• BMB Reports
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• 제29권6호
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• pp.555-563
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• 1996

A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAE-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 11.2 nmol/min/mg protein and pi value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent $K_m$, values of 190 ${\mu}M$ and 120 ${\mu}M$ for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of $37^{\circ}C$. The enzyme activity was significantly activated by $Mg^{2+}$, $Mn^{2+}$ and $Fe^{2+}$, and inhibited severely by $Ca^{2+}$. PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, chinese hamster and rat.

• Food Science and Biotechnology
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• 제14권3호
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• pp.317-322
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• 2005

A simple and convenient method of immobilizing dextransucrase via an affinity interaction is described, along with the use of this system to synthesize leucrose. Dextransucrase was produced in sucrose-free medium by fermenting a constitutive mutant of Leuconostoc mesenteroides NRRL B-512F and was separated using an ultrafiltration membrane. The purified enzyme was free of dextran polymer, which previously was always found with the sucrose-induced enzyme. Therefore, it was possible to immobilize the enzyme on dextran-based resins using an affinity interaction. Sephadex G-200 was the best resin for immobilizing the dextransucrase and gave a fast flow rate through the packed column. The immobilized dextransucrase retained more than 80% of its specific activity after immobilization ($K_m\;=\;18.1\;mM$ and $k_{cat}\;=\;450\;sec^{-1}$ vs. 13.1 mM and $640\;sec^{-1}$, respectively, for the free enzyme). The immobilized dextransucrase showed improved stability over a pH range of 4.0 to 6.5 and at moderately high temperatures over $40^{\circ}C$. When immobilized dextransucrase was used to synthesize leucrose via the transfer reaction with sucrose and fructose, about 74% of the sucrose was converted into leucrose after one day, and the half-life of the enzyme activity was 15 days. Regeneration of the resin by supplementation with dextransucrase enabled the recovery of the initial activity of the system, but both the reaction and the flow rate were lower, probably owing to the accumulation of dextran inside the resin.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.111-116
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• 1996

Polyphenol oxidase(PPO) isolated from the crude extract of ascidian, Halocynthia roretzi, showed higher affinity for catechol than tyrosine or DL-DOPA. Successful enzyme assay could be performed at $25^{\circ}C$, 10min. by mixing 0.2ml of crude enzyme extract with 2.8ml of 0.13M catechol in 0.1M sodium phosphate buffer(pH 6.4). The specific activity of PPO which had been purified with a combination of ammonium sulfate treatment, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sepharose 6B was 13-fold disc gel electrophoresis. The activity of PPO was stable from pH 5.0 to 8.0 and showed the peak activity at pH 6.4 .The optimum reaction temperature for PPO oxidation on catechol was 35$^{\circ}C$ and those enzyme were heat stable up to 4$0^{\circ}C$. Molecular weigth of the enzyme was estimated about 170kDa. One molecule was found to be composed of gour subunits. Two of them had molecular weigh of 55kDa and the others 30kDa. The {TEX}$K_{m}${/TEX} values, {TEX}$V_{max}${/TEX} and catalytic efficiency({TEX}$V_{max}${/TEX}/{TEX}$K_{m}${/TEX}) for catechol were 0.12mM, 2.5mM/liter/min. and {TEX}$0.18min^{-1}${/TEX} respectively. The substrate affinity and electrophorectic pattern suggested that the enzyme of ascidian was considered to be not tyosine but catechol oxidase.

• 생명과학회지
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• 제22권6호
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• pp.837-843
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• 2012

본 연구는 새롭게 개발된 느티만가닥버섯의 6개 품종에 대한 형태적, 생리적 특성을 조사하고 endo-, exo-cellular 효소 활성을 측정하기 위해서 수행되었다. 국내 야생종인 Hm3-10과 일본 재배종인 Hm1-1과의 단핵균사 교배를 통하여 343개의 교배 균주를 획득하여 재배를 실시하여 58개 균주를 1차 선발하고 2차로 6개 균주를 선발하였다. 6개 선발 균주를 대상으로 배양 일수 별로 재배를 실시한 결과 배양일수가 80일 이상에서는 재배일수가 19~20일로 단축되어 최적 배양일수를 80일로 결정하였다. 80일 배양일수에서 각 품종별 형태적 특성을 검증한 결과 Hm15-3, Hm15-4, Hm17-5의 3균주가 재배에 적합한 균주로 판명되었다. 각 균주의 endo-cellular 효소 활성도를 측정한 결과, ${\alpha}$-amylase의 효소 활성도가 73.9~102,2 unit/mg protein으로 가장 높았으며, chitinase 의 효소 활성도가 8.1~13.1 unit/mg protein으로 측정되었다. Exo-cellular효소 활성도를 측정한 결과, ${\alpha}$-amylase의 효소 활성도가 5,292~1,184 unit/mg protein으로 가장 높았으며, CMCase와 Xylanase의 효소 활성도가 각각 1,140~245 unit/mg protein, 94~575 unit/mg protein으로 측정되었다. 그러나 ${\beta}$-glucosidase와 chitinase의 활성도는 비교적 낮은 활성도를 나타내었다.

• 생약학회지
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• 제30권3호
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• pp.306-313
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• 1999

Polyphenol Oxidase(PPO) was purified from an extract of Ginkgo biloba leaves by ammonium sulfate fractionation followed by sephadex G-150 column chromatography, which resulted in a 18-fold increase in specific activity. The enzyme was most active at pH 8.5 and the temperature optimum for the PPO catechol oxidation reaction was $45^{\circ}C$. Heat inactivation studies showed that heating for 7, 9 and 48 min, at 80, 70 and $60^{\circ}C$ respectively caused a 50% loss in enzymatic activity and that the enzyme was completely inactivated after heat treatment at $90^{\circ}C$ for 60 min. Km values of the PPO for catechol, hydroquinone and 4-methylcatechol derived from Lineweaver-Burk plots were $6.06\;{\times}\;10^{-4}M,\;1.02\;{\times}\;10^{-3}M,\;1.41\;{\times}\;10^{-3}M$ respectively. Of the substrates tested, 4-methylcatechol was oxidized most readily and the enzyme did not oxidize monophenols. The enzyme datalyzed browning reaction was completely inhibited in the presence of reducing reagents, namely ascorbic acid, cysteine, glutathione, 2-mercaptoethanol, potassium metabisulfite at 0.5 mM level. Sodium chloride showed very little inhibition effect on Ginkgo biloba leaves PPO. Lineweaver-Burk analysis of inhibition data revealed that the inhibition by cysteine, 2-mercaptoethanol, potassium cyanide was competitive with ki values of $1.1\;{\times}\;10^{-5}M,\;2.4\;{\times}\;10^{-5}M,\;8\;{\times}\;10^{-5}M$, respectively. Among the divalent cations, $Cu^{2+}ion$ was a strong activator on PPO and $Mn^{2+}ion$ was little or no effect on PPO activity $Ni^{2+}ion$ was an inhibitor on PPO.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.412-422
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• 2019

The present study reports the morphological and molecular characterization of the fungal strain, CMSS06 and evaluates its raw starch hydrolyzing ability in four different agricultural substrates (rice bran, banana peel, cassava tubers, and coconut water). The potential use of each agricultural substrate to replace the expensive fermentation media was evaluated with six different fermentation media: rice bran (RB), banana peel (BP), cassava starch (CS), cassava in coconut water (CSCW), cassava in modified coconut water (CMCW), and pure Coconut water (CW). The fungal strain CMSS06 was identified as Thielaviopsis ethacetica by the analysis of the ITS sequences. The T. ethacetica alpha amylase enzyme exhibited maximum alpha amylase activity at 72 h, pH 7.0, and $40^{\circ}C$ on soluble starch. This species resulted in the highest enzyme activity (mU/ml) of 26.06, 10.89, 58.82, 14.2, and 54.67 with the RB, BP, CS, CSCW, and CMCW fermentation media, respectively. The results indicate that CS can be used as a carbon substrate and CMCW can be used to accelerate the fermentation by T. ethacetica. The enzyme was partially purified by 40-60% ammonium sulphate fraction, and it showed total enzyme activity, total protein content, specific activity, purification fold, and a recovery of 2400 mU, 30 mg, 80 mU/mg, 2.7, and 71.1%, respectively. The molecular mass of the T. ethacetica alpha amylase was estimated on SDS-PAGE, and two bands around 50 kDa and 70 kDa were identified. The present study implies that T. ethacetica can produce alpha amylase, and it can be used to hydrolyze raw starch during the fermentation processes.

• Food Science and Biotechnology
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• 제18권6호
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• pp.1342-1350
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• 2009

This paper investigates the production and optimization of $\beta$-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in stirred tank bioreactor. Response surface methodology was used to investigate the effects of fermentation parameters on $\beta$-galactosidase enzyme production. Maximum specific enzyme activity of 4,622.7 U/g was obtained at the optimum levels of process variables (aeration rate 2.21 vvm, agitation speed 173.4 rpm, initial sugar concentration 33.8 g/L, incubation time 24.0 hr). The optimum temperature and pH of the $\beta$-galactosidase enzyme produced under optimized conditions were $37^{\circ}C$ and pH 7.0, respectively. The enzyme was stable over a pH range of 6.0-7.5 and a temperature range of $25-37^{\circ}C$. The $K_m$ and $V_{max}$ values for O-nitrophenol-$\beta$-D-galactopyranoside (ONPG) were 1.20 mM and $1,000\;{\mu}mol/min{\cdot}mg$ protein, respectively. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in $\beta$-galactosidase enzyme production. Hence, this study fulfills the lack of using mathematical and statistical techniques in optimizing the $\beta$-galactosidase enzyme production in stirred tank bioreactor.

• BMB Reports
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• 제41권6호
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• pp.444-447
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• 2008

Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.

• Journal of Microbiology and Biotechnology
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• 제29권7호
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• pp.1043-1052
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• 2019

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with $p-NPC_{16}$ at a pH of 8.5 and $50^{\circ}C$, and the $K_m$, $k_{cat}$, and $k_{cat}/K_m$ values were 1.05 mM, $292.95s^{-1}$ and $279s^{-1}mM^{-1}$, respectively. The lipase was highly stable at $7.5{\leq}pH{\leq}10.0$. $K^+$ and $Na^+$ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.

• 한국식품영양과학회지
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• 제15권3호
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• pp.276-285
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• 1986

능이 [Sarcodon aspratus (Berk.) S. Ito]로부터 단백질가수분해(蛋白質加水分解) 효소(酵素)를 분리 정제하여 그 특성을 조사하였다. 본 효소는 Tri-acryl CM-cellulose, Ultrogel AcA 54, Hydroxy apatite column chromatography와 preparative isoelectric focusing 방법으로 순차 정제되었고, 정제된 효소는 PAGE상에서 단일 band를 나타내었으며 활성(活性)은 62.5O.D/mg.protein으로 조효소(粗酵素)보다 8.01배 증가하였다. 작용최적(作用最適) pH는 10.1로 alkaline protease였으며 작용최적(作用最適) 온도(溫度)는 $57^{\circ}C$부근이었다. $50^{\circ}C$ 이하의 온도(溫度)와 pH $4.0{\sim}10.8$ 범위에서 안정(安定)하였으나, $60^{\circ}C$에서 30분후 26%, $65^{\circ}C$에서는 65%가 실활(失活)되었다. $Mn^{++}$은 효소의 활성(活性)을 증가시켰으나 $Hg^{++}$와 $Cu^{++}$는 현저히 저해시켰다. SDS-PAGE에 의해 분자량(分子量)은 30,100으로 측정되었고, monomer임이 확인되었다. 등전점(等電点)은 9.80이었다.