• KSBB Journal
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• v.14 no.1
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• pp.96-102
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• 1999

Marine bacterium Bacillus cereus ASK202 produced an extracellular agarase (E.C.3.2.1.81) which showed a high level of enzyme activity in the presence of agar and agarose. In the optimal culture conditions, the agarase production increased 7.7 folds compared with the one obtained from the basal medium. Agarase production reached upto 160 units/L after 24hr of cultivation in a modified marine medium at $25^{\circ}C$. The degree of purification increased 31.5 folds with 27.8% yield through freeze drying, DEAE Sepharose CL-6B and Superose 6HR 10/30 column chromatography. The molecular weight of the purified agarase was determined to be 90,000 daltons by gel-permeation filteration. Optimal temperature and pH for the enzyme activity were $40^{\circ}C$ and 7.8, respectively. The enzyme was stable up to $50^{\circ}C$ and at a broad pH range of 5.0-10.0. The $\beta$-agarase was activated by $Zn(NO_3)_2$, and was inhibited by $CuSO_4$ and $SnCl_2$. The Km and Vmax values of this enzyme for agarose as a substrate was $2.4mg/m\ell$ and 13.6 mg/m$\ell$, respectively.

• Biomolecules & Therapeutics
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• v.23 no.2
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• pp.189-194
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• 2015

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 $1A2^*8$, R456H; $^*15$, P42R; $^*16$, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ~ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers ($k_{cat}$) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency ($k_{cat}/K_m$) increased up to 2.5 fold with a slight increase of its $K_m$ value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.15-21
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• 1997

We studied the characteristics of extracellular autolysins from Moraxella sp. CK-1 which has been known to lyse the cyanobacterial cell walls. This bacterium excreted autolysins from the early exponential growth phase. These enzymes showed optimal action condition of 60-$70^{\circ}C$ and pH 9.0. Whereas $Na^{+}$, $K^{+}$ and $Li^{+}$ ions exhibited positive effect on the enzyme activity, $Ba^{2+}$, $Mg^{2+}$, $Ca^{2+}$ and $Mn^{2+}$ ions exhibited negative effect. Especially, $Fe^{2+}$ and $Cu^{2+}$ ions almost completely suppressed the activity. Four extracellular autolysins of 30, 32, 38 and 41 kDa were detected in renaturing SOS-PAGE gel containing 0.2% heat-killed Micrococcus luteus cells as substrate. Among these 4 autolysins, 2 enzymes of 32 and 41 kDa distributed in the culture medium throughout the experimental time, but the 38 kOa enzyme diminished and 30 kOa began to appear at mid-exponential growth phase. When SOS-insoluble peptidoglycan of M. luteus was treated with the autolysins of Moraxella sp. CK-l, the concentration of free amino groups in reaction mixture increased. This indicates that the autolysins are N-acetylmuramyl-L-alanine amidase or endopeptidase.

• Journal of the Korean Wood Science and Technology
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• v.15 no.4
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• pp.18-25
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• 1987

The cellulolytic activities of Aspergillus fumigatus KC-1 was investigated, which showed the most active producer of cellulase among the 256 strains of cellulose-decomposing microorganisms screened in our laboratory. All the examined cellulolytic activities (filter paper-, Avicel-, cotton-, CMC-, salicin- and xylansaccharifying activity) in a culture of A. fumigatus KC-1 grown on 1% popular sawdust pretreated with peroxide alkaline reached a maximum within 4-5 days. The optimum pH and temperature for the enzymatic activity was found to be pH 4.5 and $60^{\circ}C$ respectively. The sawdust of poplar wood delignified with 1% NaOH and 20% peracetic acid succesively recorded the highest hydrolysis rate in the tests of enzymatic saccharification. The major end product of hydrolysis of poplar wood with the cellulolytic enzymes obtained from A. fumigatus KC-1 was glucose with small amount of cellobiose and xylose. It can be concluded from these results that A. fumigatus KC-1 is an advantagous source of a cellulase that is capable of hydrolyzing cellulose to glucose rapidly. The influence of degree of delignification, substrate size and its concentration on the rate of hydrolysis of poplar wood was also discussed.

• Journal of Periodontal and Implant Science
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• v.41 no.2
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• pp.73-78
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• 2011

Purpose: For periodontal tissue engineering, it is a primary requisite and a challenge to select the optimum types of cells, properties of scaffold, and growth factor combination to reconstruct a specific tissue in its natural form and with the appropriate function. Owing to fundamental disadvantages associated with using a two-dimensional substrate, several methods of seeding cells into three-dimensional scaffolds have been reported and the authors have asserted its usefulness and effectiveness. In this study, we explore the cell attachment of periodontal ligament fibroblasts on nanohydroxyapatite (n-HA) scaffold using avidin biotin binding system (ABBS). Methods: Human periodontal ligament fibroblasts were isolated from the health tooth extracted for the purpose of orthodontic procedure. HA nanoparticles were prepared and $Ca(NO_3)_2-_4H_2O$ and $(OC_2H_5)_3P$ were selected as precursors of HA sol. The final scaffold was 8 mm in diameter and 3 mm in height disk with porosity value of 81.55%. $1{\times}10^5$ periodontal ligament fibroblasts were applied to each scaffold. The cells were seeded into scaffolds by static, agitating and ABBS seeding method. Results: The number of periodontal ligament fibroblasts attached was greater for ABBS seeding method than for static or agitating method (P<0.05). No meaningful difference has been observed among seeding methods with scanning electron microscopy images. However, increased strength of cell attachment of ABBS could be deduced from the high affinity between avidin and biotin ($Kd=10^{-15}\;M$). Conclusions: The high-affinity ABBS enhances the ability of periodontal ligament fibroblasts to attach to three-dimensionally constructed n-HA scaffold.

• The Korean Journal of Mycology
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• v.15 no.1
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• pp.29-37
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• 1987

Cellulolytic mocrooganisms were isolated from the cellulose-cultural properties. Among them, Aspergillus clavatus with the highest cellulase activity was identified by the morphological characteristics and it's enzyme activities were compared on the various cultural conditions. It was found that the induction of carboxymethylcellulase(CMCase), avicelase and salicinase in CMC liquid media showed the highest enzyme acitivity on five day's cultivation at $30^{\circ}C$ and thereafter their activities were gradually decreased with time. After crude extracellular enzymes precipitated with the 70% saturated ammonium sulfate solution were dialyzed with 20 mM acetate buffer pH 6.0, each crude enzyme was examined. The optimal activities of CMCase and avicelase were both found to be at $50^{\circ}C$ and pH 6.0. Their thermal stability was appeared at the $50^{\circ}C$. CMCase and avicelase had the maxima activities with 1.5% and 2.2% substrate concentration, respectively. The concentration of 5 mM $Mg^{++}$ or $Ca^{++}$ was found to have a maximum cellulase activity and its activity was inhibited with more than 5 mM $Cu^{++}$ and $Zn^{++}$ concentration. Cellulase activity was also inhibited sensitively by the inhibitors such as 2-mercaptoethanol, iodine and sodium azide.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.250-256
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• 2003

Lactic acid bacteria with amylolytic and acid producing activities can ferment starch directly to lactic acid thereby producing a monomer for the production of biodegradable poly lactic acid (PLA). In this study, the strain producing L-lactic acid from soluble starch was isolated from Nuruk. The isolated strain was identified as Enterococcus sp. through its morphological, cultural, biochemical characteristics as well as the 16S rDNA sequence analysis, and named Enterococcus sp. JA-27. Enterococcus sp. JA-27 produced exclusively L-lactic acid from soluble starch as a carbon source. The optimal conditions for the maximum production of L-lactic acid from Enterococcus sp. JA-27 were 30 C, pH 8, 1.5 % soluble starch as a substrate and 3.5 % tryptone as a nitrogen source, 0.1 % $K_2$$HPO_4$, 0.04 % $MgSO_4$. $7H_2$O, 0.014 % $MnSO_4$$.$4$H_2O$, 0.004% $FeSO_4$$.$$7H_2$O. Batch and fed batch culture were carried out and the former was more effective. L-Lactic acid production in the optimum medium was significantly increased in a 7 L jar fermenter, where the maximum L-lactic acid concentration was 3 g/L. For the purification of lactic acid in fermented broth, two stage ionexchange column chromatographies were employed and finally identified by HPLC.

• Horticultural Science & Technology
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• v.19 no.3
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• pp.338-341
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• 2001

A new passive nutrient supplying system (PNS) was designed. The experiment was conducted to compare PNS with automatic hydroponic system (AHS) by investigating the growth characteristics of muskmelon (Cucumis melo L.) in spring and summer. No significant differences in growth characteristics of leaf area and shoot dry weight were observed between PNS and AHS. However, better netting of melon fruit was shown in PNS. Sugar content of melon fruit was also $1.0-2.0Brix^{\circ}$ higher in PNS than in AHS. The suitable substrate for melon culture with PNS was the mixture of perlite 70% and rice hull 30%. These results suggested that new PNS could be introduced to growers without any loss of fruit yield and quality of muskmelon.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.267-273
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• 1982

Among 12 kinds of ginsenosides in ginseng saponin, ginsenoside-Rb$_1$was contained the most abundantly. But ginsenoside-Rd which is similar to ginsenoside-Rb$_1$in structure, was known to be superior to ginsenoside-Rb$_1$pharmaceutically. In order to convert ginsenoside-Rb$_1$into ginsenoside-Rd by microbial enzyme treatment, a Rhizopus sp. was selected among various strais of molds found in rotten ginseng roots. Enzyme was prepared from the extract of wheat bran koji culture by ammonium sulfate precipitation (1.0 sat'd) and succeeding ammonium sulfate fractionation method (0.6-0.9 sat'd). For the purpose of use as substrate, saponins were purified by the several purification steps from alcohol extract of red ginseng roots. We obtained the total saponin which was composed of 36.5% of ginsenoside Rb$_1$, 12.2% of ginsenoside-Rd and other ginsenosides. For increase of ginsenoside-Rb$_1$ component ratio, we also obtained further purified ginsenoside-Rb group saponin containing 54.5% of ginsenoside-Rb$_1$, 1.1% of ginsenoside- Rd and other ginsenosides from purified the total saponin. In the enzymatic reaction system including the total saponin or the ginsenoside-Rb group saponin, we confirmed the specific conversion of ginsenoside-Rb$_1$to ginsenoside-Rd proportionally and no change of any other ginsenoside patterns by thin layer chromatography and high performance liquid chromatography.

• Journal of Applied Biological Chemistry
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• v.52 no.4
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• pp.157-162
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• 2009

An agar-liquefying bacteria (SC-22), which produces a diffusible agarase that caused agar softening around the colony was isolated from Daecheong lake in Korea. Chemotaxanomic and phylogenetic analyses based on 16S rRNA gene sequences revealed the strain was classified as Cellvibrio mixtus SC-22. The isolate SC-22 showed maximal extracellular agarase activity with 58.5 U/mL after 48 h cultivation in the presence of 0.2% agar. It was observed that the isolate produced two kinds of extracellular and three kinds of intracellular isoenzymes. The major agarase was purified from the culture filtrate of agarolytic bacteria by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The molecular mass of the purified enzyme was estimated to be 25 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 7.0 and $50^{\circ}C$, respectively. The agarase activity was activated by $Fe^{2+}$, $Na^+$ and $Ca^{2+}$ ions while it was inhibited by $Hg^{2+}$, $Mn^{2+}$ and $Cu^{2+}$ at 1 mM concentration. The predominant hydrolysis product of agarose by the enzyme was galactose and disaccharide on TLC, indicating the cleavage of $\beta$-1,4 linkage in a random manner. The enzyme showed high substrate specificity for only agar and agarose among various polysaccharides.