• Journal of Microbiology
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• 제42권4호
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• 한국식품과학회지
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• 제35권2호
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• pp.201-205
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• 2003

맥주 공장 부산물 효모의 용도를 다양화하기 위하여 효모 슬러리를 자가 소화시키고 상등액을 농축하여 조미료로 이용할 수 있는 효모추출물을 제조하였다. 맥주 효모의 세포벽에 부착되어 있는 호프 쓴맛 성분을 제거하기 위하여 효모 슬러리에 20%(w/v) NaOH 용액을 가하여 pH 9.8로 맞추고, 약 5분간 방치한 후 즉시 원심분리하면서 물로 세척하였다. 세척 후 효모 쓴맛 정도는 24.1 bitterness unit(BU)로서 threshold value 25.0 BU 미만이었다. 세척한 효모를 자가 소화시킨 후 수율[(수득한 효모추출물의 고형분 무게)/(투입 효모슬러리의 고형분 무게)${\times}100$]을 조사한 결과, 효모슬러리 농도를 $9{\sim}10%(w/w)$로 하고, 온도 $53^{\circ}C$, pH 6.8에서 20시간 이상 자가 소화시키면 34%(w/w)이상, 최대 38%(w/w)까지의 수율로 효모 추출물을 얻을 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.163-163
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• 2005

• 한국동물학회:학술대회논문집
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• 한국동물학회 1999년도 한국생물과학협회 학술발표대회
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• pp.224.2-224
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• 1999

No Abstract, See Full Text

• Journal of Microbiology
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• 제45권6호
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• BMB Reports
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• 제44권10호
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• pp.665-668
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• 2011

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

• 생명과학회지
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• 제24권12호
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• pp.1316-1324
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• 2014

신령버섯균사체(Agaricus blazei mycelia: ABM)를 대두박이 함유된 액체배지에 배양하고, 이것을 자가분해($53^{\circ}C$, pH 5.5, 120 rpm, 3 hr)하여 항암성이 강한 isoflavone-conjugated glycoprotein (Gluvone 이라 명명)을 분리하였다. Gluvone은 지금까지 알려진 당단백질과는 달리 분자량이 작고(9,400 Da), isoflavone이 결합되어 있다는 점이 다르다, Gluvone은 60% 탄수화물(glucose, fructose, ribose), 31% 단백질 및 2% isoflavone (daidzein, genistein)으로 구성되어 있었다. 이 Gluvone은 S-180 복수암세포, MCF-7 인체유선암세포에 대한 독성이 강하였고, S-180 세포로 유발한 mouse 복수암을 강하게 억제하였다.

• 한국식품영양학회지
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• 제14권2호
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• pp.161-166
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• 2001

맥주효모박 중의 단백질 함량은 41.7%, 탄수화물은 46.0%이었다. 주요 무기질은 K. P, Mg, Ca이었으며, 이들의 함량은 각각 1659.7mg%, 1,197.4mg%, 210.4mg% 및 105.6mg% 이었다. 맥주효모박 중의 주요 비타민은 비타민 C와 비타민 B군이었으며 이들의 함량은 각각 7.9IU/100g 및 4.2IU/100g이었다. 맥주효모박의 자가소화 최적반응농도는 10% 였으며, 자기소화 촉진 및 오염방지제로 사용된 에탄올과 NaCl의 최적 농도는 맥주효모 현탁액의 경우 NaCl 3% 첨가구에서, 빵효모현탁액의 경우 NaCl 5% 첨가구에서 가장 효과적이었다. NaCl의 첨가에 의한 자가촉진효과는 맥주효모박보다 빵효모에서 더 우수하였다. 또한 맥주 효모현탁액에 에탄올을 첨가한 경우, 에탄올 첨가 농도에 따른 자가소화 촉진효과의 차이는 나타나지 않았으며, 빵효모 현탁액에 에탄올을 첨가한 경우 에탄올 3% 첨가구에서 가장 효과적이었다. 이들의 결과로 볼 때 에탄올의 첨가에 의한 자가소화 촉진효과 또한 맥주효모박에 비해 빵효모가 더 우수하였다. 맥주 효모박을 자가소화시킨 후 glucanase의 첨가는 정미성이 강한 IMP와 GMP의 함량을 160% 정도 증가시켰다.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1997년도 학술발표회
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• pp.44-44
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• 1997

Ca$^{2＋}$ is one of the most common metal ions associated with proteins, playing more or less well-defined functional roles in biological activities. In protease, the presence of $Ca^{2＋}$ slows down autolysis and enhances thermal stability. Subtilisin, one of the best studied proteases, is known to have two $Ca^{2＋}$ -binding sites.(omitted)

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.