• 미생물학회지
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• 제24권2호
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• pp.119-126
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• 1986

A periplasmic endoprotease, named protease Pi, was purified to homogeneity from Escherkchia coli by conventional procedure with insulin as substrate. This enzyme degrades insulin and glucagon to trichloroacetic acid-soluble meterials, but shows little or no hydrolysis of bovine serum albumin, casein or globin. Its molecular weight was 110, 000 when determined by gel filtration on Sephacryl S-300 and was 105, 000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be single polypeptide. This snzyme is metalloprotease, since it is completely inhibited by o-phenanthroline and can be activated by addition of divalent metal cations, such as $Mg^{2+}\;and\;Co^{2+}$. It is destinct from protease Ci, a cytoplasmic insulin degrading enzyme, since protease Pi is localized to the periplasm. Since protease Pi selectively degrades GTP cyclohydrolase I, it appears to play a role in the regulation of pteridine biosynthesis.

• 한국미생물·생명공학회지
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• 제27권6호
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• pp.466-470
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• 1999

Protease production and its characteristics were investigated with Mucor racemosus f. racemosus PDA 103 which was isolated from Korean traditional meju. Optimum culture conditions of the strain for the production of the protease in basic medium[bean(Baektae):H2O=1:1(w/v)] were as follows: pH 6, 3$0^{\circ}C$ and 72hrs. Optimum pH and temperature for the enzyme activity of the protease produced by Mucor racemosus f. racemosus were pH 5 and 5$0^{\circ}C$, respectively. The enzyme was relatively stable a pH2.0~5.0 and at temperature below 4$0^{\circ}C$. Phenylmethane-sulfonyl fluoride and Ag+ inhibited the enzyme activity. This indicates that the enzyme is serine protease. Km value was 0.9$\times$10-4M and Vmax value was 5.93$\mu\textrm{g}$/min. This enzyme hydrolyzed casein more rapidly than bovine albumin.

• 미생물학회지
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• 제30권4호
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• pp.299-304
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• 1992

An intracellular protease form cells of Pseudomonas carboxydovorans DSM 1227 grown on carbon monoxide was purified 57-fold in six steps to homogeneity with a yield of 4.3% using azocoll as a substrate. The molecular weight of the enzyme was determined to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed the purified enzyme to be a dimer with two identical subunits of molecular weight 72,000. The enzyme was stimulated by $Mg^{2+}$ but was inhibited completely by $Cd^{2+}$ $Fe^{2+}$ $Hg^{2+}$, and $^Zn{2+}$ The enzyme activity was also inhibited by EDTA, EGTA, phenylmethylsulfonyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl)carbodiimide, iodoacetamide and dithiothereitol. The optimal pH and temperature for the enzyme reaction were found to be 7-8 and 50.deg.C, respectively. Casein and bovine serum albumin were hydrolyzed by the enzyme, but carbon monoxide dehydrogenase was not.

• 한국미생물·생명공학회지
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• 제1권2호
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• pp.99-104
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• 1973

A study on the active center of the acid protease from Penicillium sp. was conducted, and also the milk clotting activity of acid prorease was measured. 1. PCMB failed to influence the proteolytic activity of acid protease, indicating that a reactive sulfhydryl group is not required for the enzymatic activity. 2. $\varepsilon$-amino caproic acid did not show any inhibitory effect on tile proteolytic activity of acid protease. 3. Also 2, 4-dinitro phenol did not show any inhibitory effect on the enzyme activity. 4. Acid protease from Penicillium sp. showed a strong milk clotting activity in the presence of Ca ion. 5. This enzyme had a strong proteolytic activity on various substrate, such as casein, denatured hemoglobin, ovalbumin, denatured bovine muscle protein, denatured percine muscle protein and denatured chicken muscle protein.

• Asian-Australasian Journal of Animal Sciences
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• 제33권6호
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• pp.1023-1033
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• 2020

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

• Asian-Australasian Journal of Animal Sciences
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• 제16권12호
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• pp.1822-1829
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• 2003

Porcine colostrum and milk were separated into the acid-soluble and casein fractions by acidification followed by centrifuge. The acid-soluble fraction of porcine colostrum was further separated by liquid chromatography and anisotropic membrane filtration. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components was determined by incubating bovine trypsin or chymotrypsin in a medium containing their corresponding substrates with or without addition of various amounts of porcine colostrum, porcine milk or their components. The inhibition of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF with the intestinal contents with or without addition of porcine colostrum. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid (TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The inhibitory activity declined rapidly during lactation. It was also found that trypsin and chymotrypsin inhibitory activity and the inhibition on IGF-I and EGF degradation in the acid-soluble fraction were higher than that in the casein fraction. Heat-resistance study indicated that trypsin inhibitors in porcine colostrum survived heat treatments of $100^{\circ}C$ water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heatsensitive. Separation of the acid-soluble fraction of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the trypsin and chymotrypsin inhibitory capacity was mainly due to a group of small proteins with molecular weight of 10,000-50,000. In conclusion, the present study confirmed the existence of high levels of protease inhibitors in porcine colostrum, and the inhibition of porcine colostrum on degradation of milk-borne growth factors in the pig small intestinal tract was demonstrated for the first time.

• Asian-Australasian Journal of Animal Sciences
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• 제19권8호
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• pp.1164-1173
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• 2006

Cellulase from Aspergillus niger, (${\alpha}$-amylase from Bacillus sp. and protease from Bacillus globigii were used as enzyme sources in this study to examine how their respective substrates protect them in two kinds of simulated gastrointestinal tract digesting processes. Avian total digest tract simulation test showed that filter paper, Avicel and cellulose resulted in 7.7, 6.4 and 7.4 times more activity than of unprotected cellulose, respectively. Protease with addition of casein, gelatin or soybean protein showed no significant protection response. Starch protected amylase to be 2.5 times activity of the unprotected one. Monogastric animal total tract digestion simulation test showed that filter paper, Avicel and cellulose resulted in 5.9, 9.0 and 8.8 times activity of unprotected cellulase, respectively. Casein, gelatin and soybean protein resulted in 1.2, 1.3 and 2.0 times activity of unprotected protease, respectively. Starch did not protect amylase activity in monogastric animal total tract simulation. Protection of mixed enzymes by substrates in two animal total tract simulation tests showed that filter paper in combination with soybean protein resulted in 1.5 times activity of unprotected cellulose, but all substrates tested showed no significant protection effect to protease. Soybean protein and starch added at the same time protected the amylase activity to be two times of the unprotected one. Test of non-purified substrate protection in two animal total digest tract simulation showed that cellulase activity increased as BSA (bovine serum albumin) concentration increased, with the highest activity to be 1.3 times of unprotected enzyme. However, BSA showed no significant protection effect to protease. Amylase activity increased to 1.5 times as BSA added more than 1.5% (w/v). Cellulase activity increased to 1.5 times as soybean hull was added higher than 1.5%. Amylase had a significant protection response only when soybean hull added up to 2%. Protease activity was not protected by soybean hull to any significant extent.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2051-2059
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• 2016

To date, there has been no employment of a magnetoelastic (ME) biosensor method to detect Salmonella enterica serovar Typhimurium in soil. The ME biosensor method needs to be investigated and modified for its successful performance. The filtration method, cation-exchange resin method, and combinations of both methods were employed for the extraction of S. Typhimurium from soil. The number of S. Typhimurium and the resonant frequency shift of the ME sensor were then compared using a brilliant green sulfa agar plate and an HP 8751A network analyzer. A blocking study was performed using bovine serum albumin (BSA), polyethylene glycol (PEG), and casein powder suspension. Finally, the modified ME biosensor method was performed to detect S. Typhimurium in soil. The number of S. Typhimurium was significantly decreased from 7.10 log CFU/soil to 4.45-4.72 log CFU/soil after introduction of the cation-exchange resin method. The greatest resonant frequency shift of the measurement sensor was found when employing centrifugation and filtration procedures. The resonant frequency shift of the PEG-blocked measurement sensor was $3,219{\pm}755Hz$, which was significantly greater than those of the BSA- and casein-blocked ME sensor. The optimum concentration of PEG was determined to be 1.0 mg/ml after considering the resonant shift and economic issue. Finally, the modified ME biosensor method was able to detect S. Typhimurium in soil in a dose-response manner. Although these modifications of the ME biosensor method sacrificed some advantages, such as cost, time effectiveness, and operator friendliness, this study demonstrated a novel approach of the ME biosensor method to detect S. Typhimurium in soil.

• Applied Biological Chemistry
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• 제39권6호
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• pp.449-454
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• 1996

곤충 병원성 곰팡이 Beauveria bassiana(ATCC7159)로부터 곤충 cuticle 분해 효소인 extracellular protease의 최적 생산 조건을 gelatin, vine serum albumin(BSA), casein 및 polypeptone 등을 첨가하여 조사하였다. 이 효소의 최적 생산 배양 조건은 미량원소를 함유한 0.5% Polypeptone, 50mM potassium phosphate pH 6.0이었다. 이 조건에서 pretense의 생산은 배양 1일 후부터 급격히 상승하여 배양 3일 이후 최대로 생산되었으며 배양액의 pH가 7.0 이상이 되면 효소 생산효과가 거의 없었다. 이 pretenses는 phenyl methyl sulfonyl fluoride (PMSF)에 저해받는다. 효소 활성은 pH 8.5와 11.5에서 높게 나타났다. 또한 비변성 isoeletricfocusing gel 전기영동 후 각 gel 절편의 활성을 측정한 결과 protease활성이 세부분에서 나타났다. 따라서 탄소원 및 질소원으로 polypeptone을 이용하여 생산되는 pretense는 세종류 이상인 것으로 판단된다.

• 한국가축번식학회지
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• 제20권4호
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• pp.453-458
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• 1997

형질전환 동물의 후손에서 transgene은 멘델의 법칙에 따라 유전된다고 일반적으로 인식되어져 왔다. 따라서 본 연구에서는 transgene이 이러한 인식과 일치하는지를 여러 세대를 통하여 확인하고 후손에서 어떻게 유전되는지를 연구하기 위하여 형질전환 생쥐를 생산하여 본 연구의 모델로 삼았다. 수정된 생쥐의 embryo에 DNA를 microinjection하는 방법으로 MMTV-LTR (long terminal repeat), bovine ($\alpha$s1-casein cDNA, 그리고 SV 40 splicing과 polyadenylation site 등의 sequence를 포함한 3.0Kb의 DNA가 주입되었다. 여기에서 태어난 새끼는 dot blot과 Southern blot에 의하여 transgene의 존재여부가 확인되어 founder line이 만들어졌다. 그들의 자손은 PCR에 의해서 transgene이 유전되는지를 확인하였다. F0의 72마리 새끼중에서 4마리의 Founder가 transgene을 가지고 있었다(5.6%). F0에서 F1으로의 유전(transmission)은 각각 33.3, 7.7, 0, 62.5%이었다. Transgene은 F1에서 F2로 각각 63.6, 5.9, 68.8% 유전되었고, F2에서 F3로 각각 85.7, 0, 88.2% 유전되었다. 따라서 본 연구 모델에 의하면 transgene은 멘델의 법칙을 따르는 경우와 deletion이 되는 경우로 각각 관찰되었다.