Arthrobacter sp. A-6에 의한 Inulin Fructotransferase (depolymerizing)의 생산

박정복,권영만,최용진 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.1

Inulin을 분해하여 DFAIII를 생산하는 inulin fructotransferase(depolymerizing)(EC2.4.1.93)를 세포 외로 다량 생산하는 균주를 토양으로부터 분리하고 분리균(A-6)의 형태적 내지는 생화학적 특성을 조사 분석하여 Arthrobacter sp.로 동정하였다. Arthrobacter sp. A-6 분리균은 초기 pH가 5.2인 2.5% inulin, 0.1% trypton, 0.14% (NH_4)_2SO_4, 0.2% KH_2PO_4, 0.03% CaCl_2, 0.03% MgSO_4·7H_2O, 0.5 ㎎% FeSO_4·7H_2O, 0.16 ㎎% MnSO_4·H_2O, 0.2% Tween 80 조성의 배지에서 30℃, 96시간 진탕 배양했을 때 최대의 효소 생산량(약 60 units/㎖)을 나타내었다. 또한 분리균이 생산하는 조효소 형태의 inulin fructotransferase의 효소 활성 최적 pH는 5.0, 그리고 최적 온도는 60℃로 측정되었으며 본 효소에 의해 생산되는 DFA는 paper chromatography, HPLC 및 ^(13)C-NMR 분석 결과 자료가 DFAIII 표준품과 완전 일치되어 DFAIII인 것으로 확인되었다. A bacterial strain A-6 producing the high level of an extracellular inulin fructotransferase(depolycerizing)(EC 2.4.1.93) which converts inulin into di-D-fructofuranose dianhydride III(DFAIII) was isolated from soil. The isolated strain could be classified as a species belonging to the genus Arthrobacter based on its morphological and physiological characteristics identified in this work. Production of the enzyme was induced by inulin, and the highest activity was detected in the slightly acidic medium supplemented with 2.5% inulin and 0.1% trypton as a sole carbon and a nitrogen source, respectively. Under the optimal conditions, the enzyme activity in the culture supernatant reached approximately 60 units/㎖ after 96 hours of cultivation. The optimum pH and temperature for the crude enzyme preparation from Arthrobacter sp. A-6 were pH 5.0 and 60℃, respectively. The DFA produced by the action of the inulin fructotransferase was confirmed to be DFAIII by paper chromatography, HPLC and ^(13)C-NMR spectroscopy.

Arthrobactor sp. A-6의 배양과 Chicory 뿌리 추출물에서 Di-Fructofuranose Dianhydride(DFAIII)의 생산

김기은,신창훈,최용진,김찬화 한국미생물학회 2000 미생물학회지 Vol.36 No.1

Chicory 뿌리에서 Arthrobacter sp. A-6를 배양하여 inulin fructotransferase를 생산하였을 매 균주의 성장속도, 효소의 생산성을 검토하였다. 가공된 치커리 뿌리는 inulin fructotransferase의 crude enzyme solution으로 처리하였을 때 생산되는 di-fructofuranose dianhydride(DFA III)의 양을 chicory 뿌리의 가공방법에 따른 차이를 비교하였다. 우선 standard 배지에서 생산된 효소액으로 10%의 inulin이 포함된 standard inulin용액을 처리하면, 1.14mg/ml의 DFA III가 생산되었다. Chicory뿌리의 전처리방법에 따라, 같은 조건으로 반응을 진행시키는 실험을 통해 각 배지에서의 생산효율을 비교하였다. Chicory뿌리를 washing과 extraction과정을 거치지 않고 그대로 반응시켰을 경우, 2.29 mg/ml의 DFA III가 생산되어 생산성이 가장 높았는데, 이는 세척과정에서 inulin이 유실되지 않으므로, 기질로 작용하는 inulin의 양이 가장 높은 데 기인하는 것으로 생각된다. Arthrobacter sp. A-6 was cultivated and DFA III(di-fructofuranose dianhydride) was produced with inulin fructotransferase from the chicory root. The specific growth rate, yield of cell mass and yield of enzyme from the culture in variable chicory root extracts were studied and the results compared. Standard inulin solution(10%) was treated with the crude enzyme solution of inulin fructotransferase from the cell culture, 1.14mg/ml of DFA III was produced. The enzyme reactions were processed with various preparations of chicory root extracts in the same conditions. The highest yield of DFA III production(2.29 mg/ml) was obtained from the chicory roots without washing or extraction. The yield of DFA III from the washed chicory roots without extraction was at lowest(0.44 mg/ml). The production process of inulin fructotransferase and DFA III from the chicory root without prewashing or extraction steps were more efficient.

Bacillus sp. snu-7에 의한 Inulin Fructotransferase의 생산

김우표,강수일,김수일,Kim, Woo-Pyo,Kang, Su-Il,Kim, Su-Il 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.43 No.2

A bacterial strain, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose 1,2':2,3' dianhydride(DFA III), was isolated from soil and presumed as Bacillus sp.. The highest production of the enzyme was obtained by using medium containing Jerusalem artichoke extract as carbon source, peptone as organic nitrogen source, and $NH_4H_2PO_4$, as inorganic source. Under optimum condition, the enzyme activity of the culture broth supernatant reached maximal 2.61 units/ml after cultivation for 45 hrs. Inulin을 가수분해하여 di-D-fructofuranose dianhydride(DFA)를 생성하는 inulin frucroransferase를 분비하는 균주를 토양으로부터 분리하였으며, Bacillus sp로 동정하였다. 본 효소에 의하여 생성되는 DFA는 TLC 와 HPLC로 분석한 결과, fructose 두 분자가 ${\beta}\;1,2':{\alpha}2,3'$ 결합을 한 DFA III로 동정되었다. 본 균주는 탄소원으로 1.5% 돼지감자즙, 유기 질소원으로 1.0% peptone, 무기 질소원으로 $0.27%\;NH_4H_2PO_4$를 사용했을 떼 효소 생산이 2.709 units/ml로 최대였으며, inulin을 탄소원으로 사용할 경우는 1.0% yeast extract, $0.2%\;NaNO_3$를 첨가했을 때 효소 생산이 2.245units/ml로 최대를 나타내었다. 본 균주를 최적 액체 배지에서 72시간 배양한 결과 배양 45시간에 2.61 units/ml 최대 활성을 보였다.

Enterobacter sp. S45에 의한 Inulin fructotransferase의 생산

강수일,김수일 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.1

토양에서 inulin으로부터 di-D-fructofuranose dianhydride(DFA)를 생산하는 inulin fructotransferase를 생산하는 균주로 Enterobacter sp. S45를 선발, 분리하였다. 본 효소에 의하여 생산되는 DFA는 ^(13)C-nmr과 HPLC로 분석한 결과 fructose 두 분자가 β 1,2’ : α 2,3’ 결합을 한 DFA III로 동정되었다. 본 균주는 탄소원으로 inulin, 유기질소원으로 corn steep liquor, 무기질소원으로 NH_4H_2PO_4를 사용할 때 효소생산이 가장 많았으며 이 조건하에서 72시간 배양으로 최대효소생산을 보였다. 균 배양중 배양액내에 DFA III가 생성되었다가 소멸되는 것으로 보아 균체내에 DFA III를 분해, 이용하는 효소가 존재한다고 추정되었다. A bacterial strain, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose dianhydride (DFA) was isolated from soil and presumed as Enterobacter sp. The DFA isolated on Bio-gel P2 column was identified as DFA III by high performance liquid chromatography and ^(13)C-nmr spectroscopy. The enzyme production was induced by inulin and markedly enhanced by the addition of corn steep liquor and NH_4H_2PO_4 for nitrogen source. Under optimum condition, the enzyme activity in the culture broth reached at maximum, 0.22 unit/㎖ after cultivation for 72 hour.

효모 Inulin Fructotransferase (Depolymerizing)틀 이용한 Sucrose-YNB 배지로 부터의 DFAⅢ의 生成

김재근 啓明專門大學 産業開發硏究所 2000 啓明硏究論叢 Vol.18 No.2

돼지감자, 치커리, 우엉, 도라지 등의 천연식물자원에 함유되어 있는 inulin으로부터 미생물분해효소에 의한 설탕대체감미료인 DFAⅢ의 생산·개발을 위한 선행연구자료로 활용하기 위하여 inulin fructotransferase (depolymerizing) 생성능이 있는 효모균주인 Kluyveromyces marxianus var. marxianus IFO 1735를 inulin 보다 가격이 저렴한 sucrose 함유합성배지에 접종하여 어떠한 배지조성과 배양조건에서 DFAⅢ 생성이 양호한 가를 paper chromatography(PC)법으로 정성분석하였다. 이 실험에서 얻어진 결과를 요약하면 다음과 같다. DFAⅢ의 생성을 위한 sucrose-YNB 배지의 조성은 sucrose와 yeast nitrogen base(YNB)의 농도는 각각 0.5%이었으며 최적 배양조건은 초기 pH, 배양온도 및 배지량은 각각 5.0, 30℃ 및 140㎖이었다. 상기 최적조건에서 36시간 배양하였을 때 DFAⅢ의 spot가 가장 크고 뚜렷하게 나타났다. The optimal culture condition and concentration of sucrose as carbon source and of YNB as nitrogen source for the production of inulin fructotransferase (depolymerizing) by the cultivation of Kluyveromyces marxianus var. marxianus IFO 1735 in sucrose-YNB medium were investigated with spot of di-D-fructofuranose-l, 2': 2,3' dianhydride (DFAⅢ) detected by paper chromatography(PC) The results of investigation were as follows ; The culture medium for the DFAⅢ production was found to consist of 0.5% sucrose and 0.5% yeast nitrogen base(YNB). Optimal initial pH of the culture medium, culture temperature and medium volume for the DFAⅢ production were pH 5.0, 30℃ and 140㎖, respectively. When this strain was cultured for 48h in optimal conditions described above, Paper chromatogram of the DFAⅢ production was showed with the largest spot by PC

Kluyveromyces marxianus var. marxianus IFO 1735에 의한 Inulin Fructotransferase의 생산 및 이용에 관한 연구

김재근,坂井石夫 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.3

Iunlin을 di-D-fructofuranose 1,2' : 2,3' dianhydride (DFAⅢ)로 전환시키는 inulin fructotransferase (inulaseⅡ)를 생산하는 균주로 Kluyveromyces marxianus var. marxianus를 선별하였다. 공시균의 inulaseⅡ의 생성을 위한 최적 배지조성은 1.0%의 sucrose 및 0.75%의 YNB이었으며 최적 배양조건은 초기 pH, 배양온도 및 배지량은 각각 4.7, 30℃ 및 140 ㎖이었다. 상기 최적 조건으로 36시간 배양하였을 때 효소 생산량이 가장 높아 배양액 ㎖당 약 4.2 unit를 나타내었다. 본 효소의 이용성을 검토한 결과 돼지감자 추출액을 이용한 천연배지의 경우 상기 최적 배양조건에서 48시간 배양으로 DFAⅢ 생산량이 가장 높아 배양액 ㎖당 13.25 ㎎을 나타내었다. Kluyveromyces marxianus var. marxianus isolated as an inulin-assimilating microorganism produces inulin fructotransferase (inulaseⅡ) which catalyses the conversion of inulin into di-D-fructofuranose 1, 2' : 2, 3' dianhydrde (DFAⅢ). The DFA produced by the organism was isolated by using active carbon column, and identified as DFAⅢ by high performance liguid chromatography. The culture medium giving maximum inulaseⅡ producton was found to consist of 1% sucrose and 0.75% yeast nitrogen base (YNB). The inulaseⅡ production was induced by inulin or sucrose as a carbon source and increased by addition of YNB as a nitrogen source. Optimal initial pH of the culture medium, culture temperature and medium volume for the enzyme production were pH 4.7, 30℃ and 140 ㎖, respectively. Under the optimal conditions described above, the enzyme activity in the culture supernatant reached 4.2 units/㎖ after cultivation for 36 h. The DGAⅢ was accumulated at 13.25 ㎎/㎖ after 48 h of culture in the Jerusalem artichoke tuber medium.

Production of Inulin Fructotransferase(depolymerizing) from Flavobacterium sp. LC-413

Cho, Chul-Man,Lim, Young-Soon,Kang, Soo-Kyung,Jang, Kyung-Lib,Lee, Tae-Ho The Korean Society of Food Science and Nutrition 1996 Preventive Nutrition and Food Science Vol.1 No.1

A bacterial strain LC-413, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose dianhydride(DFAIII) and amount of oilgosaccharides, was isolated from soil and pre-sumed as Flavobacteium sp. LC-413. The enzyme production was induced by inulin as carbon source and enhanced by the addition of 0.3% malt extract and 0.2% {TEX}$NaNO_{3}${/TEX} as nitrogen source. The enzyme activity in the culture supernatant reached at the maximum, 78.6units/ml, after 11 hours of cultivation in the medium composition of 1.5% inulin, 0.2% {TEX}$NaNO_{3}${/TEX}, 0.05% {TEX}$K_{2}${/TEX}{TEX}$HPO_{4}${/TEX}, 0.05% {TEX}$MgSO_{4}${/TEX}.7{TEX}$H_{2}${/TEX}O, 0.05% KCI, a trace amount of {TEX}$FeSO_{4}${/TEX}.7{TEX}$H_{2}${/TEX}O, and 0.3% malt ext. at 3$0^{\circ}C$. The oilgosaccharide produced by enzyme reaction from inulin was identified as DFA III by and {TEX}${13}^C${/TEX}-NMR spectrosocpy.

Purification and Characterization of Inulin Fructotransferase (Depolymerizing) from Arthrobacter sp. A-6

( Jeong Bok Park ),( Yong Jin Choi ) 한국미생물생명공학회 1996 Journal of microbiology and biotechnology Vol.6 No.6

Microbial production of difructose anhydride III from Jerusalem artichoke tuber powder by recombinant yeast <i>Saccharomyces cerevisiae</i> and <i>Kluyveromyces marxianus</i>

Ko, Hyunjun,Bae, Jung-Hoon,Kim, Mi-Jin,Sung, Bong Hyun,Sohn, Jung-Hoon ELSEVIER 2019 INDUSTRIAL CROPS AND PRODUCTS Vol. No.

<P><B>Abstract</B></P> <P>Difructose anhydride III (DFA III), a functional sweetener, is enzymatically produced from inulin by inulin fructotransferase (IFTase; EC 4.2.2.18). Here, a recombinant yeast strain hyper-secreting IFTase from <I>Arthrobacter aurescens</I> was developed by employing the translational fusion partner technique, and the recombinant yeast produced 77.5 g (64.6% yield) of DFA III from 200 g of crude extract of Jerusalem artichoke tuber powder containing 120 g of inulin during direct fermentation. The sugar purity of DFA III was increased up to 95% by additional incubation with <I>Kluyveromyces marxianus</I>. This study is the first report of the secretory production of recombinant IFTase in <I>Saccharomyces cerevisiae</I>, and the direct conversion of DFA III from crude extract of Jerusalem artichoke tuber powder by microbial fermentation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A recombinant <I>Saccharomyces cerevisiae</I> hyper-secreting inulin fructotransferase (161.6 U/mL) was developed. </LI> <LI> The strain produced 77.5 g of DFA III from 200 g of Jerusalem artichoke tuber powder. </LI> <LI> By additional culture of <I>Kluyveromyces marxianus</I>, the DFA III content based on carbohydrates was increased up to 95%. </LI> <LI> This is the first direct production of DFA III from crude JAP by microbial fermentation. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Cloning and Expression of Inulin Fructotransferase Gene of Arthrobacter sp.A-6 in Escharichia coli and Bacillus subtilis

Kim, Hwa Young,Kim, Chan Wha,Choi, Yong Jin 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.2

The inulin fructotransferase (depolymerizing) (IFTase, EC 2.4.1.93) gene of Arthrobacter sp. A-6 was cloned and expressed in Escherichia coli and Bacillus subtilis. The IFTase gene consisted of an ORF of 1,311 nucleotides encoding a polypeptide of 436 amino acids containing a signal peptide of 31 amino acids in the N-terminus. The molecular mass of the IFTase based on the nucleotide sequence was calculated to be 46,116 Da. The recombinant E. coli DH5α cells expressing the Arthmbacter sp. A-6 IFTase gene produced most of the IFTase intracellularly. In contrast, the recombinant B. subtilis DB 104 carrying the IFTase gene on a B. subtilis-E. coli expression vector secreted the IFTase into the culture fluid efficiently.