• Journal of Applied Biological Chemistry
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• 제44권3호
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• pp.113-118
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• 2001

Xylose (glucose) isomerase was purified to homogeneity from cell-extracts of Streptomyces chibaensis J-59 via ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, and gel filtration on Sephacryl S-300. The purified enzyme is a homotetramer with a native molecular mass of 180 kDa and a subunit molecular mass of 44 kDa. The amino acid N-terminal sequence of glucose isomerase from S. chibaensis J-59 was determined to be Ser-Tyr-Gln-Pro-Thr-Pro-Glu-Asp-Arg-Phe-Thr-Phe-Gly-Leu. The first 14 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of glucose isomerase produced by other Streptomyces spp. The optimum pH and temperature for activity were 7.5 and 85, respectively. The purified enzyme required $Mg^{2+}$, $Co^{2+}$, and $Mn^{2+}$ for the activity, $Mg^{2+}$ being the most effective. The enzyme was not inhibited by $Ca^{2+}$, but was inhibited by $Hg^{2+}$, $Ag^+$, and $Cu^{2+}$. The $K_m$, $V_{max}$, and $k_{cat}$ values of S. chibaensis J-59 isomerase for glucose were 83 mM, 40.9 U/mg, and $1,843min^{-1}$, respectively. In the presence of $Co^{2+}$, cell-free enzymes retained 100% without loss of activities by the heat-treatment at $70^{\circ}C$ for 7 days. The enzyme retained 50% residual activity after heating at $85^{\circ}C$ for 13.5 h, at $90^{\circ}C$ for 126 min. The enzyme is more thermostable than any other glucose isomerases of Streptomyces spp.

• Applied Biological Chemistry
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• 제36권2호
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• pp.86-92
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• 1993

무에서 DEAE Bio-Gel, Con-A 및 Superose-6 칼럼을 이용하여 myrosinase를 정제하고 그 효소학적 특성을 검토한 결과 myrosinase(II)는 2개의 subunits를 가졌으며, 이들의 분자량은 SDS-GAGE상에서 각각 53 및 39 KD였다. 정제된 무효소의 비활성도는 37,500 units/mg이었으며, 정제도는 44배였다. 최적 활성 pH는 phosphate 및 Tris-HCl 완충액에서 $6.5{\sim}7.0$이었으며, pH7.0에서 그 효소활성이 가장 안정하였다. 활성 최적온도는 $37{\sim}38^{\circ}C$였고, 열안정성은 $30^{\circ}C$ 이하였다. 무 myrosinase에 대한 무기염의 영향은 구리 및 수은 이온은 효소 활성을 매우 저해하며, ascorbic acid의 농도별 영향은 1 mM일 때 최대활성을 나타내었다. Ascorbic acid analogue에 대한 활성은 dehydroascorbic acid에 대해서는 거의 없었으며, 나머지 analogue들도 ascorbic acid보다 상당히 활성이 낮았다. 무 myrosinase에 대한 환원제의 영향은 2-mercaptoethanol과 dithiothreitol에 의해서는 활성을 나타내지 않았으나, 이들을 ascorbic acid 등 2-mercaptoethanol과 함께 첨가하면 활성을 나타내었다.

• Applied Biological Chemistry
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• 제33권1호
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• pp.73-78
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• 1990

한국산 토마토로부터 endo-polygalacturonase를 gel filtration과 이온교환크로마토그라피에 의하여 약 24배 정제할 수 있었고, 최대 효소활성을 위한 PH는 5.0, 최적온도는 $50^{\circ}C$ 였으며 pH안정범위는 $4.0{\sim}5.0$, 열안정성은 $50^{\circ}C$에서 1시간 열처리 하였을 때 약 45 % 실활 되었다. 정제된 이 효소는 SDS-polyacrylamide gel 전기영동에 의하여 단일밴드로 확인되었으며, 그 분자량은 50,000정도였고, Km값은 $1.43{\times}10^{-1}\;mol/l이었다. 금속이온중 $Ag^+$, $Zn^{++}$이온이 효소의 활성을 촉진시켰으며, $Na^+$, $K^+$등의 이온에 의해서는 활성이 저해되었다.

• Journal of Applied Biological Chemistry
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• 제60권3호
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• pp.257-263
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• 2017

A cellulolytic strain Y2 was isolated from soil obtained in the Canadian Alpine region. The isolate was identified as Paenibacillus sp. Y2 by 16S rRNA sequencing. When grown in LB medium supplemented with carboxymethyl-cellulose (CMC), CMCase production increased to 122.0% of that observed in LB without CMC. Culture supernatant was concentrated by ultrafiltration and 80% ammonium sulfate precipitates were separated by Hi-Trap Q and CHT-II chromatography. The purified enzyme (EG-PY2) showed a homogeneous single band and the molecular mass was estimated to be 38 kDa by SDS-PAGE. Optimum pH and temperature of the enzyme were 4.5 and $30^{\circ}C$, respectively. The half-life of enzyme activity at 50 was 140.7 min, but the enzyme was drastically inactivated within 5 min at $55^{\circ}C$. The enzyme was highly activated to 135.7 and 126.7% by 5.0 mM of $Cu^{2+}$ or $Mg^{2+}$ ions, respectively, and moderately activated by $Ba^{2+}$ and $Ca^{2+}$ ions, whereas it was inhibited to 76.8% by $Fe^{2+}$, and to ${\leq}50%$ by $Mn^{2+}$, $Co^{2+}$, $Zn^{2+}$, and EDTA. The enzyme was activated to 211.5% in the presence of 0.5 M of NaCl and greatly tolerant to 3.15M of NaCl. The enzyme showed 2.98 times higher ${\beta}$-glucanase activity than CMCase activity. Based on these results, it can be concluded that EG-PY2 is an acidophilic, cold-active, and halotolerant endoglucanase. The authors suggest it is considered to be useful for various industrial applications, such as, fruit juice clarification, acidic deinking processes, high-salt food processing, textile and pulp industries, and for biofuel production from seaweeds.

• Applied Biological Chemistry
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• 제14권1호
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• pp.1-7
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• 1971

과실쥬우스용 효소제를 개발할 목적으로 펙틴분해력이 강한 미생물균주를 선택하고 이에서 얻은 효소제에 의하여 포도쥬우스 및 포도주 제조과정에 있어서의 효과를 시험하여 다음과 같은 결과를 얻었다. 1) Aspergillus 및 Rhizopus속 균주에서 펙틴분해효소 생산력이 가장 강한 Aspergillis niger를 선정 하였다. 2) 이 균주에 의한 효소의 생성과정과 정제과정을 실험하고 조효소제품의 활성과 안정도에 미치는 pH 및 온도의 영향을 조사하였다. 3) 포도쥬우스 제조시에 효소제를 사용하면 free run juice의 수율이 증가하고 투명한 쥬우스를 얻을 수 있었다. 4) 포도주 양조시에 효소제를 사용하면 적포도주에는 아무 효과를 가지지 못하나 백포도주의 경우에는 과즙의 여과촉진 및 청정효과가 있을것으로 기대된다.

• Applied Biological Chemistry
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• 제20권1호
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• pp.123-129
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• 1977

효모세포벽 분해효소를 생산하는 386주(株)의 미생물을 분리하여 그중 강한 1주(株)를 선발 동정(同定)하고 선정균의 효소생산조건을 검토하여 다음과 같은 결과를 얻었다. 1. 선정한 M-10 strain은 Humicola sp.로 동정(同定) 되었다. 2. 선정균의 효소생성은 pH 6.0 $33^{\circ}C$에서 가장 양호하였다. 3. 탄소원으로서 baker's yeast 4%가 Iytic enzyme 생산에 가장좋았고 기타 laminarin과 dextrin 등이 효과적이었으며 질소원도 peptone이 다소 효과적이나 baker's yeast로서 족하였다. 4. 효소생산에 $K_2HPO_4$ 0.1%와 $MgSO_4{\cdot}7H_2O$ 0.01%의 첨가가 가장 효과적이고 기타 염류의 첨가는 효과가 없었다. 5. 선정한 배지조성에서 72시간 진탕배양으로 최고에 달하였다.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.628-636
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• 2012

Raw-starch-digesting enzyme (RSDA) was immobilized on Amberlite beads by conjugation of glutaraldehyde/polyglutaraldehyde (PG)-activated beads or by crosslinking. The effect of immobilization on enzyme stability and catalytic efficiency was evaluated. Immobilization conditions greatly influenced the immobilization efficiency. Optimum pH values shifted from pH 5 to 6 for spontaneous crosslinking and sequential crosslinking, to pH 6-8 for RSDA covalently attached on polyglutaraldehyde-activated Amberlite beads, and to pH 7 for RSDA on glutaraldehyde-activated Amberlite. RSDA on glutaraldehyde-activated Amberlite beads had no loss of activity after 2 h storage at pH 9; enzyme on PG-activated beads lost 9%, whereas soluble enzyme lost 65% of its initial activity. Soluble enzyme lost 50% initial activity after 3 h incubation at $60^{\circ}C$, whereas glutaraldehyde-activated derivative lost only 7.7% initial activity. RSDA derivatives retained over 90% activity after 10 batch reuse at $40^{\circ}C$. The apparent $K_m$ of the enzyme reduced from 0.35 mg/ml to 0.32 mg/ml for RSDA on glutaraldehyde-activated RSDA but increased to 0.42 mg/ml for the PG-activated RSDA derivative. Covalent immobilization on glutaraldehyde Amberlite beads was most stable and promises to address the instability and contamination issues that impede the industrial use of RSDAs. Moreover, the cheap, porous, and non-toxic nature of Amberlite, ease of immobilization, and high yield make it more interesting for the immobilization of this enzyme.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권3호
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• pp.155-162
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• 2002

The cyclic amidohydrolase family enzymes, which include allantoinase, dihydroorotase, dihydropyrimidinase and (phenyl)hydantoinase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in vivo. Each enzyme has been independently characterized, and thus well documented, but studies on the higher structural traits shared by members of this enzyme family are rare due to the lack of comparative study. Here, we report upon the expression in E. coli cells of maltose-binding protein (MBP)- and glutathione S-transferase (GST)-fused cyclic amidohydrolase family enzymes, facilitating also for both simple purification and high-level expression. Interestingly, the native quaternary structure of each enzyme was maintained even when fused with MBP and GST. We also found that in fusion proteins the favorable biochemical properties of family enzymes such as, their optimal pHs, specific activities and kinetic properties were conserved compared to the native enzymes. In addition, MBP-fused enzymes showed remarkable folding ability in-vitro. Our findings, therefore, suggest that a previously unrecognized trait of this family, namely the ability to functional fusion with some other protein but yet to retain innate properties, is conserved. We described here the structural and evolutionary implications of the properties in this family enzyme.

• Journal of Applied Biological Chemistry
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• 제44권4호
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• pp.167-172
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• 2001

A cDNA fragment encoding the chloroplastic fructose-1, 6-bisphosphatase (FBPase) was cloned via PCR from the cDNA library of pea leaves. The cloned cDNA, about 1.05 kbp without signal sequence, was introduced into a pET-28a vector for expression in E. coli strain BL21(DE3)pLysS. The recombinant FBPase was purified through $Ni^+-NTA$ affinity chromatography and characterized. Molecular mass of the monomer was about 42,000. Enzymatic activity of the purified enzyme as the native pea chloroplastic FBPase was the highest at alkaline pH (pH 9.0). The recombinant enzyme was activated by a reducing agent DTT and was insensitive to AMP. The activation energy (Ea) and Arrehenius frequency factor were 42.67 kcal/mol and $2.65{\times}10^{14}/s$, respectively, slightly higher than those of the native enzyme. $K_M$ and $V_{max}$ were $99.98{\mu}M$ and $52.9{\mu}M/min$, respectively, which were comparable with the native enzyme.

• Journal of Applied Biological Chemistry
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• 제52권3호
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• pp.151-156
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• 2009

As part of an investigation to identify microorganisms that are biotechnologically interesting for industrial application, we isolated a bacterial strain from Chungkookjang that produces extracellular neutral lipase. In addition, the crude enzyme was characterized. This isolated strain, designated as JKA-3 was identified as Bacillus subtilis JKA-3 based on morphological, physiological and biochemical characteristics, as well as phylogenetic analysis using 16S rRNA gene sequence. The cells were rod-shaped and $0.6-0.8{\times}2.0-2.3\;{\mu}m$ in size. Optimal growth conditions were $35-40^{\circ}C$ and pH 6.0-8.0. The isolate was able to grow in up to 0-10.0% (w/v) NaCl. Optimal activity conditions of the crude lipase fraction of B. subtilis JKA-3 were pH of 7.0 at $35^{\circ}C$. This enzyme was stable in the pH ranging 6.0-8.0.