• Journal of Microbiology and Biotechnology
• /
• 제18권6호
• /
• pp.1081-1089
• /
• 2008

A novel ginsenoside-hydrolyzing ${\beta}$-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of Q-Sepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatography. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and $60^{\circ}C$. It was highly stable within pH 3-9 and at temperatures lower than $55^{\circ}C$. The enzyme was specific to ${\beta}$-glucoside. The order of enzyme activities against different types of ${\beta}$-glucosidic linkages was ${\beta}$-(1-6)>${\beta}$-(1-2)>${\beta}$-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at $45^{\circ}C$ and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was $Rb1{\to}Rd{\to}F2{\to}CK$. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified ${\beta}$-glucosidase proves to be a new protein that has not been reported before.

• Journal of Microbiology and Biotechnology
• /
• 제25권7호
• /
• pp.1007-1014
• /
• 2015

Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using genesplicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentrationdependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.

• 미생물학회지
• /
• 제42권4호
• /
• pp.294-298
• /
• 2006

Streptomyces somaliensis가 생산하는 phospholipase D (PLD)를 정제하기 위하여 펩티드 항체 결합 면역 친화 크로마토그래피용 칼럼을 개발하였다. 단백질 구조 예측 프로그램과 Streptomyces PLD X-선 결정구조를 참조하여, S. somaliensis PLD의 1차 구조로부터 항원특성이 높고. 표면에 위치하는 것으로 예상된 5종류의 펩티드들을 epitope로 선정한 뒤, 이에 대한 항체로 면역친화 크로마토그래피용 칼럼을 제작하였다. 배양 농축액을 칼럼에 통과시켜 정제한 활성 분획을 SDS-PAGE 및 Western blot 결과, 칼럼 종류에 따라 순수한 PLD또는 35 kDa의 단백질 불순물만을 포함하는 PLD 정제 분획을 보여 면역친화 칼럼의 높은 항원결합 특이성을 보여주었다. 그러나 수용액상에서 PLD 자체의 구조적 불안정성 때문에 정제 후 PLD의 특이적 활성 및 정제 수율은 낮았다.

• 한국식품과학회지
• /
• 제30권1호
• /
• pp.218-223
• /
• 1998

우유 casein단백질을 alcalase로 가수분해시켰을 때, 생성되는 peptide 중 철분과 결합력이 있는 peptide인 IBP가 $immobilized\;Fe^{3+}\;affinity\;chromatography$에 의하여 용이하게 분리되었다. IBP의 분자량은 2,175 dalton이었으며 $pH\;6,\;37^{\circ}C$에서 1시간동안 일정량의 철분과 항온처리하였을 경우, 25, 50, 100 g/mL의 IBP는 각각 4.2, 5.7, 7.1 g의 철분을 가용화시키는 능력을 보였다. IBP는 proline (24.5 Mol%), lysine (15.7 Mol%), glutamic 또는 glutamine (14.9 Mol%) 등의 아미노산으로 구성되어 있었으며 이들의 N-terminal sequence는Met-Ala-Pro-Lys-His의 순으로 이루어져 있었다. 분자량, 아미산 조성, N-terminal sequence등의 결과를 종합해 보면 IBP는 casein 중 -casein의 아미노산 서열 $102{\sim}119$에 해당하는 18개의 아미노산으로 구성된 peptide임을 알 수 있었다.

• BMB Reports
• /
• 제35권2호
• /
• pp.206-212
• /
• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• 농약과학회지
• /
• 제2권3호
• /
• pp.28-35
• /
• 1998

전국에서 분리한 세균으로부터 항균력이 있는 길항세균들을 먼저 분리한 다음 그중 배양배지 속으로 강력한 항균력을 나타내는 peptide를 분비하는 A405균주를 선발, Biolog System 및 rDNA 염기서열을 이용하여 Bacillus subtilis로 동정하고 이 균주가 생성하는 항균 peptide의 항균력을 조사한 바 Botrytis cinerea, Cercospora sp., Fusarium oxysporum, Penicillium digitatum, Celletotrichum gloeosporioides, Rhizoctonia solani, Pythium ultimum, Pyricularia oryzae등의 식물병원균과 Escherichia coli, Pseudomonas spp. 등의 세균 및 효모인 Candida albicans의 성장도 억제하는 강력한 항균력을 보였다. 또한 이 균주가 생산하는 항균 peptide는 알콜, 아세톤과 같은 유기용매에 내성을 가지고 있었으며 proteinase K와 phenol 처리시에는 항균력이 감소되거나 소실되는 특성을 가지고 있었다. 이 항균 peptide를 SDS-PAGE, Native-PAGE 및 Tris-Tricine 전기영동으로 분자량이 3kDa인 작은 peptide임을 확인하였고 aspartic acid, glycine, serine, glutamine, valine, leucine, isoleucine, proline, tyrocine 등의 9가지 아미노산으로 구성되어 있음을 Amino Acid Analyzer로 확인하였으며 냉동건조한 항균 peptide 50 ug/ml을 체리토마토에 처리하여 과일의 선도유지를 조사한 결과 대조구에 비하여 월등한 선도유지 효과가 있음을 입증하였다.

• BMB Reports
• /
• 제30권5호
• /
• pp.342-345
• /
• 1997

Catechol 1,2-dioxygenase $I_1$ (CD $I_1$) gene of Acinetobacter Iwoffii K24, $catA_1$ was expressed in Escherichia coli and was partially purified by using a MonoQ column. Expressed CD $I_1$ had the same molecular weight as purified CD $I_1$ from A. Iwoffii K24 on SDS-PAGE. Expressed CD $I_1$ was also identified by Western blotting and peptide sequencing of N-terminal and internal regions. When compared with purified CD $I_1$ of A. Iwoffii K24, expressed CD $I_1$ had similar substrate specificities and the effects of compounds on enzyme activity. N-terminal amino acid sequence of CD I expressed in E. coli was the same as that of purified CD $I_1$, suggesting that CD $I_1$ may be under the same posttranslational processing in E. coli and A. Iwoffii K24.

• Journal of Microbiology and Biotechnology
• /
• 제10권6호
• /
• pp.805-810
• /
• 2000

An antifungal protein antagonistic to the rice blast fungus, Pyricularia oryzae was purified from Paenibacillus macerans PM-1 by ammonium sulfate fractionation, Q Sepharose Fast Flow column chromatography, Phenyl Sepharose CL-4B column chromatography and Superose 12 gen filtration. An apparent molecular mass of the purified antifungal protein was determined as 8 kDa by SDS-PAGE and 9 kDa by analytical gel filtration, respectively, suggesting that the purified protein is a monomer. The antifungal protein was stable at pH range from 7-12 and up to $100^{\circ}C$. The protein was also stable at 0.1-1% Tween 20 and Triton X-100. The N-terminal amino acid sequence of the antifungal protein was Thr-Glu-Leu-Pro-Leu-Gly-Ile-Val-Met-Asp-Lys-Tyr-Thr-Asp-Ala-Phe-Lys-Phe-Asp-Met-Phe. Comparison of the determined sequence with other peptide and DNA sequences did not reveal homology at all. Therefore, the purified antifungal protein was speculated to be a novel protein. The condidial germination in vitro of P. oryzae KJ301:93-39 by the purified protein ($5.9{\mu} g/ml$) was limited to $9{\pm}3.2%$ only, compared with $69{\pm}2.4%$ of the control. Ungerminated conidia were swollen at basa and mid cell by the purified protein. In vivo bioassay for inhibition of conidial germination of P. oryzae KJ 301, one of the most predominating racesin Korea. the purified protein ($5.9{\mu} g/ml$)strongly inhibited the conidial germination. The conidia, even though germinated, could not develop any further to produce appressoria efficiently.

• 식물병과 농업
• /
• 제5권1호
• /
• pp.27-33
• /
• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• BMB Reports
• /
• 제31권2호
• /
• pp.111-116
• /
• 1998

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the $DRB1^*0405$ subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allelespecific region covering the ${\beta}1$ domain of $DRB1^*0405$ was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR ${\beta}-chain$. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.