• KSBB Journal
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• 제21권5호
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• pp.394-400
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• 2006

이 연구의 최종 목표는 방향성 분자진화기술(directed molecular evolution)을 이용한 음이온에 안정한 papain의 개발이다. 음이온 안정성 papain 생산을 위한 유전자의 방향성 분자진화 방법 개발이 이루어졌으며 분자진화된 음이온 안정성 papain의 스크리닝 방법 개발됐다. 분자진화된 papain의 아미노산 서열 및 특성 분석과 분자진화된 재조합 papain의 생산방법 확립되었다. 분자진화된 재조합 papain의 formulation 및 제품 적용화 기술 개발이다. 연구 결과 Papain petidase IV 유전자의 확보 및 발현 균주 개발하였고 음이온 안정성 papain 유전자를 얻기 위한 분자진화의 방법 개발 및 조건 확립하였다. 분자진화 방법으로 error prone PCR 방법 확립, DNA shuffling을 통한 mutagenesis 방법 확립, staggered extension process 방법 확립 및 분자 진화된 음이온성 안정성 papain의 효율적 스크리닝 방법 개발하였다. Skim milk agar plate 이용, 활성 및 안정성이 뛰어난 개량형 papain을 Filter paper방법을 이용하여 screening 방법을 개발하였다.

• 한국자원식물학회지
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• 제19권2호
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• pp.348-354
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• 2006

6 종(도깨비고비, 참쇠고비, 족제비고사리, 꼬리고사리, 거미고사리 및 변산일엽)의 양치식물에서 돌연변이원 처리에 따른 영향을 구하고 인위적인 돌연변이를 유도하기 위하여, 균질화한 전엽체에 감마선 및 EMS, NMU, $NaN_3$등의 회학돌연변이원과colchicine을 처리하였다. 전엽체 증식률은 전반적으로 처리농도 및 처리시간에 비례하여 감소되는 경향을 보였다. 전엽체의 증식률에 근거할 때, 감마선 처리의 최적 조건은 족제비고사리의 경우 20krad였으며, 그 나머지 종은 $5{\sim}10krad$ 수준이었다. EMS의 적정 처리조건은 50mM에서 3시간이었으며, NMU는 $5{\sim}10mM$에서 $1{\sim}3$시간 수준이었다. 그리고 $NaN_3$ 처리의 적정조건은 $0.5{\sim}1mM$ 에서 $1{\sim}3$시간이었으며, colchicine의 경우 각각의 처리조건에서 전엽체 증식률은 종에 따라 다소 차이를 보였다.

• Bulletin of the Korean Chemical Society
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• 제30권6호
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• Bulletin of the Korean Chemical Society
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• 제30권7호
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• pp.1547-1552
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• 2009

Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

• BMB Reports
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• 제45권8호
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• pp.452-457
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• 2012

Diketoreductase (DKR) from Acinetobacter baylyi contains two tryptophan residues at positions 149 and 222. Trp-149 and Trp-222 are located along the entry path of substrate into active site and at the dimer interface of DKR, respectively. Single and double substitutions of these positions were generated to probe the roles of tryptophan residues. After replacing Trp with Ala and Phe, biochemical and biophysical characteristics of the mutants were thoroughly investigated. Enzyme activity and substrate binding affinity of W149A and W149F were remarkably decreased, suggesting that Trp-149 regulates the position of substrate at the binding site. Meanwhile, enzyme activity of W222F was increased by 1.7-fold while W222A was completely inactive. In addition to lower thermostability of Trp-222 mutants, molecular modeling of the mutants revealed that Trp-222 is vital to protein folding and dimerization of the enzyme.

• The Plant Pathology Journal
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• 제19권6호
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• pp.301-304
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• 2003

Gamma-ray irradiation is known to induce various mutations in plants caused by chromosome alterations. This study investigated disease responses of selected gamma-ray induced rice mutants generated from seven Japonica-type rice cultivars against three plant diseases. Among the tested 22 mutants, three gain-of-function mutants and six loss-of-function mutants against rice blast were obtained, as well as three loss-of-function mutants against bacterial leaf blight (BLB). Two of the loss-of-function mutants were susceptible to both rice blast and BLB. Gain-of-function mutation has not been frequently observed in rice plants, thus, the mutants can be used to identify loci of novel genes for the regulation of disease resistant response.

• 미생물학회지
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• 제20권2호
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• pp.89-97
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• 1982

Mutator effect를 가진 plasmid pKMlO1을 MNNG처리와 tetrazolium-glactcse 배지에서 MMS에, 의한 Salmonella typhimuriun의 Gal-에서 Gal+로 전환시키는 능력에 따라 pKM101 변이체들을 선택하고 이들의 성질을 chemical과 UV를 사용하여 pKM101과 비교 하였다. pKM101변이체들은 Salmonella typhimurium내에서 모두 안정하였고 정상적으로 다른 균주로 전달되었으며 picillin에 대한 저항성은 원래의 plasmid보다 같거나 높아졌다. 이들 중 두 변이체는 chemical mutagen인 MMS와 4-NQO, 그리고 UV에 의한 Salmonella typhimurimlla의 돌연변이들을 모체 pKMIOI보다 증가시켰으며 다른 변이체들은 이에 대한 효과가 감소 되었거나 전혀 잃은 것이었다. Salmonella typhimurium uvr- 변이주에 있어서 이들 변이체들의 chemical과 UV에 의한 돌연변이율에 미치는 치사효과는 모체 pKMIOI과 비슷한 양상을 나타내었다. 이러한 결과는 plasmid pKMOI의 chemical 및 UV mutator effect가 한 유전인자에 의해 결정된다는 것을 시사한다.

• 한국작물학회지
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• 제42권2호
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• pp.247-253
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• 1997

콩에서 돌연변이를 효율적으로 유기시킬 수 있는 적정 EMS농도를 결정하기 위하여, 황금콩, 장엽콩, 검정콩 001에 30, 50, 70mM EMS세 수준으로 처리 한 다음 M$_1$종자의 포장발아율과 M$_2$ 세대의 돌연변이체 출현빈도율을 조사하였으며 다량 뿌리혹형성 변이체 선발을 위하여 신팔달콩002 종자 약 18,000립에 30mM EMS를 처리하여 M$_2$세대에서 선발하고, 선발된 변이체의 뿌리혹 형성능력을 미국 초다뿌리혹형성 nts계통과 비교한 결과는 다음과 같다. 1. M$_1$종자의 포장출현율은 검정콩 001가 황금콩과 장엽콩보다 높은 경향이었으며, 세 품종 모두 EMS처리 농도가 증가함에 따라 포장출현율이 낮았다. 2. M$_2$세대에서 돌연변이개체는 엽록소 결핍개체가 가장 많이 출현하였는데, 그외에도 생장점괴사, 엽이상, 엽수변이체, 단경 등 다양한 변이양상을 보였다. 3. 30mM EMS처리가 포장발아율도 높고, 돌연변이개체 출현율도 50, 70mM에 비하여 양호한 편으로 판단되었다. 4. 신팔달콩 002로부터 30mM EMS처리에 의하여 M$_2$세대에서 뿌리혹 형성 nts계통보다도 많은 뿌리혹이 형성되었으며, 뿌리혹형성도 일찍 시작되었다.

• Molecules and Cells
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• 제27권5호
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• pp.547-556
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• 2009

Parathyroid hormone is the most important endocrine regulator of calcium concentration. Its N-terminal fragment (1-34) has sufficient activity for biological function. Recently, site-directed mutagenesis studies demonstrated that substitutions at several positions within shorter analogues (1-14) can enhance the bioactivity to greater than that of PTH (1-34). However, designing the optimal sequence combination is not simple due to complex combinatorial problems. In this study, support vector machines were introduced to predict the biological activity of modified PTH (1-14) analogues using mono-substituted experimental data and to analyze the key physicochemical properties at each position that correlated with bioactivity. This systematic approach can reduce the time and effort needed to obtain desirable molecules by bench experiments and provide useful information in the design of simpler activating molecules.

• 한국미생물·생명공학회지
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• 제19권2호
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• pp.116-121
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• 1991

A bacterial strain KN, which highly produced a protease, was isolated from several soil samples and identified to to belong to the genus Bacillus. We selected mutant strain Bacillus sp. KN103N, which was hyperproducer of protease and was resistant to D-cyclowerine, from the strain KN by several steps of mutagenesis. Neutral protease productivity of mutant strain KN103N was about 55 times as much as that of the original strain KN. The optimum pH and temperature for the enzyme activity were 7.0 and 50$^{\circ}C$, respectively and the enzyme was relatively stable at pH6.0~8.0 and below 40$^{\circ}C$. The enzyme was inactivated by EDTA, but not by DFP. These results indicate that the enzyme from Bacillus sp. KN103N was a neutral (metallo-) protease.