• Journal of the Korean Chemical Society
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• v.39 no.9
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• pp.734-740
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• 1995

The primary and secondary structures of Xanthomonas palargonii 5S rRNA were determined. The higher order structure of the 5S rRNA was also analysed using several ribonucleases and chemical probes, such as ethylnitrourea, Pb2+, dimethylsulfate and diethyl pyrocarbonate. Ethylnitrourea was used for probing the phosphate groups involved in the tertiary interactions in the 5S rRNA. Nucleotides G72, A73, G75, A78, G98, G100 and A101 in unstable helical region d, nucleotides C36, C37, A39, and C41 in loop c, nucleotides A29 and G33 in stem C became resistant to ethylnitrourea modification in the presence of Mg2+. On the basis of findings from chemical and enzymatic studies, and also Pb2+-induced cleavages, it was concluded that region b-C and unstable helical region d may act as hinges in the folding of 5S rRNA.

• Journal of Microbiology and Biotechnology
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• v.32 no.7
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• pp.885-891
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• 2022

Plant-based protein sources such as soybean meal have low digestibility and are generally promoted accumulation of undigested proteins into the intestine by enzymatic treatments. Moreover, potential intestinal pathogens ferment undigested proteins, producing harmful substances, such as ammonia, amines and phenols, leading to an overactive immune response and diarrhea in weaned pigs. As a solution, dietary proteases hydrolyze soybean-based antinutritive factors, which negatively affect immune responses and gut microbiota. In this study, we investigated the effects of dietary proteases (PRO) in a low-crude protein (CP) commercial diet on the immune responses and gut microbiota of weaned pigs. The experimental design consisted of three dietary treatments: a commercial diet as a positive control (PC; phase1 CP = 23.71%; phase 2 CP: 22.36%), a lower CP diet than PC as negative control (NC; 0.61% less CP than PC), and NC diet supplement with 0.02% PRO. We found that PRO tended to decrease the frequency of diarrhea in the first two weeks after weaning compared with PC and NC. In addition, pigs fed PRO showed decreased TNF-α and TGF-β1 levels compared with those fed PC and NC. The PRO group had a higher relative proportion of the genus Lactobacillus and lower levels of the genus Streptococcus than the PC and NC groups. In conclusion, the addition of PRO to a low CP commercial weaned diet attenuated inflammatory responses and modified gut microbiota in weaned pigs.

• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Clean Technology
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• v.22 no.3
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• pp.175-180
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• 2016

Tyrosinases catalyze the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to o-quinone (diphenolase activity), which are mainly involved in the modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (DOPA) and DOPA/DOPAquinone-derived intermolecular cross-linking. Previously, we obtained a slightly acidic and cold-active tyrosinase, tyrosinase-CNK, by our recombinant protein approach. The enzyme showed optimal activity at pH 6.0 and 20 ℃ with an abnormally high monophenolase/diphenolase activity ratio and still had approximately 50% activity compared with the highest activity even in ice water. Here, we investigated reaction stability of the recombinant tyrosinase-CNK as a psychrophilic enzyme. The enzyme showed remarkable thermal stability at 0 ℃ and the activity was well conserved in repeated freeze-thaw cycles. Although water-miscible organic solvent as reaction media caused the activity decrease of tyrosinase-CNK as expected, the enzyme activity was not additionally decreased with increased concentration in organic solvents such as ethanol and acetonitrile. Also, the enzyme showed high salt tolerance in chaotropic salts. It was remarkably considered that 2⁺ metal ions might inhibit the incorporation of Cu²⁺ into the active site. We expect that these results could be used to design tyrosinase-mediated enzymatic reaction at low temperature for the production of catechols through minimizing unwanted self-oxidation and enzyme inactivation.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.345-355
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• 1991

In order to effectively utilize the by-products of sea-food, the utilization of enzyme-modified flounder(Limanda aspera) skin gelatin as an emulsifier was investigated. In the experiment, the gelatin was extracted from the flounder skin with the heat-treatment at $60^{\circ}C$ and in pH 5.0 for 3 hrs with four volumes of distilled water and emulsifiers were enzymatically modified L-leucine alkyl esters$(L-leucine-OC_n$ : n= 2, 4, 6, 8 and 10) to the gelatin$(EMFSG-C_2,\;EMFSG-C_4,\;EMFSG-C_6,\;EMFSG-C_8,\;EMFSG-C_{10})$ for improving the functional properties such as emulsifying activity, emulsifying viscosity, whippability, electric conductivity, critical micelle concentration and interface tension, etc. Also, the functional properties of the L-leucine alkyl ester modified gelatins were compared with those of Tween-60 as reference. Molecular weights of the enzymatically modified flounder skin gelatin(EMFSG) were 20.5kDa. in $EMFSG-C_2.\;19.5 kDa.\;in\;EMFSG-C_4\;and\;16.5kDa.\;in\;EMFSG-C_6,\;EMFSG-C_8$ and $EMFSG-C_{10}$. respectively. Emulsifying activity and emulsifying viscosity in the modified gelatins were risen with increase of carbon number of the introduced L-leucine alkyl esters. Among the modified gelatins, $EMFSG-C_6$ exhibited the highest emulsifying stability and foaming stability, whereas $EMFSG-C_8$ showed the highest whippability. The electric conductivities of the all $EMFSG-C_n$ were linearly risen to critical micelle concentration(CMC) , therefore $EMFSG-C_{10}$ exhibited the lowest CMC value and interface tension, and dense particles in the microscopic observation. In conclusion, the best quality in functional properties was assured on $EMFSG-C_{10}$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.38 no.4
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• pp.479-485
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• 2009

The acidolysis was performed to produce structured lipid with palm mid fraction (PMF) and stearic acid for 7, 24, and 36 hr at $70^{\circ}C$. The reaction was catalyzed by lipozyme TLIM (immobilized lipase from Thermonyces lanuginosa, amount of 10% and 20% by weight of total substrates) in the shaking water bath. The reaction conditions for maximum incorporation of stearic acid on the structured lipid were obtained when molar ratio of PMF and stearic acid was 1:2; concentration of lipozyme TLIM was 20wt%; reaction temperature was $70^{\circ}C$; and reaction time was 36 hr. After reaction under this condition, incorporation of stearic acid in the structured lipid was obtained up to 36.3% while the major components of triacylglycerol were 1,2-dipalmitoyl-3-stearoylglycerol (PPS, 28.19 area%), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS/PSO, 20.70 area%) and 1-palmitoyl-2,3-distearoylglycerol (PSS, 18.13 area%). However, the fatty acid composition at the sn-2 position suggested that the positional specificity of lipozyme TLIM was not observed due to the acyl migration.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.171-177
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• 2009

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

• Korean Journal of Heritage: History & Science
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• v.40
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• pp.387-411
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• 2007

The analysis of ancient DNA (aDNA) has become increasingly considerable anthropological, archaeological, biological and public interest. Although this approach is complicated by the natural damage and exogenous contamination of a DNA, archaeologists and biologists have attempted to understand issues such as human evolutionary history, migration and social organization, funeral custom and disease, and even evolutionary phylogeny of extinct animals. Polymerase chain reaction(PCR) is powerful technique that analyzes DNA sequences from a little extract of an ancient specimen. However, deamination and fragmentation are common molecular damages of aDNA and cause enzymatic inhibition in PCR for DNA amplification. Besides, the deamination of a cytosine residue yielded an uracil residue in the ancient template, and results in the misincorporation of an adenine residue in PCR. This promotes a consistent substitution (cytosine thymine, guanine adenine) to original nucleotide sequences. Contamination with exogenous DNA is a major problem in aDNA analysis, and causes oversight as erroneous conclusion. This report represents serious problems that DNA modification and contamination are the main issues in result validation of aDNA analysis. Now, we introduce several criterions suggested to authenticate reliance of aDNA analysis by many researchers in this field.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.147-153
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• 2004

Protein modification by N-acetyl-${\beta}$-D-glucosamine (O-G1cNAc) on the hydroxyl groups of Ser or Thr ubiq-uitously occurs in eukaryotic cells and is involved in many cellular phenomena. The level of O-G1cNAc-mod-ified protein is regulated by OGT and O-GlcNAcase enzymes. We have tried to produce recombinant O-GlcNAcase in E. coli as an effort to establish in vitro screening system for modulators of O-GlcNAcase. The culture conditions for improvement of O-GlcNAcase productivity, were as follows: induction temperature, $30^{\circ}C$; the concentration of L-arabinose, 0.02% and induction time, 5 hr. Under these culture conditions, E. coli cells containing O-GlcNAcase gene had no enzyme activity until up to 3 hr culture. However, O-GlcNAcase activity dramatically increased from 3 to 5 hr culture. It almost maintained the same level after 5 hr culture. Western blot analysis verified the amount of expressed O-GlcNAcase increased with culture time, being con-sistent with activity data. The optimal reaction condition determined in this study was as follows: protein quan-tity, $5{\mu}g$; reaction time, 30 min; reaction temperature, $45^{\circ}C$; substrate concentration, 2 mM; reaction pH, 6.5. Methanol had little effect on O-GlcNAcase activity and 90% of activity were retained at 10%. Only 15% resid-ual activity were detected at 5% of chloroform.

• Journal of Dairy Science and Biotechnology
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• v.33 no.3
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• pp.179-196
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• 2015

Because of our aging population, there is increasing concern about the impact of dementia and age-related cognitive decline. Intense research efforts on effective dietary interventions for the prevention or amelioration of dementia and age-related cognitive decline have indicated that dairy products affect physiological health and potentially healthy brain function during aging. Milk is a rich source of proteins and peptides with nutritional and immunotropic activities. The preparation of biologically active proteins and peptides generally requires enzymatic degradation, chemical modification, or the addition of specific co-factors. Milk-derived preparations are widely available in the food industry in the form of hygiene products and infant formulas. However, milk-derived products could also be applied as preventive or therapeutic measures for a wide-range of pathological conditions not only in neonates and infants but also in adults, including the elderly. Because they have no adverse side effects, milk-derived proteins and peptides could be used as a supplementary treatment for dementia and age-related cognitive decline.