${\gamma}-Cyclodextrin$ Glycosyltransferase 생산균주의 분리, 동정 및 효소 생산조건

김명희,임영희,배경숙,오태광,손천배,Kim, Myung-Hee,Lim, Young-Hee,Bae, Kyung-Sook,Oh, Tae-Kwang,Sohn, Cheon-Bae 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.4

A cyclodextrin glycosyltransferase-producing bacterium was newly isolated from soil using alkaline pH medium containing 1% $Na_2CO_3$. The isolated strain was identified as Bacillus brevis by morphological and biochemical characteristics, and fatty acid composition and designated Bacillus brevis CD162. The strain showed the best enzyme production of 0.9 unit/ml after 96 hrs of culture at $30^{\circ}C$ in a medium of 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% $K_2HPO_4$ 0.05% $MgSO_4{\cdot}7H_2O$, and 1.5% $Na_2CO_3$ at initial pH 10.2. Cyclodextrin glycosyltransferase 생산균주를 토양으로부터 분리하여 형태학적, 생화학적 그리고 균주의 세포벽 지방산 조성분석에 의해 Bacillus brevis로 동정하였고, Bacillus brevis CD162로 명명하였다. 또한 배지조성에 따른 cyclodextrin glycosyltransferase의 최적생산조건을 검토한 결과, 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% $K_2HPO_4$, 0.05% $MgSO_4{\cdot}7H_2O$, 1.5% $Na_2CO_3$ (pH 10.2)의 배지 조건에서 $30^{\circ}C$에서 96시간 배양하였을 때 가장 높은 효소생산인 0.9 unit/ml을 얻을 수 있었다.

Γ-Cyclodextrin Glycosyltransferase 생산균주의 분리, 동정 및 효소 생산조건

김명희,손천배,임영희,배경숙,오태광 대전대학교 이과대학 기초과학연구소 1997 自然科學 Vol.- No.-

Cyclodextrin glycosyltransferase 생산균주를 토양으로부터 분리하여 형태학적, 생화학적 그리고 균주의 세포벽 지방산 조성분석에 의해 Bacillus brevis로 동정하였고, Bacillus brevis CD162로 명명하였다. 또한 배지조성에 따른 cyclodextrin glycosyltransferase의 최적생산조건을 검토한 결과, 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% KHPO₄, 0.05% MgSO₄·7H₂O, 1.5% Na₂CO₃ (pH 10.2)의 배지 조건에서 30℃에서 96시간 배양하였을 때 가장 높은 효소생산인 0.9 unit/ml을 얻을 수 있었다.(1997년 7월 10일 접수, 1997년 11월 21일 수리) A cyclodextrin glycosyltransferase-producing bacterium was newly isolated from soil using alkaline pH medium containg 1% Na₂CO₃, The isolated strain was identified as Bacillus brevis by morphological and biochemical characteristics, and designated Bacillus brevis CD162. The strain showed the best enzyme production of 0.9 unit/ml after 96 hrs of culture at 30℃ in a medium of 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% K₂HPO₄, 0.005% MgSO₄·7H₂O and 1.5% Na₂Co₃at initial pH 10.2

Bacillus brevis CD162 Cyclodextrin Glycosyltransferase의 정제 및 특성

김명희,임영희,오태광,손천배,Kim, Myung-Hee,Lim, Young-Hee,Oh, Tae-Kwang,Sohn, Cheon-Bae 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.4

The cyclodextrin glycosyltransferase (CGTase, EC 3.2.1.19) from Bacillus brevis CD162 was purified by precipitating with ammonium sulfate, DEAE-Sepharose CL-6B column chromatography and Sephadex G-150 column chromatography. The molecular mass and pI of the purified enzyme were estimated to be 74,000 and 6.3 by SDS-PAGE and isoelectric focusing, respectively. The purified enzyme was clearly identified as the CGTase by zymogram after SDS-PAGE. The optimum pH and temperature for the enzyme activity were 8.0 and $55^{\circ}C$, respectively. The enzyme was stable at the range of pH $5.5{\sim}9.0$, and up to $50^{\circ}C$. The amino acid sequence from the $NH_2-terminal$ of the purified CGTase was Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln. The yields of the products from starch as the substrate were 1.3% for ${\alpha}-$, 33.9% for ${\beta}-$, and 9.7% for ${\gamma}-cyclodextrin$. Bacillus brevis CD162가 생산하는 cyclodextrin glycosyltransferase (CGTase)를 ammonium sulfate 침전, DEAE-Sephadex CL-6B 및 Sephadex G-150 컬럼 크로마토그래피를 수행하여 분리정제하였다. 정제된 CGTase는 분자량이 약 74,000, 등전점은 약 6.3인 단백질이었고, 정제된 단백질을 SDS-PAGE한 후 변성된 단백질을 재활성시켜 zymogram을 수행한 결과 cyclodextrin glycosyltransferase임을 확인할 수 있었다. 이 효소의 최적활성 pH와 온도는 각각 8.0과 $55^{\circ}C$이었으며, pH $5.5{\sim}9.0$과 $50^{\circ}C$까지 안정한 활성을 보였다. 또한, CGTase의 $NH_2$-말단 부위의 아미노산서열은 Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln 이었으며, 전분으로부터 cyclodextrin으로의 전환률을 분석한 결과, ${\alpha}$-cyclodextrin은 1.3%, ${\beta}$-cyclodextrin은 33.9% ${\gamma}$-cyclodextrin은 9.7% 이었다.

Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in <i>Streptomyces peucetius</i>

Malla, Sailesh,Niraula, Narayan Prasad,Liou, Kwangkyoung,Sohng, Jae Kyung Elsevier 2009 Journal of bioscience and bioengineering Vol.108 No.2

<P><B>Abstract</B></P><P>To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes <I>desIII</I> and <I>desIV</I> from <I>Streptomyces venezuelae</I> ATCC 15439 and the glycosyltransferase pair <I>dnrS/dnrQ</I> from <I>Streptomyces peucetius</I> ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong <I>ermE⁎</I> promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of <I>dnrS</I> and the <I>dnrS/dnrQ</I> pair, whereas pDesSD25 and pDesQS25 were constructed to express <I>desIII/desIV</I> and <I>dnrS/dnrQ–desIII/desIV</I>, respectively. All of these recombinant plasmids were introduced into <I>S. peucetius</I> ATCC 27952. The recombinant strains produced more DXR than the <I>S. peucetius</I> parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.</P>

Functional carbohydrate microarray-based screening for substrate specificity of Pasteurellamultocida sialyltransferase

Chang Sup Kim,Jeong Hyun Seo,Jae Kyung Sohng,Hyung Joon Cha 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Carbohydrate microarray might have powerful potential for assaying substrate specificity or activity of carbohydrate-processing enzymes such as glycosyltransferase and glycosidase. However, carbohydrate microarray system has been rarely utilized because complex chemical reactions are demanded for microarray preparation. Recently, we have been developed a simple and cost-effective method for fabrication of carbohydrate microarray which uses the reaction of amine-conjugated carbohydrates with glass surface. In the present work, we employed the developed carbohydrate microarray system to rapidly determine substrate specificity or activity of glycosyltransferase. Pasteurella multocidaoriginated sialyltransferase was used as a target glycosyltransferase because it is a wonderful target to prove the potential of carbohydrate microarray. Collectively, the functional carbohydrate microarray would be a powerful analytical tool to screen substrate specificity or other properties of carbohydrate-processing enzymes.

Bacillus brevis CD162 Cyclodextrin Glycosyltransferase의 정제 및 특성

김명희,손천배,임영희,오태광 대전대학교 이과대학 기초과학연구소 1997 自然科學 Vol.- No.-

Bacillus brevis CD162가 생산하는 cyclodextrin glycosyltransferase (CGTase)를 ammonium sulfate 침전, DEAE-Sephadex CL-6B 및 Sephadex G-150 컬럼 크로마토그래피를 수행하여 분리정제하였다. 정제된 CGTase는 분자량이 약74.000, 등전점은 약 6.3인 단백질이었고, 정제된 단백질을 SDS-PAGE한 후 변성된 단백질을 재활성시켜 zymogram을 수행한 결과 cyclodextrin glycosyltransferase임을 확인할 수 있었다. 이 효소의 최적활성 pH와 온도는 각각 8.0과 55℃이었으며, pH 5.5~9.0과 50℃까지 안정한 활성을 보였다. 또한, CGTase의 NH₂-말단 부위의 아미노산서열은 Ser-Val-Thr-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln 이었으며, 전분으로부터 cyclodextrin으로의 전환률을 분석한 결과, α-cyclodextrin은 1.3%, β-cyclodextrin은 33.9%, γ-cyclodextrin은 9.7% 이었다(1997년 7월 10일 접수, 1997년 9월 25일 수리) The cyclodextrin glycosyltrasferase (CGTase, EC 3.2.1.19) from Bacillus brevis CD162 was purified by precipitating with ammonium sulfate, DEAE-Sepharose CL-6B column chromatography and Sephadex G-150 column chromatography. The molecular mass and pI of the purified enzyme were estimated to be 74,000 and 6.3 by SDS-PAGe and isoelectric focusing, respectively. The purified enzyme was clearly identified as the CGTase by zymogram after SDS-PAGE. The optimum pH and temperature for the enzyme activity were 8.0 and 55℃, respectively. The enzyme was stable at the range of pH 5.5~9.0, and up to 50℃. The amino acid sequence from the NH₂-terminal of the purified CGTase was Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln. The yields of the products from starch as the substrate were 1.3% for α-,33.9% for β-, and 9.7% for γ-cyclodextrin.

호알칼리성 Bacillus sp. No. 4의 Cyclodextrin Glycosyltransferase에 의한 Glycosyl Sucrose의 생산과 저충치성 당으로서의 응용

손천배,유미경,김명희,문숙경 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Action of a cyclodextrin glycosyltransferase (CGTase) produce from alkalophilic Bacillus sp. No. 4 was studied in a solution containing starch and sucrose to prepare glycosyl sucrose syrup with good sweetness and antidecaying properties of teeth. In the initial stage of the reaction the CGTase produced cyclodextrin, however, the cyclodextrin disappeared and glycosyl sucrose was formed with the lapse of reaction time. The best proportion of sucrose to starch for prodution of glycosyl sucrose was about 1 : 1. The optimum pH and temperature of the coupling reaction was pH 6.0 and 60℃, respectively. Main composition of glycosyl sucrose syrup prepared with 20% starch and 20% sucrose was sucrose 18%, glucosyl sucrose (G_2F) 15.3% and amltosyl sucorse (G_3F) 11.3%. And glucose, maltose and maltotriose were produced very little. Smaller amounts of acid and insoluble glucan were formed in the syrup by Streptococcus mtans OMZ176 than in the sucrose. Therefore, the prepared glycosyl sucrose sucrose syrup is expected to prevent teeth from decaying.

Bacillus stearothermophilus KY-126가 생산하는 Cyclodextrin glycosyltransferase의 정제 및 특성

강상모,유시형,Kang, Sang-Mo,Yoo, Si-Hyung 한국식품과학회 1994 한국식품과학회지 Vol.26 No.4

토양을 대상으로 하여 CGTase를 생산하는 균주를 분리, 선별하여 Bacillus stearothermophilus KY-126을 얻었다. CGTase의 정제는 ammonium sulfate precipitation, ion exchange chromatography, gel filtration의 과정을 통해 분리 정제하여 단일 효소를 얻었으며, 분자량은 약 67,000이었다. 효소 반응의 최적 온도는 $65^{\circ}C$였으며, $55^{\circ}C$에서 30분간 열처리에도 비교적 열에 안정하였다. 최저 활성 pH는 5.5였고 pH5.5에서 10.5까지 비교적 안정하였다. $HgCl_{2}$에 의해 저해를 받았으며, 그 외의 금속 이온에는 저해를 받지 않았다. Soluble starch로부터 CD의 전환율은 43%이었으며, ${\alpha}-:,\;{\beta}-:,\;{\gamma}-$, CD의 생성 비율은 2.9 : 2.1 : 1이었다. A bacterial strain No. KY-126, which produced extracellular cyclodextrin glycosyltransferase(CGTase), was isolated from soil and identified as Bacillus stearothermophilus KY-126. The enzyme was purified by the treatments of ammonium sulfate precipitation, DEAF-Sephadex, Sephadex G-100 column chromatography. The optimal pH and temperature for the enzyme activity were pH 5.5 and $65^{\circ}C$, respectively. And the enzyme was stable at pH values from 6.0 to 11.0 at $55^{\circ}C$ for 30 min and stable up to $60^{\circ}C$ for 30 min.. The enzyme was inhibited by $HgCl_{2}$. The molecular weight of the enzyme was estimated to be 67,000 by using SDS-PAGE. The maximum conversion from starch to cyclodextrin (CD) by CGTase was 43% and obtained at 6 hr reaction and the ratio of ${\alpha}-,\;{\beta}-,\;{\gamma}-$, CD production at this time was 2.9 : 2.1 : 1.0.

Bioactive Compounds / Food Microbiology : < Note >: Biochemical Characterization of Recombinant UDP-Glucose:Sterol 3-O-Glycosyltransferase from Micromonospora rhodorangea ATCC 31603 and Enzymatic Biosynthesis of Sterol-3-O-β-Glucosides(s)

( Nguyen Huu Hoang ),( Sung Yong Hong ),( Nguyen Lan Huong ),( Je Won Park ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.3

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3- O-β-D-glucoside, campesterol-3-O-β-D-glucoside, and cholesterol-3-O-β-D-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

Codon optimized expression of difficult to express first glycosyltransferase–KanF gene of Kanamycin biosynthetic pathway in E. coli

Hue Thi NGUYEN,Amit Kumar CHAUDHARY,Anaya Raj POKHREL,Jae Kyung SOHNG 한국생물공학회 2014 한국생물공학회 학술대회 Vol.2014 No.10