• Applied Biological Chemistry
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• v.39 no.6
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• pp.494-500
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• 1996

Uptake of hen metal ions by water dropwort (Oenanthe stolonifera DC.) and its cadmium-binding protein were studied to probe for good method to remove heavy metal contaminants from environments. The plant was cultured in the culture medium (pH 7.0) containing the various concentrations of $Cd^{2+}$, $Cr^{3+}$ or $Pb^{2+}$, for 3 and 7 days. The residual heavy metals deposited in roots linearly increased as the metal ions concentration increased up to 17 ppm for $Cd^{2+}$, 20 ppm for $Cr^{3+}$ and 50 ppm for $Pb^{2+}$. Above these concentrations, the plant growth was inhibited and the uptake rates of the metal ions decreased. The heavy metals absorbed by the plant were mostly deposited in roots. In particular, the residual concentration of lead in roots was about four times higher than those of cadmium and chromium. When cultured in the medium containing 20 ppm of each metal ion, 80% of cadmium, 90% of cromium and 96% of lead were deposited in roots out of the total residual metal ions in the plant. These values correspond to 6.1 mg of cadmium, 5.2 mg of chromium and 23.6 mg of lead per one gram of roots tissue on a dry weight basis. A cadmium-binding protein was partially purified by extraction, gel filtration and DEAE-Cellulose chromatography from water dropworts that was grown in the medium containing 20 ppm $Cd^{2+}$. The purified protein was a single band on SDS- and non-denaturing- polyacrylamide gel electrophoresis. Its molecular mass was estimated to be ca. 5,000 dalton by gel filteration. Analysis of amino acid composition of the protein indicated that it had a typical amino acid composition of heavy metal-binding protein in that it contained 27% of acidic amino acids and 9.9% of cysteine. However, it is likely that the protein is a new plant metal-binding protein, since its amino acid composition is somewhat different from those of phytochelatins that have been known so far.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.95-100
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• 2007

The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

• Microbiology and Biotechnology Letters
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• v.42 no.4
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• pp.317-323
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• 2014

Violacein has received much attention due to its various important biological activities, including broad-spectrum antibacterial and antifungal activity, anti-malarial, anti-tumoral, anti-oxidant, and anti-diarrheal activities. EP15224 strain isolated from forest soils in Korea was found to be a new species belonged to the genus Massilia based on its 16S ribosomal DNA sequences. The 16S ribosomal DNA of strain EP15224 displayed 97% homology with Massilia sp. BS-1, the nearest violacein-producing bacterium. Strain EP15224 produced bluish-purple pigment well in a synthetic MM2 medium containing glucose, $(NH_4)_2SO_4$, $Na_2HPO_4{\cdot}7H_2O$, $KH_2PO_4$, $MgSO_4{\cdot}7H_2O$, and 1 mM $\small{L}$-tryptophan. The chemical analysis of the pigment by LC/MS/MS showed that it is violacein with molecular weight of 343.34. This is the second report on the production of violacein by a Massilia species. In this study, the optimal culture conditions for violacein production were established under which 280 mg/l crude violacein was produced : glucose 2 g/l, $(NH_4)_2SO_4$ 1 g/l, $Na_2HPO_4{\cdot}7H_2O$ 2 g/l, $KH_2PO_4$ 1 g/l, $MgSO_4{\cdot}7H_2O$ 0.1 g/l, L-tryptophan 0.24 g/l, 25 ml medium in a 250 ml flask, with an inoculumn size of 10% (v/v), 72 h of cultivation with 250 rpm at $25^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.63-70
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• 2017

A functional screen of 60,672 fosmid metagenomic clones amplified from marine sediment obtained from the Dokdo islets in Korea identified the gene EstES1, whose product, EstES1, displayed lipolytic properties on tributyrin-supplemented media. EstES1 is a 576 amino acid protein with a predicted molecular weight of 59.4 kDa including 37 N-terminal leader amino acids. EstES1 exhibited the highest sequence similarity (44%) to a carboxylesterase found in Haliangium ochraceum DSM14365. Phylogenetic analysis indicated that EstES1 belongs to a currently uncharacterized family of lipases. Within the conserved domain, EstES1 retains the catalytic triad that consists of the consensus penta-peptide motif, GESAG. EstES1 demonstrated a broad substrate specificity toward the long acyl group of ethyl esters (C2-C12), and its optimal activity was recorded toward p-Nitrophenyl butyrate (C4) at pH 9.0 and $40^{\circ}C$ (specific activity of 255.4 U/mg). The enzyme remained stable in the ranges of $60-65^{\circ}C$ and pH 9.0-10.5 and in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, EstES1 has potential for use in industrial applications involving high temperature, organic solvents, and/or alkaline conditions.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.2
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• pp.170-174
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• 1999

Proessing conditions for fermented and dried sauces with the underutilized fishes were investigated. Hair tail, gizzard shad, and kangdale were hydrolyzed at $60^{\circ}C$ for 6 hours using $4\%$ Alcalase, and their hydrolysates were separted by molecularporous membrane. The hydrolytic ratios of hair tail, gizzard shad, and kangdale were estimated to be $84.2\%$, $83.6\%$ and $85.1\%$, respectively. Amino nitrogen recoveries were determind to be $73.1\~73.9\%$ by a membrane with molecular weight cutoff 100 dalton and $91.7\~92.5\%$ by a membrane with 500 dalton. Ultrafiltration was very efficient means for removing bitter taste. With the additions of $2\%$ glucose, $4\%$ lactose and $4\%$ skim milk, product yields of hair tail, gizzard shad, and kangdale were determind to be $16.4\%,\;17.2\%$ and $17.0\%$, respectively. Water adsorption rates of hair tail and kangdale showed $5.0\~9.2\%$ and $5.5\~9.6\%$, respectively, under Aw 0.52$\~$0.94. Contents of total nitrogen in the fermented and dried sauces prepared with hair tail, gizzard shad and kangdale were $3.9\%,\;4.1\%$ and $3.7\%$, respectively, and those of amino nitrogen were $3.2\%,\;3.4\%$ and $3.1\%$, respectively. In the fermented and dried sauces prapared with hair tail, gizzard shad and kangdale, the hygroscopities at Aw 0.88 were $6.9\%,\;7.5\%$ and $6.8\%$, respectively, and solubilities under dissolved in water for 30 minutes were $84.6\%,\;83.6\%$ and $93.8\%$, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.6
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• pp.547-557
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• 1986

The characteristics of the three alkaline proteinases, Enz. A, B and C, from the pyloric caeca of mackerel have been investigated. The optimum condition for the activity of the Enz. A, B and C was pH 9.4, 9.8 and 9.8 at $45^{\circ}C$ for $2\%$ casein solution, and was pH 9.2 10.2 and 9.8 at $45^{\circ}C$ for $5\%$ hemoglobin denatured by urea, respectively. Enz. A, B and C by heat treatment at $50^{\circ}C$ for 5 min were inactivated 90, 33 and $37\%$, respectively, over the original activity. The reaction rate of the three alkaline proteinases was constant to the reaction time to 40 min in the reaction condition of $2{\mu}g/ml$ of enzyme concentration and $2\%$ casein solution. The reaction rate equation and Km value against casein substrate determined by the method of Lineweaver and Burk were: Enz. A, Y=3.6X and $Km=5.0{\times}10^{-3}\%$; Enz. B, Y=6.0X and $Km=1.0{\times}10^{-3}\%$; Enz. C, Y=4.2X and $Km=3.6{\times}10^{-3}\%$. The three alkaline proteinases were inactivated by $Ag^+$ and $Hg^{2+}$, but activated by $Mn^{2+},\;Sn^{2+}\;and\;Pb^{2+}$, Enz. B and C were remarkably inhibited by the soybean trypsin inhibitor. Molecular weight of Enz. A, B and C determined by SDS-polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration was in the range of $27,500{\pm}2,500,\;20,500{\pm}1,500\;and\;15,250{\pm}250$, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.8
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• pp.1265-1273
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• 2005

The purpose of this study was to prepare accelerated salt-fermented anchovy sauce using a shrimp processing byproducts (head, shell and tail) as a fermenting accelerator, and to investigate its physicochemical and enzymatic properties. Four types of sauces were prepared with 0, 10, 20, and 30$\%$ (w/w) addition of shrimp byproduct and fermented at 24$\pm$2$^{\circ}C$ for 360 days. During fermentation, all four type sauces decreased moisture content (67.5$\%$68.0$\%$ to 64.0$\∼$64.8$\%$) and pH (5.52$\∼$7.10 to 5.03$\∼$6.58), but showed increase in their crude protein (7.0$\∼$8.2 to 10.8$\%$) and volatile basic nitrogen contents (40$\∼$75 to 180$\∼$200 mg/100 g of sauce). The ratio of amino nitrogen to total nitrogen contents of control (0$\%$) and sauce with 10$\%$ shrimp byproducts (10$\%$ sauce) were maximized at 270 days, whereas 20$ \% $ and 30$\%$ added sauces were at 180 days. Endoprotease and exoprotease activities of anchovy sauces added with 20$\%$ and 30$\%$ of shrimp byproducts tend to be higher than those of control (0$\%$) and 10$\%$ addition. Proteolytic activities of sauces at pH 9 were about 2 times higher than those at pH 6. Amidolytic activities for LeuPNA decreased remarkably during fermentation, and control (0$\%$) almost lost their activity at 180 days, while additional sauces were relatively stable. These suggest that alkaline pretense of anchovy and shrimp byproducts as a endoprotease mainly contributed to the fermentation of salt-fermented sauces. The protein molecular weight distribution of sauces indicated 2 groups of peaks (peak 1,>70,000 da and peak 2, 3,000$\∼$29,000 da). As the fermentation proceeded, peak 1 tended to decrease in all of sauces, but peak 2 increased rapidly from 30 to 270 days. Optimum fermentation periods of control and 10$\%$ sauces were 270 days and those of 20$\%$ and 30$\%$ sauce were 180 days. The results suggest that shrimp byproduct can be used as accelerator of salt-fermented sauce.

• Tuberculosis and Respiratory Diseases
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• v.47 no.3
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• pp.347-355
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• 1999

Background: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate($PIP_2$) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate($IP_3$) and diacylglycerol. PLC-${\gamma}1$, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-yl have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by $IP_3$, phosphatidic acid, etc. Very recently, another PLC-${\gamma}1$ activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-${\gamma}1$ activator, AHNAK, in lung cancer specimens and their paired normal. Methods: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract Immunohistochemical stain was performed with primary antibody against AHNAK protein. Results: Twenty-two among twenty-eight lung cancer tissues showed overexpression of AHNAK protein (eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomas). The resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot Conclusion: The overexpression of PLC-${\gamma}1$ activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-${\gamma}1$ act in concerted manner in carcinogenesis.

• KSBB Journal
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• v.13 no.2
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• pp.181-186
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• 1998

In order to obtain high-level production of recombinant human thrombopoietin (rhTPO) in insect cell line, HTI-TN-5B1-4 (TN5), conditions for optimal rhTPO expression such as multiplicity of infection (MOI), the cell density at infection, harvesting time and type of culture method as well as growth media were determined. When TN5 cells were cultured as anchorage-dependent state in 60-mm dish, cell density $2\times^6$ cells,MOI of 10 and Garvesting the culture media at 72 hr post-infection wrere the cinditions for highest rh TPO production. High production of rhTPO was also achieved by using EXPRESS FIVE serum free media rather than SF900II serum free media-1. Anchorage-dependent TN5 cells were adapted as a suspension culture when they were grown in the presence of heparin. TN5 cells were successfully cultured at 0.2 L scale in suspension culture without having aggregation. When TN5 cells were cultured as suspension state, cell density of $0.6\times10^6$ cells/mL, MOI of 1 and harvesting the culture media at 72 hr post-infection, gave the highest yield of rhTPO.