• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.206-211
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• 2004

Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter ＋ exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1115-1119
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• 2001

Eight Escherichia coli cells with aerobic growth deflects were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created transcriptional fusion to lacZY. Two of these mutants, CLIO and CLl2, were irradiated with UV to obtain specialized transducing phages. The phages that took out the neighboring chromosomal DNA of the related gene responsible for deflective aerobic growth were identified. The in vivo cloned chromosomal sequence revealed that the mutated gene of CLIO was located at min 34.5 on the Escherichia coli linkage map and 1,599,515 on the physical map. The physical map indicated that there were 7 cistrons in the operon. We named this operon oxg10. The promoter sequence of oxg10 exhibited a possible binding site far SoxS, a transcriptional regulator that activates the transcription of various SoxRS regulon genes. Transferring the oxg10:: ${\lambda}placMu53$ mutation into the wild-type strain, RZ4500, resulted in the inhibition of normal aerobic growth, while the salute mutation in strain MO inhibited aerobic cell growth completely. The full operon sequences of oxg10 were cloned from the Excherichia coli genomic library. The mutated gene of CLl2 was identified to be a sucA gene encoding the ${\alpha}$-ketoglutarate dehydrogenase El component in the TCA cycle.

• Food Science and Biotechnology
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• v.18 no.2
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• pp.500-505
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• 2009

The fibrinolytic enzyme, subtilisin D5, was purified from the culture supernatant of the isolated Bacillus amyloliquefaciens DJ-5. The molecular weight of subtilisin D5 was estimated to be 30 kDa. Subtilisin D5 was optimally active at pH 10.0 and $45^{\circ}C$. Subtilisin D5 had high degrading activity for the A$\alpha$-chain of human fibrinogen and hydrolyzed the $B{\beta}$-chain slowly, but did not affect the $\gamma$-chain, indicating that it is an $\alpha$-fibrinogenase. Subtilisin D5 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the serine protease. The specific activity (F/C, fibrinolytic/caseinolytic activity) of subtilisin D5 was 2.37 and 3.52 times higher than those of subtilisin BPN' and Carlsberg, respectively. Subtilisin D5 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first 15 amino acid residues of the N-terminal sequence of subtilisin D5 are AQSVPYGISQIKAPA; this sequence is identical to that of subtilisin NAT and subtilisin E.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.976-983
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• 2003

Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1047-1053
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• 2005

Cyclosporin A binding protein type-19 kDa peptidyl-prolyl cis/trans isomerase (PPIases, EC 5.2.1.8) of Euglena gracilis was purified and some of its biochemical characters were elucidated. Purification of the PPIase was achieved by employing a series of steps involving ammonium sulfate precipitation, Superdex G-75 gel filtration chromatography, Mono­Q anion and Mono-S cation exchange chromatographies, and Superdex S-200 gel filtration chromatography on FPLC. Purified PPIase had a specific activity of 8,250 units/mg, showing a 27-fold increase compared with that of cell-free extract of Euglena gracilis. The enzyme consisted of a single polypeptide chain with a molecular mass of 19 kDa. It showed high substrate specificity to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and $k_{car}/K_{m}$, for this substrate was found to be $61.19{\times}10^5/sec$. The isomer distributions were investigated at an equilibrium of seven different peptide substrates, varying Xaa in Suc-Ala-Xaa-Pro-Phe-p-nitroanilide in dimethylsulfoxide. The cis/trans equilibrium constants were estimated to be from 0.14 (Ile) to 0.63 (Gly), which correspond to $12.00\%\;to\;38.52\%$ of the cis population, respectively, under experimental condition. The enzyme was highly sensitive to the immunosuppressive ligand cyclosporin A, but not to other immunosuppressants such as FK506 and rapamycin. Thus, it appears to belong to the class of cyclophilin.

• Korean Journal of Food Science and Technology
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• v.22 no.3
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• pp.285-289
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• 1990

Scoring difference test was applied for determining the organoleptic characteristics of mushrooms which was ${\gamma}-irradiated$ in connection with the shelf-life extension. Irradiation at 1 to 3kGy caused a significant softening of mushroom tissue (p<0.05), but it showed no immediate influence on the overall appearance, flavor and taste of mushrooms. After 17 days of storage at $9{\pm}1^{\circ}C\;and\;80{\pm}7%$ RH, 2 to 3 kGy-irradiated mushrooms were significantly better than the nonirradiated control in the sensory properties such as appearance, texture, flavor and taste (p<0.01). Organoleptic scores have shown that the irradiated samples are acceptable more than 17 days, compared to 7 days in the nonirradiated control.

• BMB Reports
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• v.29 no.6
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• pp.519-524
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• 1996

Peptidylprolyl cis-trans isomerase (PPlase) catalyzes the cis-trans isomerization of prolyl peptide and facilitates the folding of cellular proteins and peptides. PPlase consists of two distinct immunophilins, each specifically binding to the immunosupressive drug cyclosporin A (CsA) or FK506, respectively. A PPlase was isolated and partially purified from porcine spleen. The molecular weight of porcine spleen PPlase was determined to be ~14,000 on the basis of SDS-PAGE. The purified enzyme was strongly inhibited by FK506, but not by CsA. The inhibition constant and the true concentration of enzyme preparations were determined by active site titration using the tight binding inhibitor FK506: $K_{i}=18.7$ nM and $E_{t}=172$ nM. The equilibrium ratio of conformer. [cis]/[trans], of prolyl peptide substrates (N-Suc-Ala-Xaa-Pro-Phe-p-NA) in anhydrous trifluoroethanol/LiCl solvent system varied from 0.24 to 0.85 depending on the nature of Xaa. Overall. in this solvent-salt system, the populations of the cis conformer of substrates in equilibrium are higher than in an aqueous solution so that the substantial error caused by high background absorption can be reduced. The reactivities of porcine spleen PPlase are shown to be highly sensitive to changes in the structure of substrates. Thus, $k_{cat}/K_m$ value for the most reactive substrate (Xaa Leu) is $4.007+10^{6}M^{1}s^{1}$ and, is 2,636 fold higher than that for the least reactive peptide substrate tested, Xaa=Glu.

• Journal of the Korean Chemical Society
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• v.27 no.6
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• pp.449-456
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• 1983

The facile route of preparing S-2-(${\omega}$-aminoalkylamino) ethyl dihydrogen phosphorothioates, potential chemical radioprotectants, have been studied. Intermediate 3-(2-phthalimidoethyl)-2-oxazolidinone was prepared by a reaction of potassium phthalimide and 3-(2-bromoethyl)-2-oxazolidinone, which was obtained through the alkaline ring closure of a mixture of carbonate and 2,2'-dibromo diethylamine prepared from diethanolamine. This was converted to N-[2-(2-bromoethylamino)ethyl] phthalimide hydrobromide by 30% HBr(gas) in acetic acid and N-(2-bromoethyl)-1,2-ethanediamine dihydrobromide was obtained by reacting the hydrobromide with a solution of HBr-HOAc. N-(2-bromoethyl)-1,3-propanediamine dihydrobromide could be prepared through the Cortese treatment of 2-(3-aminopropylamino) ethanol, which was prepared by a reaction of 1,3-diaminopropane and 2-chloroethanol. These dihydrobromides were treated by sodium thiophosphate in DMF to result S-2-(${\omega}$-aminoalkylamino) ethyl dihydrogen phosphorothioates. The characteristics of each reaction path were discussed in regards to reaction conditions and overall yields and a facile route of preparing each derivative was proposed.

• Korean journal of food and cookery science
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• v.4 no.2
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• pp.21-30
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• 1988

To study the optimum fermentation and aging conditions for preparation of Andong Sikhe which is consumed in the northern Kyungbuk province as a traditional beverage, Sikhe were fermented and aged at $12^{\circ}C$ for 48, 60 and 72hrs and fermented at 40, 50. and $60^{\circ}C$ for 2, 3, and 4hrs and then aged at $12^{\circ}C$ for 20 hrs, Non-volatile organic acids and free sugars were analyzed by GC and HPLC, respectively, and sensory test was performed. Sikhe fermented at $40^{\circ}C$ for 3hrs and aged at $12^{\circ}C$ for 20hrs was evaluated the most favorable one by sensory test. This Sikhe had the lowest pH 4.6~4.7 among sample and 14.6% of sugar. The sugar content was lower than those of Sikhe Fermented at $50^{\circ}C$ and $60^{\circ}C$. In organic acid analysis, the concentration of malate 67.53%/mg in this Sikhe was the highest. Lactate, succinate and maleate were produced more in this Sikhe than in any other ones. Oxalate which was existed before fermentation was not found in the Sikhe, and a small amounts of citrate and fumarate were produced. In sugar analysis, maltose was dominant 11.56%, glucose 1.25% and fructose 0.45%.

• Journal of Aquaculture
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• v.11 no.2
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• pp.133-140
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• 1998

A 20-week growth trial was conducted in flow-through aquarum system to investigate the practical dietary carbohydrate sources for juvenile abalone (Haliotis discus hannai). Four replicate grops of the abalone averaging 0.125g were fed one of eight diets containing 24.2% wheat flour (WF), 20% dextrin (DEX), 20% sucorse (SUC), 10% $^{\alpha}$-potato starch+10% $^{\beta}$-potato starch (ab-S), 15% $^{\alpha}$-potato starch (a-S15), 20% $^{\alpha}$-potato starch (a-S20), 25% $^{\alpha}$-potato starch (a-S25), or mixture (MIX) with practical ingredients such as soybean meal, corn gluten meal, cotton seed meal and heat flour. In addition, these formulated diets were compare with macroalgae such as dried sea mustard Undaria (D-SM) or dried sea tangle Laminaria(D-ST). Survival rate, weight gain, shell growth and soft body weight of abalone were not significantly affected by the different dietary carbohydrate sources (P>0.05), whereas those fed a-S15 diet were slightly low. These values of abalone fed D-ST were lowest (P<0.05), followed by those fed D-SM. Lipid contents of soft body from abalones fed a-S25, D-ST or D-SM were significantly lower than those of abalone fed other diets (P<0.05). These data indicate that abalone can equally utilize any carbohydrate sources used in this study.