• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.901-910
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• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.730-738
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• 2016

est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60℃ and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

• Microbiology and Biotechnology Letters
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• v.41 no.4
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• pp.391-397
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• 2013

A putative cyclomaltodextrinase (LLCD) gene was cloned from the genome of Lactococcus lactis subsp. lactis KCTC 3769 (ATCC 19435), which encodes 584 amino acids with the predicted molecular mass of 68.7 kDa. KCTC 3769 shares approximately 40% of amino acid sequence identity with the CDase-family of enzymes. The dimeric enzyme with C-terminal six-histidines was heterologously expressed and purified from recombinant E. coli. LLCD showed the highest activity against ${\beta}$-cyclodextrin (CD) at pH 7.0 and $37^{\circ}C$. In particular, LLCD exhibited extremely low activity against starch and pullulan, while its CD-hydrolyzing activity was about 80 times higher than starch. Due to its much higher activity on CD over starch, LLCD has been identified as a member of CDases. However, LLCD can be distinguished from the other common CDases on the basis of its extremely low hydrolyzing activity against starch, pullulan, and acarbose.

• KSBB Journal
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• v.11 no.4
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• pp.445-452
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• 1996

To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) as a reporter under the control of GAL10, GAL7, GAL1, and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : they dropped from $0.24 h^{-1}$ during the glucose-consuming period to 0.04 -$0.10 h^{-1}$ during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7 and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

• Molecules and Cells
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• v.40 no.12
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• pp.954-965
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• 2017

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Numerous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.494-498
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• 1988

The optimum conditions for glucoamylase production, and ethanol productivity of the transformant TSD-14 were investigated as compared with the parental strains. The properties of TSD-14 were comparatively similar to the donor S. diastaticus IFO 1046 as regards the conditions of glucoamylase production and ethanol productivity. The soluble starch was the most effective carbon source for the glucoamylase production. While inorganic nitrogen sources did not prompt cell growth and enzyme production, the organic nitrogen sources generally enhanced both cell growth and glucoamylase production. The metal salts such as FeSO$_4$, MgSO$_4$, MnCl$_2$, and NiSO$_4$were favorable to the enzyme production. And the optium temperature and initial pH for glucoamylase production were 3$0^{\circ}C$ and 5. The transformant TSD-14 produced 8.3%(v/v) ethanol from 15% sucrose medium, 4.8%(v/v) ethanol from 15% soluble starch medium, and 7.5%(v/v) ethanol from 15% liquefied potato starch medium. The corresponding fermentation efficiency were 84% , 45% and 70%, respectively.

• KSBB Journal
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• v.23 no.1
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• pp.44-47
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• 2008

Anthracycline antibiotics doxorubicin (DXR) is clinically important cancer therapeutic agent produced by Streptomyces peucetius. DXR result by further metabolism of rhodomycin D (RHOD) and require a deoxy-sugar component for their biological activity. In this study, production of TDP-L-daunosamine and its attachment to ${\varepsilon}$-rhodomycinone (RHO) to generate RHOD has been achieved by bioconversion in Streptomyces venezuelae that bears eleven genes. S. peucetius seven genes (dnmUTJVZQS) were transformed by plasmid and S. venezuelae two genes desIII, IV and two more S. peucetius drrA, B genes were integrated into chromosomal DNA. To generate the feeding substrate RHO, 6L S. peucetius grown on agar plate was harvested, extracted with organic solvent and then purified using preparative HPLC. Recombinant S. venezuelae grown on agar plate containing RHO was harvested and its n-butanol soluble components were extracted. The glycosylated product of aromatic polyketide RHO using heterologous host S. venezuelae presents the minimal information for TDP-L-daunosamine biosynthesis and its attachment onto aglycone. Moreover, the structure of auxiliary protein, DnrQ, was predicted by fold recognition and homology modeling in this study. This is a general approach to further expand of new glycosides of antitumor anthracycline antibiotics.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• The Journal of the Korean Society for Microbiology
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• v.13 no.1
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• pp.17-24
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• 1978

Modulating effects of Candida albicans on the immune responses of mice immunized with sheep red blood cells(SRBC) were assessed both by footpad tests for anaphylactic, Arthus and delayed type hypersensitivity rections against homologous and heterologous antigenic challenges and by serum antibody titrations for hemagglutinin and hemolysin against SRBC. The results are summarized as follows: 1. In the mice simultaneously immunzed with C. albicans and SRBC, anaphylactic type and Arthus type footpad reactions to C. albicans challenge were enhanced, and extents of the enhancements were proportional to the concentration of SRBC administered for immunization, reaching peak in mice immunized with 0.2ml($10^8$) of 5% SRBC suspension. Although a little enhancement of delayed type hypersensitivity to C. albicans was observed in those mice, there was no significant difference between the mice groups immunized either with SRBC alone or SRBC and C. albicans simultaneously. 2. Simultaneous immunization of mice with C. albicans and SRBC resulted in the suppression of both anaphylactic type and Arthus type footpad reactions to SRBC, and the extent of such suppressions was inversly proportional to the numbers of C. albicans administered for immunization. Delayed type reaction of the mice to SRBC varied little in regards to the different numbers of C. albicans injected. 3. Hemagglutinin titers differed little between the mice groups immunized with SRBC alone or with SRBC and C. albicans simultaneously. Hewever, hemolysin titers were lower in the mice immunized simultaneously with SRBC and C. albicans. 4. In the peripheral blood of mice immunized simultaneously with SRBC and C. albicnas. there observed increases in the percents of monocyte and polymorphonuclear leukocytes and decrease in the numbers of lymphocytes and pyroninophilic lymphocytes. These results indicated that C. albicans is an immunosuppressant of the mice to SRBC when both anteigns were administered simultaneously for immunization, and that SRBC acted as an enhancer of anaphylactic type and Arthus type reaction of mice to C. albicans when administered simultaneously.