• Applied Biological Chemistry
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• v.36 no.2
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• pp.105-110
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• 1993

Inulin fructotransferase from Enterobacter sp. S45 was purified with DEAE-cellulose column chromatography and fast protein liquid chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 42,800 by SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for the enzyme reaction were pH 5.5 and $55^{\circ}C$, respectively. $Mg^{2+}$ activated the enzyme activity, but $Fe^{3+}$, $Cu^{2+}$, $Hg^{2+}$ significantly inhibited. After exhaustive digestion of inulin by the enzyme, DFA III, sucrose, 1-kestose and nystose were produced. Sucrose, 1-kestose, raffinose and melezitose can't be used as substrates by the enzyme, but nystose and 1-F-fructofuranosyl nystose were hydrolysed. The Km and Vmax for inulin of the enzyme were 1.4 mM and $0.196\;{\mu}mole/min$, respectively.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.178-185
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• 2008

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

• 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.

• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.05a
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• pp.17-17
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• 2010

Ethylene, known as a stress hormone regulate wide developmental processes including germination, root hair initiation, root and shoot primordial formation and elongation, leaf and flower senescence and abscission, fruit ripening. The acceleration of ethylene biosynthesis in plant associated with environmental and biological stresses. 1-Aminocycloprophane-1-carboxlyate deaminase(ACCD) is an enzyme that cleaves ACC into and ammonia, a precursor of the plant hormone ethylene. Plant growth-promoting rhizobacteria (PGPR) having ACCD can decrease endogenous ACC level of tissue, resulting in reduced production of ethylene in plants. ACC deaminse was a key enzyme for protect stressed plants from injurious effects of ethylene. ACCD gene was encoded from Pseudomonas flourescens, PGPR and was cloned in Escherichia coli. We expressed the recombinant ACCD(rACCD) containing 357 amino acids with molecular weight 39 kDa that revealed by SDS-PAGE and western blot. The rACCD was purified by Ni-NTA purification system. The active form of rACCD having enzyme activity converted ACC to a-ketobutyrate. The optimal pH for ACC deaminase activity was pH 8.5, but no activity below pH 7.0 and a less severe tapering activity at base condition resulting in loss of activity at over pH 11. The optimal temperature of the enzyme was $30^{\circ}$ and a slightly less severe tapering activity at 15 - 30$^{\circ}$, but no activity over $35^{\circ}$. P. flourescens ACC deaminase has a highly conserved residue that plays in allowing substrate accessibility to the active sites. The enzymatic properties of this rACCD will provide an important reference for analysis of newly isolated ACCD and identification of newly isolated PGPR containing ACCD.

• 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.19 no.11
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• pp.1295-1300
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• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

• Journal of Nutrition and Health
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• v.19 no.6
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• pp.363-373
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• 1986

This study was devised to elucidate whether soused shrimp exhibits a digestive action on boiled pork meats. and the mechanism by which sousing with a high concentration of sodium chloride preserves nutrients in foods for a prolonged pe\ulcornerriod. Protease was isolated from soused shrimp using a combination of ammonium sulfate fractionation. DEAE - cellulose ion exchange chromatography and gel filtra\ulcornertion. The isolated protease had specific activity of 1.560 units. 210 purification fo\ulcornerld with an yield of 38%. Its optimum pH and temperature were 8.0 and $43^{\circ}C$ respectively. The molecular weight of the enzyme was 35.000. The Km value of the enzyme for casein was 1.6 x $10^{-6}$ M The e=yme required the presence of cu\ulcornerpric ion to exhibit its full activity. Eighty eight percent of the enzyme activity was in\ulcornerhibited by 3.5M NaCI showing a reversibly linear decrease of the enzyme activity as NaCI concentration increased. The nature of the inhibition by NaCl was rever\ulcornersible and noncompetitive. The protease activity in soused shrimp was well preser\ulcornerved with the elapse of time at least in part due to NaCI induced suppression of autodigestion. The enzyme was denatured by acid easily. i.e. 1% of the original activity remained after staying at pH 2 for 10 minutes. which is within the norm\ulcorneral range of pH of the human stomach. Soused shrimp was observed to be one of those containing the highest protease activity compared with the other soused foo\ulcornerds such as soused oyster. squid. clam. and Pollack intestine with respect to spec\ulcornerific activities of dialized 1:4 whole homogenates(w/v) in 5 mM sodium phospha\ulcornerte - 2.4 mM j3 - mercaptoethanol buffer. pH 8.0. Casein and boiled meats including pork, beef, and chicken appeared to be the good substrates for the protease. Casein was the best. Therefore. the ingestion of boiled meats including pork together with soused sh\ulcornerrimp would help digestion of boiled pork in human not only by increasing appe\ulcornertite also by the direct proteolytic digestion of boiled meats by soused shrimp to\ulcorner some extent. And a high concentration of sodium chloride inhibited the protease activity reversibly in a remarkable degree, which ensued in a significant retardat\ulcornerion of autodigestion of protein in foods by proteases, and hereby contributed to the preservation of foods for an extended period.

• Journal of Microbiology and Biotechnology
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• v.21 no.10
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• pp.1012-1020
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• 2011

A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of ${\beta}$-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of $55^{\circ}C$ and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of $30-37^{\circ}C$ and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants ($K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$) determined for rEglA with ${\beta}$-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 $min^{-1}$, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 $min^{-1}$, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward ${\beta}$-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.11
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• pp.1647-1653
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• 2010

Glycosaminoglycan (GAG) was purified from polysaccharide extracted from sea hare muscle on DEAE-Sepharose column and investigated for the functional groups, distribution of sugars, composition of disaccharide and structure of GAG. Purified GAG was composed of disaccharide above 55% of total sugar. Purified GAG showed amide I peak in 1648/cm and C-O stretch peak as properties of carbohydrate, amino acid peak in 1457/cm, and peak in 866/cm as properties of monosaccharide by FT-IR. Fucose, N-acetylgalactosamine, N-acetylglucosamine, glucose, galactose, mannose and xylose were found in MALDI-TOF MS/MS spectra of hydrolysates by chondroitin sulfate ABC lyase and heparanase I. Purified GAG was expected to be heparan sulfate including N-acetylgalactosamine and N-acetylglucosamine above 70% of total sugar. The structure of GAG was supposed as GlyUA(2S)-GlcNS and GlyUA-GlcNS(6S) with O-linkage on protein core.