Zoogloea ramigera 115SLR의 생고분자물질 생합성에 관여하는 pyruvyl transferase gene의 cloning 및 염기서열 결정

이삼빈 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4

다당류와 탄수화물이 식품및 의약품등에 이용의 증대로 중요한 관심의 대상이 되고 있으나, 미생물이 생합성하는 다당류의 연구 및 생합성에 관여하는 유전자들의 확보를 통한 유전학적인 연구는 미비한 상태이다. 미생물들로부터 유용한 novel 다당류의 생합성을 위해서는 무엇보다도 생합성에 관여된 유전자들의 클로닝 및 분석이 필요하다. 이 연구에서 Z. ramigera 115 SLR 균주로 부터 다당류의 생합성에 관여하는 유전자중에서 다당류의 side 잔기로 존재하는 glucose 잔기에 pyruvate를 치환시키는 역할을 하는 ketal pyruvyl transferase는 다당류의 물성(rheological properties)에 미치는 중요한 영향 때문에 이 효소를 인지하는 유전자의 분리는 탄수화물 및 다당류의 생합성 연구에 큰 기여를 할 수 있다고 본다. 먼저 xanthan gum을 생합성하는 X. campestris의 ketal pyruvyl transferase 유전자를 probe로 사용하여 Z. ramigera 115 SLR의 genomic DNA에서 염기서열의 동질성을 갖는 4.5kb BamH1 DNA 단편을 분리하였다. 이중에서 2.6kb Pst1/HindIII 단편은 사용한 probe와 강한 염기서열의 동질성을 보여주는 영역으로서 M13mp19 vector와 universal primer를 이용하는 기준방법으로 염기서열을 결정하였다. 이로부터 단백질을 인지할 수 있는 2개의 완전한 ORF와 2개의불완전한 ORF를 확인할 수 있었다. 이중에서 완전한 ORF를 갖는 두 개의 영역(2 and 3)의 염기서열의 결정과 아미노산 서열상의 동질성 분석을 통해서 ORF3는 아미노산 서열상에서 X. campestris의 GumL과 21.6%의 동질성을 보였다. 특히 ORF2는 GumL과 23.2% ExoV과는 37.4%의 높은 동질성을 나타내었으며, 아미노산 서열의 동질성이 전체적인 부분에서 나타남으로서 Z. ramigera 115SLR의 다당류 생합성에서 관여하는 pyruvyl transferase를 인지함을 판단할 수 있었다. ORF2에 존재하는 유전자로 부터 전사되는 단백질의 분자량 크기를 확인하기 위해서 T7 RNA polymerase/promoter를 이용한 in vitro transcription/translation을 실시한 결과 ORF2로 부터 33.5 kDa에 해당하는 polypeptide를 확인할 수 있었다. A gene coding or a pyruvyl transferase enzyme involved in exopoloysac-charide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of Bam HI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindⅢ DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORF1 and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X. campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (exoV product) of Rhizobium meliloti (36%) as well as the ketalase of X. campestris (23%) than that of ORF3. A gene produce of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

조, 기장, 수수의 지방질과 지방산 조성

소한섭,이삼빈,하영득 동아시아식생활학회 2002 동아시아식생활학회지 Vol.12 No.2

Fatty acid compositions of Setaria italica, Panicum miliaceum and Sorghum bicolor were determined with total lipids extracted. Total lipid content of Setaria italica, Panicum miliaceum and Sorghum bicolor were 3.9%(w/w). 2.7%(w/w) and 2.3%(w/w), respectively. Total lipids were fractionated into neutral lipids, glycolipids and phospholipids by the silicic acid column chromatography. Neutral lipids were the most abundant component. Among fatty acids separated by GC, linoleic acid was determined as a major fraction in Setaria italica, Panicum miliaceum and Sorghum bicolor. Palmitic acid and oleic acid were also separated as second major components. In Setaria italica, behenic acid was separated from the phospholipid fraction and myristic acid from the neutral lipid fraction. Linolenic acid was not detected in all the samples.

Development and validation of analytic method for determination of aflatoxins analysis in fermented food

Sam-Pin Lee,Hyun-Jeong Kim,Jung-Woo Chae,Ji-Eun Kim 한국식품영양과학회 2022 한국식품영양과학회 학술대회발표집 Vol.2022 No.10

[Review]Biopolymer Engineering : Genetic Control of Microbial Exopolysaccharide Biosynthesis

Sam-Pin Lee 한국식품영양과학회 1998 식품산업과 영양 Vol.3 No.1

Cloning and Sequencing of an Gene Involved in the Biosynthesis of Exopolysaccharide in Zoogloea Ramigera 115LR

Sam-Pin Lee(이삼빈),Min Yoo(유민) 대한의생명과학회 2000 Biomedical Science Letters Vol.6 No.1

Zoogloea ramigera 115SLR로부터 다당류 생합성에 관여하는 유전자를 분리하기 위해서 균주의 genomic DNA로부터 제조된 gene bank로부터 plasmid pLEX3이 얻어졌다. 이로부터 재조합된 5.0 kb DNA fragment를 포함하는 plasmid pLEX10은 다당류의 형태를 변환시키는 유전자를 포함하고 있으며, 이중에서 upstream 영역에 해당하는 1.7 kb DNA fragment가 분리되었다. 1.7 kb DNA 염기서열의 결과로부터 단백질을 인지할 수 있는 2개의 ORF가 존재하였으며, 50 kDa 단백질을 인지할 수 있는 ORF3은 X. campestris의 다당류 생합성 유전자들인 gumC와 R. meliloti의 exoP와 아미노산의 동질성을 나타내었다. ORF4는 N-terminal 영역이 결여된 단백질을 인지하며, Thermotoga maritima의 다당류 export에 관여하는 단백질과 동질성을 보였다. Z. ramigera 115SLR and Z. ramigera 115SLR/pLEX10은 각각 slime 또는 capsule 형태의 다당류를 생합성하며 이들로부터 생합성된 다당류양은 각각 0.26% (w/v) and 0.16% (w/v)였다. To identity the genes responsible for the biosynthesis of exopolysaccharide, recombinant plasmids pUEX10 and pLEX10 were constructed from plasmid pLEX3 which was isolated from the recombinant cosmid library of Zoogloea ramigera 115. The complete nucleotide sequence of the 1.7 kb genomic DNA insert in plasmid pUEX10 was determined. Its analysis identified two open reading frames (ORF3 & ORF4) which could encode two proteins. The amino acid sequence derived from ORF3 showed the homology with gumC protein in Xanthomonas campestris as well as exoP protein in Rhizobium meliloti. The partial amino acid sequence of ORF4 showed the homology with polysaccharide export protein in Thermotoga maritima. Z. ramigera 115SLR and Z. ramigera 115SLR/pLEX10 showed the similar pattern for EPS production. Yield of exopolysaccharides produced by Z. ramigera 115SLR and Z. ramigera 115SLR/pLEX10 was 0.26% (w/v) and 0.16% (w/v), respectively.

Optimization of Poly-γ-glutamic Acid and γ-Aminobutyric Acid by Co-fermentation of Rice Rran Using Bacillus subtilis and Lactobacillus plantarum

Sam-Pin Lee,Min-Ju Kim,Soon-Young Kwon,Eun-Jin Park 한국식품영양과학회 2014 한국식품영양과학회 학술대회발표집 Vol.2014 No.10

Alcohol Fermentation of Opuntia ficus Fruit Juice

Sam-Pin Lee,Suk-Kyung Lee,Young-Duck Ha 한국식품영양과학회 2000 Preventive Nutrition and Food Science Vol.5 No.1

Prickly pear juice (PPJ) extracted from prickly pear fruit (Opuntia ficus-indica) was used as a raw material for the production of alcoholic beverages. Prickly pear juice (PPJ) had 0.88 °Brix of soluble solid, pH 3.96 and 0.14% of total acidity. Alcohol fermentation of 25% PPJ including 22 °Brix of sugar was not successful, but was promoted by addition of grape juice (GJ). In the presence of GJ, the 22 °Brix of sugar and 1×10^(6) of inoculum was suitable for alcohol fermentation indicating the rapid decrease of sugar content. The 22 °Brix of sugar in PPJ (25%)/GJ (50%) mixture was changed to 6.5 °Brix after fermentation at 30℃ for 7 days. The alcohol content was 9.2% (w/v). PPJ (70%)/GJ (30%) mixture produced alcoholic beverage with 6.9% alcohol content resulting in the gradual decrease of soluble solid for 7 days. On the other hand, PPJ (50%)/GJ (50%) mixture carried out completely the alcohol fermentation at 22℃ for 6 days and enhanced the red color of alcoholic beverages.

Production of Fibrinolytic Enzyme from Soybean Grits Fermented by Bacillus firmus NA-1

Sam-Pin Lee,Ji-Hyun Seo 한국식품영양과학회 2004 Journal of medicinal food Vol.7 No.4

Bacillus firmus NA-1 producing firbinolytic enzyme was isolated from Japanese traditional fermented soybean (Natto). Seed starter was cultured in 5% soy milk prepared with micronized soybean powder. To optimize the production of fibrinolytic enzyme, the soybean grits were mixed with one volume of water and sterilized at 121℃ for 5 minutes, and then used for the medium for solid state fermentation at 42℃. The fibrinolytic enzyme activity of the fermented grits was 1120 plasmin unit per 100 g (wet wt.) after fermentation for 24 hours. The water extract of the fermented grits showed the highest viscosity after fermentation for 12 hours. However, the tyrosine content was the highest (962 mg%) after fermentation for 60 hours. The color of raw soybean grit was affected by heat-treatment. The activity of fibrinolytic enzyme was stable after freezing drying, but was completely destroyed by heating at 70 ℃ for 10 minutes. The color of soybean grit was greatly darkened by increasing fermentation time. Soybean grits were completely converted into valuable functional ingredient containing fibrinolytic enzyme, peptide and mucilage by the solid state fermentation.

A New In Vitro Assay of Cholesterol Adsorption by Food and Microbial Polysaccharides

Sam-Pin Lee,Han-Sup Soh,Chan-Shick Kim 한국식품영양과학회 2003 Journal of medicinal food Vol.6 No.3

The adsorption of total cholesterol by polysaccharides was measured in vitroby enzymatic reactions, includ-ing the polysaccharide precipitation procedure. Total cholesterol adsorption capacities, in a mixture of polysaccharide and to-tal cholesterol, were compared for apple pectin, gelrite gellan gum, xanthan gum, high-methoxyl pectin, citrus pectin, high-viscous alginate, low-viscous alginate, dextran, and zooglan. Acidic polysaccharides such as pectins, alginate, and xanthangum at concentrations of 0.1% (wt/vol) were able to adsorb over 90% of the total cholesterol when dissolved in distilled wa-ter, sodium acetate buffer (pH 4.6), or sodium phosphate buffer (pH 7.0). However, total cholesterol adsorptions by gellanand zooglan were dependent upon the salt concentration and pH value, which decreased cholesterol adsorption in the fol-lowing order by degree: distilled water, acidic pH, and alkaline pH. In particular, total cholesterol adsorption of zooglan wasgreatly decreased by the addition of sodium chloride. With 0.1% (wt/vol) polysaccharide dissolved in distilled water, the ad-sorption capacities of alginate, pectins, gellan gum, xanthan gum, and zooglan were 2.9, 2.88, 2.5, 2.9, and 2.4 mg/dL, re-spectively. However, 0.2% of zooglan was able to completely adsorb the cholesterol (3 mg/dL), whereas dextran did not ad-sorb cholesterol at all, producing no precipitate with hexadecyltrimethylammonium bromide.

2016 Mineral analysis for Food Nutrients Database Construction

Sam-Pin Lee,Hyun-Jeong Kim,Ji-Hyun Seo,Se-Young Kong,Young-Eun Kim,Jinbong Hwang,Dong Won Seo,Stephen Min,Timothy Jeon,Jun Soo Lee 한국식품영양과학회 2016 한국식품영양과학회 학술대회발표집 Vol.2016 No.10