• Asian-Australasian Journal of Animal Sciences
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• 제21권4호
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• pp.582-589
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• 2008

This study attempted to determine effects of recombinant porcine epidermal growth factor (pEGF) and glutamine (Gln) supplement on the growth performance and intestinal development of piglets weaned at 14 days of age. A total of ninety-six piglets were allotted to one of four dietary treatments which comprised inclusion of 1.0 mg pEGF supernatant/kg diet or 0.5% Gln both alone and in combination. Each treatment consisted of four replicates with six pigs per pen for a 28 days experimental period. Two pigs per replicate were sacrificed and gastrointestinal tract samples were collected on day 14. Data showed that dietary treatment failed to promote growth performance. On day 14, diets supplemented with pEGF elevated pancreatic chymotrypsin, jejunal alkaline phosphatase, sucrase, lactase and maltase activities (p<0.05), but failed to alter the small intestinal villus morphology, DNA, or protein content of gastrointestinal mucosa. Diets supplemented with Gln increased pancreatic chymotrypsin activity, tended to enhance the protein contents of gastric (p = 0.08) and jejunal mucosa (p = 0.09) but did not influence the serum IgA level or the enzyme activity in the gastrointestinal tract. On day 28, the diets supplemented with Gln increasedt (p<0.05) serum IgA and the proliferation of peripheral blood mononuclear cells by PHA stimulation. However, a combination of pEGF and Gln did not have a synergistic effect on these biomarkers in early-weaned piglets. The results demonstrate that diets supplemented with recombinant pEGF supernatant indeed improve intestinal digestive enzyme activity and diets supplemented with Gln increases the immune response in early-weaned piglets.

• 한국식품조리과학회지
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• 제15권1호
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• pp.73-80
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• 1999

This study was carried out to investigate the effects of enzyme treatments on the functional properties of soy protein isolate (SPI) and to examine the quality attributes of soy yogurt prepared by different enzyme treatments, sweeteners and starter cultures. Enzyme treatment increased the solubility and emulsifying capacity of soy proteins, but decreased the emulsifying stability; the enzymatic activity of ${\alpha}$-chymotrypsin was higher than that of trypsin. Enzyme treatments decreased the pH of soy yogurts prepared by both culture methods, the culture of L. bulgaricus and S. thermophilus and the culture of L. bulgaricus and K. fragilis, but increased the titratable acidity, total numbers of lactic acid bacteria and yeast. Trypsin was more effective than ${\alpha}$-chymotrypsin in decreasing pH and increasing titratable acidity and total numbers of lactic acid bacteria and yeast. Fructose decreased the pH of soy yogurts more than sucrose in the culture of L. bulgaricus and S. thermophilus, and vice versa in the culture of L. bulgaricus and K. fragilis. Fructooligosaccharides were more effective in the culture of L. bulgaricus and K. fragilis than in the culture of L. bulgaricus and S. thermophilus in increasing the titratable acidity, total count of lactic acid bacteria and yeast. In sensory evaluation, soy yogurts containing trypsin treated SPI, fructose and fructooligosaccharides (75%:25%) were more acceptable than those containing untreated or trypsin treated SPI and fructose. This was because of more smooth and less sour, in which the values of pH, titratable acidity, microbial growth, and viscosity were in the range of commercial yogurts. Soy yogurts fermented by L. bulgaricus and K. fragilis showed more smooth mouthfeel than those fermented by L. bulgaricus and S. thermophilus.

• Journal of Nutrition and Health
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• 제19권6호
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• pp.374-381
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• 1986

CCK ( Cholecystokinin)와 carbachol (carbamylchline chloride)이 췌장의 효소분비능력을 촉진시킨다는 사실은 잘 알려져있다. 생대두식이를 먹인 쥐에서 췌장세포의 hypertrophy 현상이 두드러지게 나타나고 소화효소의 과다분비 현상이 나타나므로 이번 실험에서는 익힌 대두와 생대두를 먹인 후 정상적인 췌장세포와 hypertrophy 가 일어난 췌장세포에 CCK 와 carbachol 로 자극을 준후 효소분비 능력을 비교하였다. 췌장세포에서 분해되는 단백질 분해효소들이 세포자체에 미치는 영향을 최소한으로 줄이기 위하여 췌장의 acinar cell을 분리한 후 cell column을 만들어 superfusion technique 에 의해 stimulus-enzyme secretion coupling 방법을 사용하였다. 분리한 췌장세포를 CCK와 carbachol 로 자극을 주었을대 chymotrysin 분비는 생대두를 먹인 쥐의 세포에서 익힌 대두를 먹인 군보다 높게 나타났고 amylase의 분비는 chymotrypsin 과는 달리 익힌 대두를 먹인 쥐의 세포에서 생대두를 먹인 군보다 높게 나타났다. 이번 실험의 결과는 지금까지 논쟁중에 있는 췌장의 acinar cell에서의 여러 가지 효소분비 비율은 항상 일정한 것이 아니라 자극의 종류에 따라 효소분비량의 구성비율이 달라진다는 것을 알려주고 있다.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• Asian-Australasian Journal of Animal Sciences
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• 제12권3호
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• pp.411-416
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• 1999

Ninety-six, 35 day old, crossbred pigs, were fed either a basal diet based on com, soybean meal, fish meal and whey or one of three similar diets supplemented with either 0.5% organic acid or enzyme both alone and in combination. Neither organic acid nor enzyme produced any significant (p<0.05) improvements in daily gain or feed conversion. Organic acid, both alone and in combination with enzyme, significantly (p=0.04) decreased the pH in the lower colon. None of the treatments produced any effects on E. Coli or Lactobacillus numbers in any part of the gastrointestinal tract. Feeding enzyme increased trypsin (p=0.01), chymotrypsin (p=0.03) and amylase (p=0.08) levels in the jejunum. Chymotrypsin levels were higher (p=0.04) in the ileum of pigs fed enzyme. Serum glucose levels were lower (p=0.01) on day 14 in pigs fed enzyme either alone or in combination with acid. Under the conditions of this experiment (10% dietary whey, pigs weaned at 35 days of age), neither organic acid nor enzyme were effective in improving starter pig performance. Therefore, there would appear to be little justification for the routine inclusion of these products in diets fed to pigs weaned at 35 days or later.

• 미생물학회지
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• 제45권2호
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• pp.185-192
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• 2009

Bacillus subtilis 028-1 균주는 청국장 발효에 사용하는 균주로 Staphylococcus sp. LS2 뿐만 아니라 여러 yeast 균주의 생장을 억제하는 항생물질을 생산하며, soybean meal 2%와 maltose와 같은 이당류를 1% 첨가하여 15~18시간 진탕 배양하였을 때 최대의 항생물질 생산을 보였으며 배지의 pH는 6.5 이하였다. 항생물질의 활성은 약염기성 조건에서 극대화되었으며, $100^{\circ}C$에서 20분간 가열하여도 활성은 크게 감소하지 않았고, 실온 보관 시 한 달 이상 효과가 지속되며 chymotrypsin과 papain 같은 단백질 분해효소 처리에 의해 서서히 활성이 줄어들었다. 투석에 의해 항생물질의 분자량을 측정한 결과 1,000에서 500 dalton 사이인 것으로 나타났으며 항미생물 효과는 있으나 fibrin 분해 능력이 없으므로 surfactin이 아닌 iturin 계열의 peptide성 항생물질로 보인다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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• pp.236-236
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• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Journal of Microbiology
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• 제39권4호
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• pp.305-313
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• 2001

The spr D gene encoding Strptomyces griseus protease D(SGPD); a chymotrypsin-like proteae, was cloned from Strptomyces griseus IFO13350 and sequence. Most of the amino-acid sequence deduced from the nucleotide sequence is idential to that Strptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp 369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The spr D gene was overexpressed in Streptomyce liv-idans TK24 as a heterologous host. Various media with different compositions were also used to max-imize the productivity of SGPD inthe heterologous host. The SGPD productivity was best when the transformant S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-scuccinyl-ala-ala-pro-phe-p-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM medial but it was relatively lower that in R2YE medium and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reacted the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increase in till the 10$^{th}$ day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8days of cultivation. The introduction of the spr D gene into S. lividans TK24 triggered biosyntheis of the pigmented antibiotic , actinorhodin, which implies some protease may paly a very improtant role in secondary-metabolite formation in sStreptomyces.

• 대한화학회지
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• 제38권6호
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• pp.449-455
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• 1994

효소를 이용한 생리활성 펩티드의 합성 연구에 관한 기초자료를 얻기 위하여, Kyotorphin(진통작용을 가진 펩티드) 유도체가 $\alpha-chymotrysin$를 이용한 이상계(two phase system;유기상과 수용액상)조건하에서 Ac-Tyr-OH 와 $Arg-NH_2$로부터 합성되었다. Ac-Tyr-OH(10 mM)과 $Arg-NH_2$ (20 mM)와의 Kyotorphin 유도체 합성에 대한 유기용매의 효과에서 ethyl acetate계에서의 합성 수율이 다른 유기용매(dichloromethane, n-butanol, n-hexane, chloroform)에서 보다도 더 높았다. Kyotorphin 유도체의 합성에 미치는 최적조건을 보면, 효소 농도는 10 ${\mu}M$, 온도 및 pH는 각각 $35^{\circ}C$ 및 7.0이었으며, 유기상/수용액상의 비$(\alpha)$는 15였다. Kyotorphin 유도체 합성의 최적 반응조건하에서 수율은 70.2%였으며, 이때의 반응은 24시간 후에 평형에 도달하였다.

• 한국임상수의학회지
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• 제25권5호
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• pp.317-323
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• 2008

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.