• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.304-308
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• 2005

The response of IPTG induction was investigated through the monitoring of the alkali consumption rate and buffer capacity during the cultivation of recombinant E. coli BL21 (DE3) harboring the plasmid pRSET-LacZ under the control of lac promoter. The rate of alkali consumption increased along with cell growth, but declined suddenly after approximately 0.2 h of IPTG induction. The buffer capacity also declined after 0.9 h of IPTG induction. The profile of buffer capacity seems to correlate with the level of acetate production. The IPTG response was monitored only when introduced into the mid-exponential phase of bacterial cell growth. The minimum concentration of IPTG for induction, which was found out to be 0.1 mM, can also be monitored on-line and in-situ. Therefore, the on-line monitoring of alkali consumption rate and buffer capacity can be an indicator of the metabolic shift initiated by IPTG supplement, as well as for the physiological state of cell growth.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1343-1349
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• 2004

A native strain of Pasteurella multocida was isolated from pigs suffering from severe atrophic rhinitis at domestic farms in Gyeonggi Province, Korea, and was identified as capsular serogroup 'D' and somatic serotype '4' by disc diffusion decapsulation and gel diffusion precipitation tests, respectively. The P. multocida (D:4) induced atrophic rhinitis in healthy pigs by the secondary infection. The gene for outer membrane protein H (ompH) of P. multocida (D:4) was cloned in Escherichia coli DH5$\alpha$ by PCR. The open reading frame of the ompH was composed of 1,023 bp, possibly encoding a protein with 341 amino acid residues containing a signal peptide of 20 amino acids at N-terminus, and the gene product with molecular mass of ca. 38 kDa was identified by SDS-PAGE. Hydropathy profiles indicated that there are two variable domains in the OmpH. To express the ompH in E. coli, the gene was manipulated in various ways. Expression of the truncated as well as full-length forms of the recombinant OmpH was fatal to the host E. coli BL21 (DE3). However, the truncated OmpH fused with GST was consecutively expressed in E. coli DH5$\alpha$. A large quantity of the fused polypeptide was purified through GST-affinity chromatography.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.347-356
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• 2019

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods. B. velezensis BS2 grows well at 15% NaCl and at $10^{\circ}C$. When B. velezensis BS2 was cultivated in TSB broth for 96 h at $37^{\circ}C$, the culture showed the highest fibrinolytic activity ($131.15mU/{\mu}l$) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and $37^{\circ}C$, respectively. $K_m$ and $V_{max}$ were 0.15 mM and $39.68{\mu}M/l/min$, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong ${\alpha}$-fibrinogenase and moderate ${\beta}$-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at $10^{\circ}C$, B. velezensis BS2 can be used as a starter for jeotgal.

• Fisheries and Aquatic Sciences
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• v.12 no.1
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• pp.54-59
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• 2009

Carotenoids such as $\beta$-carotene and astaxanthin are used as food colorants, animal feed supplements and for nutritional and cosmetic purposes. In a previous study, an astaxanthin biosynthesis gene cluster was isolated from the marine bacterium, Paracoccus haeundaensis. Geranylgeranyl pyrophosphate (GGPP) synthase (CrtE), encoded by the ortE gene, catalyzes the formation of GGPP from farnesyl pyrophosphate (FPP), which is an essential enzyme for the biosynthesis of carotenoids in early steps. In order to study the biochemical and enzymatic characteristics of this important enzyme, a large quantity of purified GGPP synthase is required. To overproduce GGPP synthase, the crtE gene was subcloned into a pET-44a(+) expression vector and transformed into the Escherichia coli BL21(DE3) codon plus cell. Transformants harboring the crtE gene were cultured and the crtE gene was over-expressed. The expressed protein was purified to homogeneity by affinity chromatography and applied to study its biochemical properties and molecular characteristics.

• Genomics & Informatics
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• v.4 no.3
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• pp.133-136
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• 2006

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1681-1691
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• 2023

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• Journal of Life Science
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• v.18 no.12
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• pp.1764-1770
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• 2008

The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ${\mu}g$/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ${\mu}g$/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.