• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제25권3호
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• pp.435-446
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• 2012

The present study identified volatile flavor components of Hanwoo longissimus muscle and other Korean foods (Doenjang, Chungukjang, sesame oil) and their traits were compared in relation with flavor precursors that include fatty acids and protein degradation products. Hanwoo longissimus muscle was purchased from a commercial abattoir while the other foods were sampled from three separate households. The results showed totals of 68 ($9.94{\mu}g/g$), 60 ($15.75{\mu}g/g$), 49 ($107.61{\mu}g/ml$) and 50 ($7.20{\mu}g/g$) volatile components for Doenjang, Chungukjang, sesame oil and Hanwoo beef longissimus, respectively (p<0.05). Aldehydes were the most predominant components in beef, but alcohols, acids and esters, and pyrazines are probably the major contributors to the flavor characteristics of other foods. SDS-PAGE revealed that beef longissimus muscle and Doenjang showed higher protein degradation than other foods which could be likely related to chiller ageing and ripening process. The total polyunsaturated fatty acids were approximately 50, 60, 41 and 5% for Doenjang, Chungukjang, sesame oil and beef longissimus muscle, respectively. Based on the mechanism(s) of generation of the volatile compounds and the chemical composition of each food sample, differences and traits of volatile flavor components among the four food types are likely due to fatty acid profiles, proteolytic activity and processing conditions. Aroma intense compounds like pyrazines and sulfur-containing compounds were limited in cooked beef in the current experimental condition (i.e., relatively low heating temperature). This suggests that higher heating temperature as in the case of roasting is needed for the generation of high aroma notes in meat. Furthermore, proteolytic activity and stability of fatty acids during ageing have a great influence on the generation of flavor components in cooked beef.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.440-446
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• 1989

Penicillium sp. CB-20의 polygalacturonase 생성을 위한 최적조건은 탄소원으로 펙틴을 사용하여 16 시간 배양시 최대 활성을 나타내었으며, Sephadex G-25, G-75 및 G-150을 사용한 gel filtration과 DEAE-cellulose, DEAE-Sephadex A-50에 의 한 ion exchange chromatography를 통하여 이 효소를 약 30배 가량 정제할 수 있었고, 수율은 2.31%였다. 정제효소는 polyacrylamide gel electrophoresis에 의하여 단일 밴드로 확인 되었으며, 분자량은 SDS PAGE에 의하여 21,000 정도로 측정되었다. 효소의 결정구조는 마름모꼴을 형성하고 있었으며 아미노산 조성은 17종류로써 glutamic acid, glycine, hlstidine의 함량이 비교적 많았다.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1726-1736
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• 2013

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of $P_{tac}$, whereas AspC was efficiently expressed by $P_{sod}$ originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli $DH5{\alpha}$ expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared with wild-type $DH5{\alpha}$ harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

• 한국작물학회지
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• 제43권3호
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• pp.172-178
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• 1998

Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16$^{\circ}C$ and 5/2$^{\circ}C$, day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 $mgmL^{-1}$ to 0.448 $mgmL^{-1}$, with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 $\mu\textrm{g}$ $(gFW)^{-1}$ to 120 $\mu\textrm{g}$ $(gFW)^{-1}$ with an averaged value of 77 $\mu\textrm{g}$ $(gFW)^{-1}$, and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two $\beta$-1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient ($R^2$ =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.

• Parasites, Hosts and Diseases
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• 제29권2호
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• pp.113-120
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• 1991

혈청내 특이 항체가를 측정하여 폐흡충증을 면역학적으로 진단할 경우 그 민감도와 특이도가 뛰어난 것은 이미 잘 알려져 있다. 이때에 항원으로 사용하는 폐홉충 추출액의 구성 단백질 중 항원성과 특이성이 높은 단백질을 알면 항원을 질적으로 향상시킬 수 있을 것으로 기대하고 있다. 최근 SDS-PAGE/immuoblot을 이용하여 환자 혈청내의 특이 항체(IgG)와 민감하게 반응하는 폐흡충의 수용성(수준성) 항원 단백질 분획이 보고되고 있다. 그러나 이 단백질 분획은 SDS-PAGE 후 특이항체와 반응한 polypeptide 수준의 subunit이어서 이들이 분해되기 전 성분 단백질 어느 것에서 유래한 것인지를 전혀 알 수 없는 단점이 있다. 따라서, 이와 같은 문제를 해결하고 나아가 종특이(종특리) 항원 단백질을 찾기 위해서는 분해, 변성되기 전의 성분 단백질의 성질을 파악할 필요가 있다. 이 실험에서는 폐흡충 성충 생리식염수 추출액내의 구성 단백질을 파악하고 그 분자량을 측정하기 위하여 Disc-PAGE를 이용한 Ferguson plot과 column chromatography를 실시하였다. 우선 폐홉충 추출액을 4.5~10%까지의 각기 다른 gel 농도에서 전기영동을 실시한 결과 추출액은 최소 8개 이상의 주요 성분 단백질로 구성되어 있었고 각각의 분자량은 band 1은 440kDa, band 2는 386kDa, band 3은 17.4kDa, band 4는 17k3a, band 5는 14.3kDa, band 6은 46kDa, band 7은 38kDa 그리고 band 8은 23k3a 이었다. 이어 추출액을 $1.6({\Phi}){\times}70cm$의 Sephacryl S-300 Supcrfne column을 통과시켜 7분획을 얻은 후 각 분 획을 다시 85 gel에서 전기영동으로 확인한 결과 band 1은 1~3분회, band 2는 2,3분회, band 3은 5분획, band 4는 5,6분획, band 5는 5분획, band 6,7은 3,4분획, band 8은 5분회에서 각각 관찰되어 Ferguson plot에 의해 계산한 분자량과 유사한 결과를 보이고 있었다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Asian-Australasian Journal of Animal Sciences
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• 제15권2호
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• pp.290-296
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• 2002

Serum immunoglobulins (Igs) from Israeli carp were purified using affinity chromatography. Fish were immunized with purified mouse IgG, and the specific fish antibodies were purified from the immune serum on a mouse IgG-immobilized agarose gel. Rabbit anti-Israeli carp Igs (R $\alpha$ I. carp Igs) antibodies were produced following hyperimmunization with mouse IgG specific carp antibodies. SDS-PAGE analysis under reducing condition showed that Israeli carp Igs were composed of two $\mu$-like heavy chains with about 82 and 50 kd, respectively, and one light chain with about 25 kd. On immunoblotting analysis, however, R $\alpha$ I. carp Igs failed to react with the light chain. When both protein A and protein G-purified normal carp Ig were compared with mouse IgG-specific Israeli carp Ig, no significant structural differences among them were observed. To investigate if there is any homology between other fish Ig molecules, cross-reactivity of R $\alpha$ I. carp Igs against Ig molecules from 6 different fish sera and mouse control serum was checked on immunoblotting analysis. As a result, R $\alpha$ I. carp Igs responded to Israeli carp, common carp, and tilapia Ig molecules. In flow cytometry study, however, R $\alpha$ I. carp Igs appeared to recognize 42.0%, 35.8% and <5% of Israeli carp, common carp and tilapia $Ig^+$ head kidney cells, respectively. The result suggests the heterogeneity between receptor Igs on B-like lymphocytes and soluble Igs in serum. It is crucial to obtain pure fish Igs to produce reagent antibodies as tools for the study on their specific immune responses.

• 대한한방내과학회지
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• 제24권1호
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• pp.113-122
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• 2003

Objective : The aim of this study was to evaluate the efficacy of Gagamchunggan-tang on anti-inflammation reaction with lipopolysaccharide (LPS)-induced HepG2 cell. Method : We examined the effects of the Gagamchunggan-tang, a traditional drug for liver inflammation, on the process of lipopolysaccharide(LPS)-induced nuclear factor-${\kappa}Bp65(NF-{\kappa}Bp65)$ activation in HepG2 cell. SDS-PAGE, Western blotting, Immunofluorescence staining were studied. Results : Immunoblot analysis showed that the level of nucleic $NF-{\kappa}Bp65$ was rapidly up-regulated and cytosolic inhibitory $I-{\kappa}B{\alpha}$ was down-regulated by LPS challenge. While Gagamchunggan-tang inhibited an increase of $NF-{\kappa}Bp65$ and degradation of $I-{\kappa}B{\alpha}$ in HepG2 cell. Besides LPS-induced expression of a group of genes, such as tumor necrosis factor-${\alpha}(TNF-{\alpha})$, inducible nitric oxide synthase(iNOS) and cyclooxygenase-2 (COX-2), are repressed by Gagamchunggan-tang. It may be concluded that Gagamchunggan-tang attenuates the progress of LPS-induced inflammation by reduction of $NF-{\kappa}Bp65$ activation. Conclusion : The Gagamchunggan-tang would be useful as a therapeutic agent for endotoxin-induced liver disease.

• 한국식품영양학회지
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• 제29권5호
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• pp.760-766
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• 2016

To increase the collagen recovery rate, bromelain (PB) and a microbial enzyme (PM) were used to treat to pork skin with single agent or combinations. The quality of collagen from the pork skin was evaluated by enzymatic treatments. The highest results for the solid contents and pork skin recovery rate obtained with the microbial-enzyme-bromelain mixtue (PMB) were 13.60% and 18.05% respectively. The result also showed that the color was affected by different types of enzyme treatments. Although PM treatment showed the highest result in the protein content of 251.30 mg/100 g, PMB treatment was the highest in the test of collagen content of 37.73 g/100 g among the treatments. However bands of the pork skin were detected widely at 130 kDa and 170 kDa ranges in SDS-PAGE. The band of PB treatment showed at the range of below 17 kDa, changed into a smaller molecular weight. The collagen content test of the pork skin by the treatments, collagen contents with combination treatment of pork skin with PMB (0.5%) resulted the highest in 43.76 g/100 g. Also the fat content at the above treatment was reduced to 11.12% compared to the other treatments. With these results of this experiment, we conclude that the enzymatic treatments were effective for the processing property of pork skin like enhancing the yield of collagen.