• Journal of Microbiology and Biotechnology
• /
• 제8권3호
• /
• pp.251-257
• /
• 1998

Cycloinulooligosaccharide fructanotransferase (CFTase) which produces cyclofructan from inulin was purified 332-fold from a culture broth of Bacillus macerans CFCl. The molecular mass of the CFTase was estimated to be 110 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration, indicating that the enzyme has a monomer structure. The maximal level of enzyme activity was observed at pH 7.5 and $45^{\circ}C$. The enzyme was stable in the pH range 6.0 to 9.5, and at temperatures up to $45^{\circ}C$ for 1 h. The enzyme activity was completely inhibited in the presence of 0.5 mM $Ag^+\;or\;Cu^2+$ ion. None of sucrose (GF), l-kestose (GF2), or nystose (GF3) were found to be substrates for the CFTase, but inulooligosaccharides larger than nystose were attacked by the enzyme. The CFTase catalyzes not only the cyclization as the major reaction, but also disproportionation and coupling reactions involving intermolecular transfructosylation in the same manner as cyclodextrin glucanotransferase (CGTase) (EC 2.4.1.19).

• 한국식품과학회지
• /
• 제48권6호
• /
• pp.542-547
• /
• 2016

The thermal properties of ramie leaf ${\beta}$-amylase (RBA) were examined to develop a novel process for enzyme purification. The thermostability of RBA extract prepared from ramie leaf powder was examined at various temperatures. RBA activity decreased slightly, whereas other carbohydrate-active enzymes, such as $\small{D}$-enzyme, were rapidly inactivated during 30 min incubation at $60^{\circ}C$. When the heat-treated extract was incubated with various substrates, maltose was produced exclusively as the major product, whereas the untreated crude extract produced maltose and other maltooligosaccharides. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, fewer protein bands were observed for the heat-treated extract than the untreated extract, indicating that the thermostable RBA was partially purified and other thermolabile enzymes were eliminated. Thus, the treatment of the RBA extract at $60^{\circ}C$ for 30 min resulted in 5.4-fold purification with a recovery yield of 90%.

• KSBB Journal
• /
• 제19권5호
• /
• pp.363-367
• /
• 2004

Leuconostoc mesenteroides NRRL B-1149 produces various glucoseyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/mg) with $16\%$ yield. The 1149SPase showed a molecular size of 56 kDa on denatured $10\%$ SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2~6.5 and 37^{circ}C, respectively. It had an apparent K_{m} of 6.0 mM and K_{cat} of 1.62/s for sucrose. 1149SPase crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and $30\%$ 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

• 한국식품과학회지
• /
• 제23권1호
• /
• pp.93-97
• /
• 1991

호알칼리성 Bacillus sp. YC-335가 생산하는 CGTase의 효소학적 특성 및 작용반응을 살펴보았다. ${\alpha}-CD,\;{\beta}-CD$ 및 ${\gamma}-CD$로부터 glucosyl residues를 설탕으로 전이시키는 반응에 대한 효소의 최대 반응속도, Vmax 값은 각각 $16.13,\;21.8,\;9.8{\mu}moles glucose/min/mg\;protein$이었으며 Km 값은 각각 1.68, 0.33, 0.37 mM이었다. 효소의 전분 가수분해활성은 여러 당류에 의해 촉진되었으며 특히 전분 가수분해 산물인 maltose와 glucose에 의한 효과가 가장 좋았다. 이 효소는 ${\beta}CD$에 의해 효소의 전분 분해활성이 저해되었으며 비경쟁적 저해형식을 보였다. 또한 전분으로부터 효소작용에 의해 생성된 산물을 총당량법 및 HPLC 분석을 통해 조사한 결과 이 효소는 cyclization 작용 뿐만 아니라 transglycosylation 작용과 disproportionation 작용을 가지는 것으로 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제16권11호
• /
• pp.1809-1813
• /
• 2006

A gene corresponding to 4-${\alpha}$-glucanotransferase (${\alpha}GTase$) was cloned from the thermophilic bacterium Thermus brockianus. The nucleotide sequence analysis showed that the ${\alpha}GTase$ gene is composed of 1,503 nucleotides and encodes a polypeptide that is 500 amino acids long with a calculated molecular mass of 57,221 Da. The deduced amino acid sequences of Thermus brockianus ${\alpha}GTase$ (TBGT) exhibited a high level of similarity to the amino acid sequence of ${\alpha}GTase$ of Thermus thermophilus (86%), but low level of homology to that of E. coli (26%). The TBGT gene was overexpressed in E. coli BL21, and the corresponding recombinant enzyme was efficiently purified by Ni-NTA affinity chromatography. The enzymatic characteristics revealed that optimal pH and temperature were pH 6 and $70^{\circ}C$, respectively. Most interestingly, TBGT reacted with small oligosaccharides, especially maltotriose, to form various maltooligosaccharides by using its disproportionation activity.

• 한국잠사학회:학술대회논문집
• /
• 한국잠사학회 2003년도 International Symposium of Silkworm/Insect Biotechnology and Annual Meeting of Korea Society of Sericultural Science
• /
• pp.66-70
• /
• 2003

The first line of antioxidant defense against reactive oxygen species includes the enzymatic activity of the superoxide dismutase (SOD) that catalyzes the disproportionation of superoxide to hydrogen peroxide and water. The SOD mainly removes highly toxic $O_2$$^{[-10]}$ and also prevents $O_2$$^{[-10]}$ mediated reduction of iron and subsequent OH$^{[-10]}$ generation. Along with an interest in SOD as a first line of defense against damage mediated by the superoxide anion, the SOD1 enzyme has been subjected to investigation in the molecular and cellular level. (omitted)

• 한국과학교육학회지
• /
• 제29권8호
• /
• pp.923-933
• /
• 2009

본 연구에서는 고등학교 과학 교과서에 제시된 'pH가 효소의 활성에 미치는 영향'에 대한 실험의 문제점을 분석하였다. 본 실험은 16종의 교과서 중 5개의 교과서에 소개되어 있으며, 실험 조건을 분석하였다. 교과서 분석 결과 산성 조건은 pH 3 이하이고, 염기성 조건은 pH 11이상이었다. 우선 교과서에 제시된 실험조건을 토대로 pH 조건을 다양하게 하여 실험을 실시하였다. 교과서에 제시된 것처럼 완충 용액을 사용하지 않고 pH 조건을 맞춰주었을 때, 침의 완충 작용으로 pH 범위로 pH가 맞춰지는 현상이 발견되었다. 그래서 본 연구에서는 pH 2에서 13인 완충 용액을 이용하여 효소 활성에 대한 실험을 수행하였다. pH 2에서 4사이에서 시료는 파란색을 나타내었고 pH 5부터 pH 8 에서는 색이 없어졌다. 이는 효소활성으로 인해서 녹말이 소화된 것을 나타낸다. pH가 9에서는 옅은 파란색이 나타났는데 이는 효소활성이 감소한 것을 나타낸다. 그러나 pH가 10이상으로 더 증가했을 때 효소의 비활성으로 인해 파란색이 더 짙어질 것이라는 기대와 달리, pH 10일 때 파란색이 더 옅어졌고, pH 11이상에서는 색이 없어졌다. 이는 이렇게 강한 염기성 조건에서 효소가 활성화된다고 학생들이 잘못해석하도록 영향을 미칠 수 있는 사안이다. 분석 결과 ${I_3}^-$, ${I_5}^-$는 녹말-요오드 화합물에서 녹말 나선 안에 존재하는 폴리요오드화 이온의 기초가 되며 이들이 색을 띄게 되는데, 이들이 $OH^-$와 반응하여 $I^-$, HOI, ${IO_3}^-$로 분해되기 때문으로 해석되었다.

• Journal of Microbiology and Biotechnology
• /
• 제12권6호
• /
• pp.921-928
• /
• 2002

Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.