half-life was 2 h at
. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce
-1,4-linked maltotriose and
-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce
-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.
Purification of Caudal-Related Homeodomain Transcription Factor and Its Binding Characterization
Jeong, Mi-Suk ; Hwang, Eun-Young ; Kim, Hyun-Tae ; Yoo, Mi-Ae ; Jang, Se-Bok ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1557~1564
DOI : 10.4014/jmb.0905.05021
Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.
Helicobacter pylori Urease May Exist in Two Forms: Evidence from the Kinetic Studies
Gang, Jin-Gu ; Yun, Soon-Kyu ; Hwang, Se-Young ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1565~1568
DOI : 10.4014/jmb.0906.06021
Purified Helicobacter pylori urease displayed a sigmoid curve in the plot of velocity versus [S] at urea concentrations less than 0.1mM. Under conditions where preservatives, glycerol, or polyethylene glycol (PEG) were added to the enzyme reaction, the substrate hydrolysis was consistent with Michaelis-Menten kinetics, with a
. However, at saturating substrate concentrations, the kinetic parameters of H. pylori urease were unaffected by the presence of the preservatives, and enzyme catalysis conformed to Michaelis-Menten kinetics. The Hill coefficients of the enzyme-catalyzed urea hydrolysis in the presence and absence of PEG were 1 and 2, respectively. Based on these findings, we suggest that H. pylori urease may exist in aggregated and dissociated forms, each with intact function but differing kinetics that may be of importance in maximizing urea breakdown at varying urea concentrations in vivo.
Absorption, Distribution, Metabolism, and Excretion of Decursin and Decursinol Angelate from Angelica gigas Nakai
Kim, Kang-Min ; Kim, Myo-Jeong ; Kang, Jae-Seon ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1569~1572
DOI : 10.4014/jmb.0905.05028
The pharmacokinetics of decursin and decursinol angelate (D/DA) were investigated in male SD rats following oral and intravenous administration. D/DA and metabolites obtained from in vitro samples were evaluated by LC/MS. The levels of D/DA and metabolized decursinol in the blood following oral and intravenous administrations declined according to first-order kinetics, with
values of 56.67, 58.01, and 57.22 h, respectively, being observed after administration of a dose of 2 mg/kg body weight. The large intestine was the major site of disposition following oral administration. These data indicate that D/DA is rapidly absorbed from the gastrointestinal tract. In in vitro experiment utilizing liver microsomal protein, the major metabolic reaction of D/DA occurred to change decursinol. The cumulative biliary, urinary, and fecal excretions of D/DA in bile duct-cannulated rats was
, respectively, at 72 h after administration. These results indicate that the absorption of D/DA is almost complete, and that its metabolites are primarily excreted into feces through the bile. These results indicate that D/DA is subject to enterohepatic circulation.
Reticulone, a Novel Free Radical Scavenger Produced by Aspergillus sp.
Ryoo, In-Ja ; Xu, Guang-Hua ; Kim, Young-Hee ; Choo, Soo-Jin ; Ahn, Jong-Seog ; Bae, Ki-Hwan ; Yoo, Ick-Dong ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1573~1575
DOI : 10.4014/jmb.0906.06033
Bioassay-guided fractionation of the culture broth of Aspergillus sp. FN070449 (KCTC 26428) using a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay led to the isolation of two compounds: reticulone (1) and reticulol (2). Their chemical structures were elucidated on the basis of UV, IR, NMR, and MS spectroscopic analyses. Compound 1 exhibited more potent free radical scavenging activity on
(2,2'-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]) and DPPH radicals than did butylated hydroxyanisole (BHA) and caffeic acid.
Synergistic Effects of the Combination of 20-Hydroxyecdysone with Ampicillin and Gentamicin Against Methicillin-Resistant Staphylococcus aureus
Kim, Eun-Sook ; Jeong, Seung-Il ; Kim, Jung-Hoon ; Park, Channy ; Kim, Shin-Moo ; Kim, Jin-Kyung ; Lee, Kang-Min ; Lee, Sang-Heon ; So, Hong-Seob ; Park, Ra-Kil ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1576~1581
DOI : 10.4014/jmb.0903.03015
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has led to an urgent need for the discovery and development of new antibacterial agents. As part of an ongoing investigation into the antibacterial properties of natural products, 20-hydroxyecdysone (20E), isolated from the roots of Achyranthes japonica Nakai, was found to be active against MRSA, either alone or in combination with ampicillin (AM) or gentamicin (GM), via checkerboard assay. This study investigated the antibacterial activity of 20E, which exhibited poor antibacterial activity (
) against MRSA tested. The combined activity of AM or GE plus 20E against MRSA resulted in fractional inhibitory concentractions (FICs) ranging from 4.00 to
, respectively. Meanwhile, the FIC index ranged from 0.16-4.50, indicating a marked synergistic relationship between AM, GE, and 20E against MRSA. Time-kill assays also showed a remarkable decrease between the combination and the more active compound. Therefore, this study demonstrated that AM, GE, and 20E can act synergistically in inhibiting MRSA in vitro.
Inhibitory Mechanism of Novel Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Haemophilus influenzae
Jin, Bong-Suk ; Han, Seong-Gu ; Lee, Won-Kyu ; Ryoo, Sung-Weon ; Lee, Sang-Jae ; Suh, Se-Won ; Yu, Yeon-Gyu ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1582~1589
DOI : 10.4014/jmb.0905.05036
Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Haemophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations (
) were found to range from 0.1 to
. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium at a concentration range of
. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.
Activity of Essential Oils Against Bacillus subtilis Spores
Lawrence, Hayley A. ; Palombo, Enzo A. ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1590~1595
DOI : 10.4014/jmb.0904.04016
Alternative methods for controlling bacterial endospore contamination are desired in a range of industries and applications. Attention has recently turned to natural products, such as essential oils, which have sporicidal activity. In this study, a selection of essential oils was investigated to identify those with activity against Bacillus subtilis spores. Spores were exposed to 13 essential oils, and surviving spores were enumerated. Cardamom, tea tree, and juniper leaf oils were the most effective, reducing the number of viable spores by 3 logs at concentrations above 1%. Sporicidal activity was enhanced at high temperatures (
) or longer exposure times (up to 1 week). Gas chromatography-mass spectrometry analysis identified the components of the active essential oils. However, none of the major oil components exhibited equivalent activity to the whole oils. The fact that oil components, either alone or in combination, did not show the same level of sporicidal activity as the complete oils suggested that minor components may be involved, or that these act synergistically with major components. Scanning electron microscopy was used to examine spores after exposure to essential oils and suggested that leakage of spore contents was the likely mode of sporicidal action. Our data have shown that essential oils exert sporicidal activity and may be useful in applications where bacterial spore reduction is desired.
Lipase-catalyzed Esterification of (S)-Naproxen Ethyl Ester in Supercritical Carbon Dioxide
Kwon, Cheong-Hoon ; Lee, Jong-Ho ; Kim, Seung-Wook ; Kang, Jeong-Won ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1596~1602
DOI : 10.4014/jmb.0905.05051
A lipase-catalyzed esterification reaction of (S)-naproxen ethyl ester by CALB (Candida antarctica lipase B) enzyme was performed in supercritical carbon dioxide. Experiments were performed in a high-pressure cell for 10 h at a stirring rate of 150 rpm over a temperature range of 313.15 to 333.15 K and a pressure range of 50 to 175 bar. The productivity of (S)-naproxen ethyl ester was compared with the result in ambient condition. The total reaction time and conversion yields of the catalyzed reaction in supercritical carbon dioxide were compared with those at ambient temperature and pressure. The experimental results show that the conversion and reaction rate were significantly improved at critical condition. The maximum conversion yield was 9.9% (216 h) at ambient condition and 68.9% (3 h) in supercritical state. The effects of varying amounts of enzyme and water were also examined and the optimum condition was found (7 g of enzyme and 2% water content).
High-Cell-Density Fed-Batch Culture of Saccharomyces cerevisiae KV-25 Using Molasses and Corn Steep Liquor
Vu, Van Hanh ; Kim, Keun ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1603~1611
DOI : 10.4014/jmb.0907.07027
High-cell-density cultivation of yeast was investigated using the agricultural waste products corn steep liquor (CSL) and molasses. The Saccharomyces cerevisiae KV-25 cell mass was significantly dependent on the ratio between C and N sources. The concentrations of molasses and CSL in the culture medium were statistically optimized at 10.25% (v/v) and 16.87% (v/v), respectively, by response surface methodology (RSM). Batch culture in a 5-l stirred tank reactor using the optimized medium resulted in a cell mass production of 36.5 g/l. In the fed-batch culture, the feed phase was preceded by a batch phase using the optimized medium, and a very high dried-cell-mass yield of 187.63 g/l was successfully attained by feeding a mixture of 20% (v/v) molasses and 80% (v/v) CSL at a rate of 22 ml/h. In this system, the production of cell mass depended mainly on the agitation speed, the composition of the feed medium, and the glucose level in the medium, but only slightly on the aeration rate.
Production of Genistein from Naringenin Using Escherichia coli Containing Isoflavone Synthase-Cytochrome P450 Reductase Fusion Protein
Kim, Dae-Hwan ; Kim, Bong-Gyu ; Jung, Na-Ri ; Ahn, Joong-Hoon ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1612~1616
DOI : 10.4014/jmb.0905.05043
Isoflavonoids are a class of phytoestrogens. Isoflavonone synthase (IFS) is responsible for the conversion of naringenin to genistein. IFS is a cytochrome P450 (CYP), and requires cytochrome P450 reductase (CPR) for its activity. Additionally, the majority of cytochrome P450s harbor a membrane binding domain, making them difficult to express in Escherichia coli. In order to resolve these issues, we constructed an inframe fusion of the IFS from red clover (RCIFS) and CPR from rice (RCPR) after removing the membrane binding domain from RCIFS and RCPR. The resultant fusion gene, RCIFS-RCPR, was expressed in E. coli. The conversion of naringenin into genistein was confirmed using this E. coli transformant. Following the optimization of the medium and cell density for biotransformation,
of genistein could be generated from
of naringenin. This fusion protein approach may be applicable to the expression of other P450s in E. coli.
Production of c9,t11- and t10,c12-conjugated Linoleic Acids in Humans by Lactobacillus rhamnosus PL60
Lee, Ki-Eun ; Lee, Yeon-Hee ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1617~1619
DOI : 10.4014/jmb.0907.07010
Lactobacillus rhamnosus PL60 was tested for whether it can produce c9,t11- and t10,c12-conjugated linoleic acids (CLAs) in human. After consumption of L. rhamnosus PL60, L. rhamnosus was detected in feces 1 week after the start of intake. Analysis by gas chromatography showed that concentrations of c9,t11- and t10,c12-CLAs in serum had increased and concentrations of serum leptin had significantly decreased. Results showed that L. rhamnosus PL60 can survive in human intestines and produce CLAs in humans. This is the first report that bacteria can produce CLAs in humans.
Design and Expression of Recombinant Antihypertensive Peptide Multimer Gene in Escherichia coli BL21
Rao, Shengqi ; Su, Yujie ; Li, Junhua ; Xu, Zhenzhen ; Yang, Yanjun ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1620~1627
DOI : 10.4014/jmb.0905.05055
The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.
Inhibition of Citrate Synthase Thermal Aggregation In Vitro by Recombinant Small Heat Shock Proteins
Gong, Weina ; Yue, Ming ; Xie, Bingyan ; Wan, Fanghao ; Guo, Jianying ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1628~1634
DOI : 10.4014/jmb.0901.0046
Small heat shock proteins (sHSPs) function as molecular chaperones that protect cells against environmental stresses. In the present study, the genes of hsp17.6 and hsp17.7, cytosolic class I sHSPs, were cloned from a tropical plant, Ageratina adenophorum. Their C-terminal domains were highly conserved with those of sHSPs from other plants, indicating the importance of the C-terminal domains for the structure and activity of sHSPs. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at
and the irreversible inactivation of CS at
at stoichiometric levels. The prior presence of HSP17.7 was assumed to suppress the thermal aggregation of the model substrate CS. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.
Lactobacillus plantarum 299v Surface-Bound GAPDH: A New Insight Into Enzyme Cell Walls Location
Saad, N. ; Urdaci, M. ; Vignoles, C. ; Chaignepain, S. ; Tallon, R. ; Schmitter, J.M. ; Bressollier, P. ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1635~1643
DOI : 10.4014/jmb.0902.0102
The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.
Buffering Effects of Calcium Salts in Kimchi: Lowering Acidity, Elevating Lactic Acid Bacterial Population and Dextransucrase Activity
Seo, Eun-Chae ; Moon, Jin-Seok ; Jung, Jee-Yun ; Kim, Ji-Sun ; Eom, Hyun-Ju ; Kim, So-Young ; Yoon, Hyang-Sik ; Han, Nam-Soo ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1644~1649
DOI : 10.4014/jmb.0906.06046
This study investigates the buffering effects of calcium salts in kimchi on the total acidity, microbial population, and dextransucrase activity. Calcium chloride or calcium carbonate was added to dongchimi-kimchi, a watery radish kimchi, and the effects on various biochemical attributes were analyzed. The addition of 0.1% calcium chloride produced a milder decrease in the pH after 24 days of incubation, which allowed the lactic acid bacteria to survive longer than in the control. In particular, the heterofermentative Leuconostoc genus population was 10-fold higher than that in the control. When sucrose and maltose were also added along with the calcium salts, the dextransucrase activity in the kimchi was elevated and a higher concentration of isomaltooligosaccharides was synthesized when compared with the control. Calcium chloride was determined as a better activator compound of dextransucrase than calcium carbonate, probably because of its higher solubility. Therefore, the results of this study confirm the ability of the proposed approach to modulate the kimchi fermentation process and possibly enhance the quality of kimchi based on the addition of dietary calcium salts.
Cloning and Expression of
-Glucuronidase from Lactobacillus brevis in E. coli and Application in Bioconversion of Baicalin and Wogonoside
Kim, Hyun-Sung ; Kim, Jin-Yong ; Park, Myeong-Soo ; Zheng, Hua ; Ji, Geun-Eog ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1650~1655
DOI : 10.4014/jmb.0904.04053
-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1,812 bp, encoding a 603-amino-acid protein belonging to glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the
-glucuronidases of various microorganisms, yet less than 58% with the
-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in
-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1,284 U/mg of specific activity at optimum conditions of pH 5.0 and
, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at
and more than 2 h at
. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones,
of baicalin and
of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.
Stability of Partial Nitrification and Microbial Population Dynamics in a Bioaugmented Membrane Bioreactor
Zhang, Yunxia ; Xu, Yanli ; Jia, Ming ; Zhou, Jiti ; Yuan, Shouzhi ; Zhang, Jinsong ; Zhang, Zhen-Peng ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1656~1664
DOI : 10.4014/jmb.0906.06006
Bioaugmentation of bioreactors focuses on the removal of numerous organics, with little attention typically paid to the maintenance of high and stable nitrite accumulation in partial nitrification. In this study, a bioaugmented membrane bioreactor (MBR) inoculated with enriched ammonia-oxidizing bacteria (AOB) was developed, and the effects of dissolved oxygen (DO) and temperature on the stability of partial nitrification and microbial community structure, in particular on the nitrifying community, were evaluated. The results showed that DO and temperature played the most important roles in the stability of partial nitrification in the bioaugmented MBR. The optimal operation conditions were found at 2-3 mgDO/l and
, achieving 95% ammonia oxidization efficiency and nitrite ratio (
) of 0.95. High DO (5-6 mg/l) and low temperature (
) had negative impacts on nitrite accumulation, leading to nitrite ratio drop to 0.6. However, the nitrite ratio achieved in the bioaugmented MBR was higher than that in most previous literatures. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were used to provide an insight into the microbial community. It showed that Nitrosomonas-like species as the only detected AOB remained predominant in the bioaugmented MBR all the time, and coexisted with numerous heterotrophic bacteria. The heterotrophic bacteria responsible for mineralizing soluble microbial products (SMP) produced by nitrifiers belonged to the Cytophaga-Flavobacterium-Bacteroides (CFB) group, and
- Proteobacteria. The fraction of AOB ranging from 77% to 54% was much higher than that of nitrite-oxidizing bacteria (0.4-0.9%), which might be the primary cause for the high and stable nitrite accumulation in the bioaugmented MBR.
Enrichment of Hydrogenotrophic Methanogens in Coupling with Methane Production Using Electrochemical Bioreactor
Jeon, Bo-Young ; Kim, Sung-Yong ; Park, Yong-Keun ; Park, Doo-Hyun ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1665~1671
DOI : 10.4014/jmb.0904.04002
Anaerobic digestion sludge was cultivated in an electrochemical bioreactor (ECB) to enrich the hydrogenotrophic methanogens. A modified graphite felt cathode with neutral red (NR-cathode) was charged with electrochemical reducing power generated from a solar cell. The methane and carbon dioxide collected in a Teflon bag from the ECB were more than 80 ml/l of reactant/day and less than 20 ml/l of reactant/day, respectively, whereas the methane and carbon dioxide collected from a conventional bioreactor (CB) was around 40 ml/l of reactant/day, respectively. Moreover, the maximal volume ratios of methane to carbon dioxide (M/C ratio) collected in the Teflon bag from the ECB and CB were 7 and 1, respectively. The most predominant methanogens isolated from the CB on the
days of incubation were hydrogenotrophs. The methanogenic diversity analyzed by temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region was higher in the ECB than in the CB. The DNA extracted from the TGGE bands was more than 95% homologous with hydrogenotrophic methanogens in the ECB, but was an aceticlastic methanogen in the CB. In conclusion, the ECB was demonstrated as a useful system for enriching hydrogenotrophic methanogens and increasing the M/C ratio of the gas product.
Inhibitory Effect of Aged Petroleum Hydrocarbons on the Survival of Inoculated Microorganism in a Crude-Oil-Contaminated Site
Kang, Yoon-Suk ; Park, Youn-Jong ; Jung, Jae-Joon ; Park, Woo-Jun ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1672~1678
DOI : 10.4014/jmb.0903.03001
We studied the effects of aged total petroleum hydrocarbons (aged TPH) on the survival of allochthonous diesel-degrading Rhodococcus sp. strain YS-7 in both laboratory and field investigations. The aged TPH extracted from a crude-oil-contaminated site were fractionized by thin-layer chromatography/flame ionization detection (TLC/FID). The three fractions identified were saturated aliphatic (SA), aromatic hydrocarbon (AH), and asphaltene-resin (AR). The ratio and composition of the separated fractions in the aged TPH were quite different from the crude-oil fractions. In the aged TPH, the SA and AH fractions were reduced and the AR fraction was dramatically increased compared with crude oil. The SA and AH fractions (2 mg/l each) of the aged TPH inhibited the growth of strain YS-7. Unexpectedly, the AR fraction had no effect on the survival of strain YS-7. However, crude oil (1,000 mg/l) did not inhibit the growth of strain YS-7. When strain YS-7 was inoculated into an aged crude-oil-contaminated field and its presence was monitored by fluorescent in situ hybridization (FISH), we discovered that it had disappeared on 36 days after the inoculation. For the first time, this study has demonstrated that the SA and AH fractions in aged TPH are more toxic to an allochthonous diesel-degrading strain than the AR fraction.
Genetic and Phenotypic Diversity of Parathion-Degrading Bacteria Isolated from Rice Paddy Soils
Choi, Min-Kyeong ; Kim, Kyung-Duk ; Ahn, Kyong-Mok ; Shin, Dong-Hyun ; Hwang, Jae-Hong ; Seong, Chi-Nam ; Ka, Jong-Ok ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1679~1687
DOI : 10.4014/jmb.0905.05057
Three parathion-degrading bacteria and eight pairs of bacteria showing syntrophic metabolism of parathion were isolated from rice field soils, and their genetic and phenotypic characteristics were investigated. The three isolates and eight syntrophic pairs were able to utilize parathion as a sole source of carbon and energy, producing p-nitrophenol as the intermediate metabolite during the complete degradation of parathion. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Burkholderia, Arthrobacter, Pseudomonas, Variovorax, and Ensifer. The chromosomal DNA patterns of the isolates obtained by polymerasechain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from one another. Ten of the isolates had plasmids. All of the isolates and syntrophic pairs were able to degrade parathion-related compounds such as EPN, p-nitrophenol, fenitrothion, and methyl parathion. When analyzed with PCR amplification and dot-blotting hybridization using various primers targeted for the organophosphorus pesticide hydrolase genes of previously reported isolates, most of the isolates did not show positive signals, suggesting that their parathion hydrolase genes had no significant sequence homology with those of the previously reported organosphophate pesticide-degrading isolates.
Characterization of a Phenazine and Hexanoyl Homoserine Lactone Producing Pseudomonas aurantiaca Strain PB-St2, Isolated from Sugarcane Stem
Mehnaz, Samina ; Baig, Deeba Noreen ; Jamil, Farrukh ; Weselowski, Brian ; Lazarovits, George ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1688~1694
DOI : 10.4014/jmb.0904.04022
A novel strain of fluorescent pseudomonad (PB-St2) was isolated from surface-sterilized stems of sugarcane grown in Pakistan. The bacterium was identified as Pseudomonas aurantiaca on the basis of 16S rRNA gene sequence analysis and results from physiological and biochemical characteristics carried out with API50 CH and QTS 24 bacterial identification kits. Assays using substrate-specific media for enzymes revealed lipase and protease activities but cellulase, chitinase, or pectinase were not detected. The bacterium was unable to solubilize phosphate or produce indole acetic acid. However, it did produce HCN, siderophores, and homoserine lactones. In dual culture assays on agar, the bacterium showed antifungal activity against an important pathogen of sugarcane in Pakistan, namely Colletotrichum falcatum, as well as for pathogenic isolates of Fusarium oxysporium and F. lateritium but not against F. solani. The antifungal metabolites were identified using thin-layer chromatography, UV spectra, and MALDI-TOFF spectra and shown to be phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), and N-hexanoyl homoserine lactone (HHL) (assessed using only TLC data). The capacity of this bacterium to produce HCN and 2-OH-PHZ, as well as to inhibit the growth of C. falcatum, has not been previously reported.
A High-Yielding, Generic Fed-Batch Process for Recombinant Antibody Production of GS-Engineered Cell Lines
Fan, Li ; Zhao, Liang ; Sun, Yating ; Kou, Tianci ; Zhou, Yan ; Tan, Wen-Song ;
Journal of Microbiology and Biotechnology, volume 19, issue 12, 2009, Pages 1695~1702
DOI : 10.4014/jmb.0904.04054
An animal-component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of by-products (lactate and osmolality). The proportionalities of nutritional consumption were determined by direct analysis. The robust, metabolically responsive feeding strategy was based on the offline measurement of glucose. The fed-batch process was shown to perform equivalently in GS-CHO and GS-NS0 cultures. Compared with batch cultures, the fed-batch technology generated the greater increase in cell yields (5-fold) and final antibody concentrations (4-8-fold). The majority of the increase in final antibody concentration was a function of the increased cell density and the prolonged culture time. This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines.