• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.129-134
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• 1987

Aspergillus sp. (MK-24) producing a biological active substance that inhibited the venom proteinase activity was isolated from soil. The substance also inhibited the activity of trypsin and coagulation of blood, but did not inhibit papain, $\alpha$-chymotrypsin and pepsin. The substance was partially purified from culture filtrate by precipitaion with acetone, and by chromatography of DEAE-Sepadex A-50 column and Amberlite IRC-50 ion exchange. The inhibitory substance was stable in the wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but not stable at $65^{\circ}C$ in the alkaline pH. Only 12% of the activity was decreased by the heat treatment at 10$0^{\circ}C$ for two hours. The inhibition on venom proteinase (Agkistrodon bromohoffi brevicaudus) was a mixed type. The inhibitory activity depended on the preincubation time and completely depressed by cupric, zinc and cobalt ions. The inhibition on the venom proteinase was appeared strongly on casein but not on ovalbumin or hemoglobin as a substrate.

• Journal of the East Asian Society of Dietary Life
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• v.7 no.3
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• pp.289-300
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• 1997

Newborn dog chymosin was extracted from the stomachs of dogs of 2 weeks of age, and was purified by ion exchange chromatography. Half of the sequence was determined by amino acid sequencing and the complete sequence was deduced from a cloned chymosin cDNA Results showed that the zymogen showed 79% sequence identity with calf prochymosin and 54% identity with porcine pepsinogen A The size of the propart and location of the residue which becomes the amino-terminus in the active enzyme was the same in the prochymosins. The maximum general proteolytic activity at pH 3.2 of newborn dog chymosin was 3-4% of that of porcine pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of newborn dog chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.2 of stomach extracts of individual dogs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or amino-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and newborn dog chymosin differ in the following positions (porcine pepsin numbering, subsites in parentheses) : Ser 12 Thr(S$_4$), Leu 30 Val(S$_1$/S$_3$), His 74 Gln(S'$_2$), Val 111 Ile(S$_1$/S$_3$), Lys 220 Met(S$_4$). With regard to the low general proteolytic activity of newborn dog chymosin, the substitution Asp303 Val relative to calf chymosin may contribute to an explanation of this.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.1
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• pp.97-100
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• 1992

The optimum condition for the extraction of antimicrobial substance from Lithospermi radix was investigated. For the purpose of obtaining basic data for the development of natural preservatives and for the prevention of food poisoning accidents, its antimicrobial activity was tested against several kinds of saprophytic microbes and food poisoning bacteria. The optimum condition for extraction of antimicrobial substance was to steep Lithospermi radix into 95% ethanol for 24 hours at room temperature. Antimicrobial activity was observed at the pH range $5.0{\sim}8.0$, but its activity became stronger at acidic condition. The result of ion exchange chromatography was showed that the antimicrobial activity of anionic portion was more apparent than that of cationic portion. The antimicrobial activity against Gram positive bacteria was stronger than that of Gram negative bacteria and the growth of food poisoning bacteria such as S. aureus and V. parahaemolyticus was inhibited in the concentration of 0.1% for 48 hours. As for mold and yeast, the growth of some kinds of these organisms was inhibited in the concentration of 0.1 % for 48 hours and the growth of nearly all the fungi was inhibited in the concentration of 0.15% for 96 hours.

• YAKHAK HOEJI
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• v.52 no.6
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• pp.419-425
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• 2008

A qualitative and quantitative analytical method was developed for detection of methamphetamine (MA) and its main metabolite amphetamine (AM) in oral fluid. Oral fluids of eleven drug abusers were provided by Police, specimens were collected by stimulation with a cotton swab treated with 20 mg of citric acid ($Salivette^{(R)}$; Sarstedt, USA). As the preliminary test, oral fluid samples were screened for amphetamines by Fluorescence Polarization Immunoassay (TDxFLx, Abbott Co.). Extraction for MA was performed using solid-phase extraction (SPE) by $RapidTrace^{TM}$ (Zymark, USA) with mixed mode cation exchange cartridge, CLEAN $SCREEN^{(R)}$ (130 mg/3 ml, UCT) after dilution with phosphate buffer. Samples were evaporated and derivatized by pentafluoropropionic acid anhydride (PFPA). Quantitation of MA and AM was performed by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring (SIM), the quantitation ions were m/z 204 (MA), 208 (MA-$D_5$), 190 (AM) and 194 (AM-$D_5$). The selectivity, linearity of calibration, limit of detection (LOD) and quantification (LOQ) within- and between day precision, accuracy and recoveries were examined as parts of the method validation. All oral fluid samples gave positive results to immunoassay for MA (cut-off level, 50 ng/ml as d-amphetamine). Concentrations of MA and AM by GC-MS in eleven samples were ranged 104.2${\sim}$4603.3 ng/ml and 32.4${\sim}$268.6 ng/ml, respectively. Extracted calibration curves of MA and AM were linear over the two concentration range of 1${\sim}$100 and 50${\sim}$1000 ng/ml with correlation coefficient of above 0.999. LOQ of MA and AM was 1 and 3 ng/ml, respectively. The intraand inter-day run precisions (CV) for MA and AM were less than 10%, and the accuracies (bias) for MA and AM were also less than 10% at the two different concentrations 5 and 100 ng/ml at low calibration range, 50 and 1000 ng/ml at high calibration range. The absolute recoveries of MA and AM at low and high calibration ranges were more than 82% and 75%, respectively. In this study the qualitative and quantitative analytical method of MA in oral fluid was established. Oral fluid testing may detect drug use in past hours because of its shorter detection window than urine, and be useful in post-accident situations. So oral fluids will be most useful for testing drug abuse in the driving under the influence of drug (DUID) as the alternative specimens of urine.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.133-148
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• 1987

Gibberellin(GA) 3-$\beta$ hydroxylation is very important for the shoot elogation in the higher plants, since only 3$\beta$-hydryoxylated GAs promote shoot elogation in several plants. Fluctuation of 3$\beta$-hydryoxylase activity was examined during seed maturation using two cultivars of , P. vulgaris, Kentucky Wonder (normal) and Masterpiece (dwarf). Very immature seeds of both cultivars contain high level of 3$\beta$-hydroxylase activity (per mg protein). Both cultivars showed maximum of enzyme activity (per seed) in the middle of their maturation process. Gibberellin 3$\beta$-hydroxylase catalyzing the hydroxylation of GA20 to GA1 was purified 313-fold from very early immature seeds of P. vulgaris. Crude soluble enzyme extracts were purified by 15% methanol precipitation, hydrophobic interaction chromatogrphy, DEAE ion exchange column chromatography and gel filtration HPLC. The 3$\beta$-hydroxylase activity was unstable and lost much of its activity duting the purification. The molecular weight of purified enzyme was extimated to be 42, 000 by gel filtration HPLC and SDS-PAGE. The enzyme exhibited maximum activity at pH 7.7. The Km values for [2.3-3H] GA20 and [2.3-3H]GA9 were 0.29 $\mu$M and 0.33 $\mu$M, respectively. The enzyme requires 2-oxoglutarate as a cosubstrate; the Km value for 2-oxoglutarate was 250 $\mu$M using 3H GA20 as a substrate. Fe2+ and ascorbate significantly activated the enzyme at all purification steps, while catalase and BSA activated the purified enzyme only. The enzyme was inhibited by divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Effects of several GAs and GA anaogues on the putrified 3$\beta$-hydroxylase were examined using [3H]GA9 and GA20 as a substrates. Among them, GA5, GA9, GA15, GA20 and GA44 inhibited the enzyme activity. [13C, 3H] GA20 was converted by the partially purified enzyme preparation to [13C, 3H]GA1, GA5 and GA6, which were identified by GC-MS, GA9 was converted only GA4, GA15 and GA44 were converted to GA37 and GA38, respectively. GA5 was epoxidized to GA6 by the preparation. This suggests that 3$\beta$-hydroxylation of GA20 and epoxidation of GA5 are catalyzed by the same enzyme in P, vulgaris.

• Korean Journal of Food Science and Technology
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• v.16 no.2
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• pp.235-241
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• 1984

Two fractions of pectinesterase from Chinese cabbage were isolated by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The fraction F-A and F-B were purified approximately 340- and 10-fold. The similar salt effects and pH optima (pH 7.5-8.0) were obtained for the two pectinesterase fractions. The maximum activity of both two. fractions were obtained at 20-50mM of divalent rations and at 250mM of monovalent rations. The apparent Michaelis constant of the F-A was 0.01% for citrus pectin. The temperature optima for F-A and F-B were $48^{\circ}$ and $55^{\circ}C$, respectively and both fractions were stable in the region of pH 5.0-8.0 at room temperature. The thermal inactivation of the two fractions followed the first order reaction kinetics. From D and Z-values obtained the thermal resistance of the two fractions were characterized.

• Journal of Life Science
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• v.17 no.10
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• pp.1341-1346
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• 2007

An intracellular ${\beta}-xylosidase$ from Paenibacillus sp. DG-22 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel-filtration chromatography. The molecular weight of the enzyme was measured to be 156,000 by gel filtration and 80,000 by SDS-PAGE, indicating that the enzyme consisted of two identical subunits. The purified enzyme exhibited maximum activity at $65^{\circ}C$ and pH 5.5. It retained 89% of its initial activity up to 60 min at $60^{\circ}C$ and had a half-life of 25 min at $65^{\circ}C$. The enzyme was highly specific for pNPX as the substrate. It showed little or no activity against other p-nitrophenyl glycosides and xylans. The $K_m\;and\;V_{max}$ for pNPX was 0.53 mM and 3.18 U/mg protein, respectively. The ${\beta}-xylosidase$ was strongly inhibited by $Ag^+,\;Fe^{2+},\;Hg^{2+}\;and\;Zn^{2+}$ and slightly activated by DTT. The hydrolysis product from xylobiose, xylotriose, and xylotetraose was xylose.

• KSBB Journal
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• v.25 no.5
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• pp.429-436
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• 2010

Keratinase is widely used in certain industrial applications. The present study sought to improve the culture conditions of Bacillus subtilis SMMJ-2 to facilitate mass production of keratinase. Strain SMMJ-2 was irradiated by ultraviolet light and the resulting isolates were tested for keratinase activity. Isolates displaying elevated keratinase activity were selected and used to determine the optimum temperature (24, 30, 37, 45, $55^{\circ}C$) for bacterial keratinase production during a 4 day incubation period. The highest enzyme activity (55 units/mL/min), from a Bacillus subtilis SMMJ-2 mutant (mutant No. 2) was demonstrated following incubation at $30^{\circ}C$. The effects of carbon and nitrogen sources on keratinase production were confirmed by measuring the enzyme activity from the culture broth of the mutant strain cultured in various media containing different carbon source and nitrogen sources during a 4 day period. The optimal medium composition for producing keratinase consisted of 1% glucose, 0.7% $K_2HPO_4$, 0.2% $K_2HPO_4$, and 1.2% soybean meal. Optimal initial pH and temperature for producing keratinase were 7.0 and $30^{\circ}C$, respectively. Keratinases produced by B. subtilis SMMJ-2 and the mutant No. 2 were purified from the culture broth which used soybean meal as a nitrogen source. Membrane ultrafiltration, DEAE-sephacel ion exchange and Sephadex G-100 gel chromatography were used to purify the enzymes. The purified keratinases from both B. subtilis SMMJ-2 and the mutant No. 2 showed single bands and their molecular weights were estimated as 28 kDa and 42 kDa, respectively on SDS-polyacrylamide gel electrophoresis.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• The Korean Journal of Pharmacology
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• v.31 no.2
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• pp.241-250
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• 1995

Protein B23 is one of the major nucleolar phosphoproteins associated with pre-ribosomal particles, and is localized in the granular region of the nucleolus. Recent studies suggest that protein B23 shuttles between nucleus and cytoplasm and also interacts with HIV Rev. These findings indicate that protein B23 is important in nucleocytoplasmic relationship and viral replication. However, the exact function of protein B23 is not clear yet. In acute nucleolar hypertrophy of rat liver, treated with thioacetamide, there was observed an increase of not only protein B23 but also B23-like protein p45 when anti-B23 monoclonal antibody (MAb) was used for identification. On the basis of the large B23 specific epitope structure composed of 68 amino acids, a hypothesis was formulated to examine that p45 is the pre-B23 resulting from excessive production of B23. In an attempt to investigate the precursor of B23, we analyzed the subcellular fractions and microsomal subfractions. Subsequently, we analyzed the finger printings of B23-like proteins using the tryptic peptide mapping. The results are summarized: 1) Using B23 MAb, we observed the presence of B23-like proteins in nucleolar fraction, nucleoplasmic fraction and microsomal fraction. 2) In the further microsomal subfractionation, we could partially purify B23-like protein in 2M layer of sucrose gradient. 3) When ion exchange chromatography was employed, there were protein species 80kDa(p80), 65kDa(p65) and 60kDa(p60). 4) Based on the tryptic map analysis of $^{125}I$ labeled proteins, the similarity between B23 and p80 was found only in 9 out of 14(B23) and 21(p80) peptides, and difference was found in the remaining peptides. p80 and p60 had 18 common peptides, and all the peptides of p60 were similar to those of p80. From these results, it is proposed that p45 is an abnormal metabolite resulting from carcinogenesis by thioacetamide, and it is not the precursor of B23. In addition, we suggest that p80 may be a precursor of p45.