• Journal of Life Science
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• v.18 no.6
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• pp.878-883
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• 2008

Aeromonas hydrophila DA33 was isolated from cultivated soils as a bacteria having high abilities to solubilize inorganic phosphate. Glucose dehydrogenase gene (gdh) was cloned from Escherichia coli. The recombinant plasmid, pGHS containing glucose dehydrogenase gene was introduced into A. hydrophila DA33 in order to improve the activity of phosphate-solubilizing. The transformant harboring the gdh gene, A. hydrophila pGHS/DA33 increased enzyme activity. The strain also increased the gluconic acid generation that was effective for phosphate solubilization. It was possible that the strain containing pGHS produced higher solubilized phosphate with tri-calcium phosphate as the unique (P) source, in comparison with that of wild type without plasmid. These results suggest that the strain, A. hydrophila pGHS/DA33 is expected as effective biofertilizer for phosphate solubilization.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.336-343
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• 2016

A mannanase gene was cloned into Escherichia coli from Cellulosimicrobium sp. YB-43, which had been found to produce two kinds of mannanase, and sequenced completely. This mannanase gene, designated manB, consisted of 1,284 nucleotides encoding a polypeptide of 427 amino acid residues. Based on the deduced amino acid sequence, the ManB was identified to be a modular enzyme including two carbohydrate binding domains besides the catalytic domain, which was highly homologous to mannanases belonging to the glycosyl hydrolase family 5. The N-terminal amino acid sequence of ManB, purified from a cell-free extract of the recombinant E. coli carrying a Cellulosimicrobium sp. YB-43 manB gene, has been determined as QGASAASDG, which was correctly corresponding to signal peptide predicted by SignalP4.1 server for Gram-negative bacteria. The purified ManB had a pH optimum for its activity at pH 6.5~7.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum (LBG), konjac and guar gum, while it did not exhibit activity towards carboxymethylcellulose, xylan, starch, and para-nitrophenyl-${\beta}$-mannopyranoside. The activity of enzyme was inhibited very slightly by $Mg^{2+}$, $K^+$, and $Na^+$, and significantly inhibited by $Cu^{2+}$, $Zn^{2+}$, $Mn^{2+}$, and SDS. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose, which was the most predominant product resulting from the ManB hydrolysis for mannooligosaccharides and LBG.

• Journal of Life Science
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• v.22 no.11
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• pp.1545-1551
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• 2012

Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

• Journal of Life Science
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• v.26 no.10
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• pp.1113-1120
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• 2016

Two bacterial strains producing extracellular man nanase were isolated from doenjang, a traditionally fermented soybean paste in Korea. The isolates, WL-6 and WL-11, were identified as Bacillus subtiis on the basis of their 16S rRNA gene sequences, morphological, and biochemical properties. Two genes encoding the mannanase of both B. subtilis WL-6 and B. subtilis WL-11 were each cloned into Escherichia coli, and their nucleotide sequences were determined. Both mannanase genes consisted of 1,086 nucleotides, encoding polypeptides of 362 amino acid residues. The deduced amino acid sequences of the two WL-6 and WL-11 mannanases, designated Man6 and Man11, respectively, differed from each other by eight amino acid residues, and they were highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The 26 amino acid stretch in the N-terminus of Man6 and Man11 was a predicted signal peptide. Both Man6 and Man11 were localized at the level of 94–95% in an intracellular fraction of recombinant E. coli cells. The enzymes hydrolyzed both locust bean gum and mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, forming mannobiose and mannotriose as predominant products. The optimal reaction conditions were 55°C and pH 6.0 for Man6, and 60°C and pH 5.5 for Man11. Man11 was more stable than Man6 at high temperatures.

• Microbiology and Biotechnology Letters
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• v.39 no.3
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• pp.238-244
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• 2011

Biodiesel, mono-alkyl esters of long chain fatty acids, is one of the alternative fuels derived from renewable lipid feedstock, such as vegetable oils or animal fats. For decade, various lipases have been used for the production of biodiesel. However, the production of biodiesel by enzymatic catalyst has profound restriction in industry application due to high cost. To overcome these problems, many research groups have studied extensively on the selection of cheap oil sources, the screening of suitable lipases, and development of lipase immobilization methods. In this study, we produced biodiesel from plant oil using Proteus vulgaris lipase K80 expressed in Escherichia coli cells. The recombinant lipase K80 was not only expressed in high level but also had high specific lipase activity and high stability in various organic solvents. Lipase K80 could produce biodiesel from olive oil by 3-stepwise methanol feeding method. The immobilized lipase K80 also produced biodiesel using the same 3-stepwise method. The immobilized lipase could produce biodiesel efficiently from various plant oils and waste oils.

• Journal of Life Science
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• v.27 no.9
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• pp.1003-1010
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• 2017

An open reading frame coding for mannanase predicted from the partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by polymerase chain reaction amplification, and completely sequenced. This mannanase gene, designated man26AT, consisted of 3,162 nucleotides encoding a polypeptide of 1,053 amino acid residues. Based on the deduced amino acid sequence, Man26AT was identified as a modular enzyme, which included a catalytic domain belonging to the glycosyl hydrolase family 26 and two carbohydrate-binding modules, CBM27 and CBM11. The amino acid sequence of Man26AT was homologous to that of several putative mannanases, with identity of 81% for P. ihumii and identity of less than 57% for other strains of Paenibacillus. A cell-free extract of recombinant E. coli carrying the man26AT gene showed maximal mannanase activity at $55^{\circ}C$ and pH 5.5. The enzyme retained above 80% of maximal activity after preincubation for 1 h at $50^{\circ}C$. Man26AT was comparably active on locust bean gum (LBG), galactomanan, and kojac glucomannan, whereas it did not exhibit activity on carboxymethylcellulose, xylan, or para-nitrophenyl-${\beta}$-mannopyranoside. The common end products liberated from mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, or LBG by Man26AT were mannose, mannobiose, and mannotriose. Mannooligosacchrides larger than mannotriose were found in enzymatic hydrolyzates of LBG and guar gum, respectively. However, Man26AT was unable to hydrolyze mannobiose. Man26AT was intracellularly degraded into at least three active proteins with different molecular masses by zymogram.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1115-1123
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• 2016

_L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the _L-asparaginase gene (_L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of _L-ASPG86 (_L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The _L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant _L-asparaginase (r-_L-ASPG86) showed optimum conditions at 37-40℃, pH 9. Moreover, r-_L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-_L-ASPG86 was 687.1 units/mg under optimum conditions (37℃, pH 9, and 5 mM MnSO₄).

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.253-260
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• 2005

Mitotic centromere-associated kinesin (MCAK), which is a member of the Kin I (internal motor domain) subfamily of kinesin-related proteins, is known to play a role in mitotic segregation of chromosome during M phase of the cell cycle. In the present study, we have produced a rat polyclonal antibody using human MCAK (HsMCAK) expressed in E. coli as the antigen. The antibody specifically recognized the HsMCAK protein (81 kDa), and could detect its nuclear localization in human Jurkat T cells and 293T cells by Western blot analysis. The specific stage of the cell cycle was obtained through blocking by either hydroxyl urea or nocodazole and subsequent releasing from each blocking for 2, 4, and 7 h. While the protein level of HsMCAK reached a maximum level in the S phase with slight decline in the $G_{2}-M$ phase, the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$ began to be induced in the late S phase and reached a maximum level in the $G_{2}/M $ phase, and then it disappeared as the cells enter into the $G_{1}$ phase. Immunocytochemical analysis revealed that HsMCAK protein localized to centrosome and nucleus at the interphase, whereas it appeared to localize to the spindle pole, centromere of the condensed mitotic DNA, spindle fiber, or midbody, depending on the specific stage of the M phase. These results demonstrate that a rat polyclonal antibody raised against recombinant HsMCAK expressed in E. coli specifically detects human MCAK, and indicate that the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$, which may be associated with its differential intracellular localization during the cell cycle, fluctuates with a maximum level of the shift at the $G_{2}-M$ phase.