• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.8-12
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• 1997

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive ${\lambda}cI857$ gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.

• 생명과학회지
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• 제14권4호
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• pp.636-643
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• 2004

김치에서 분리된 Lactococcus sp. JK-8 균주에 의해 생산되는 D-xylose isomerase를 17배 정제하여 물리 화학적인 특성을 조사하였다 D-Xylose isomerase의 N 말단 아미노산 서열을 결정한 결과 Ala-Tyr-Phe-Asn-Asp-Ile-Ala-Pro-Ile-Lys로 확인되었고 이것은 Lactococcus 속의 D-xylose isomerase와는 유사하지 않았다. 정제된 효소의 분자량은 gel filtration의 경우 180 kDa, subunit의 분자량은 SDS-PAGE에서 45 kDa으로 측정되었고 이 효소는 4개의 subunit으로 구성된 homotetramer였다. 최적 반응 pH는 pH 7부근이었고 pH 6∼8 사이에서 안정하였다. 최적 반응온도는 7$0^{\circ}C$였고 1 mM $Mn^{2+}$의 존재 하에서는 7$0^{\circ}C$ 이상에서도 안정하였다. 이 효소도 다른 D-xylose isomerase처럼 활성과 열 안정성을 위해서 $Mg^{2+}$, $Co^{2+}$또는 $Mn^{2+}$와 같은 2가의 양이온을 필요로 하였으며, 이중 $Mn^{2+}$가 효소 활성에 가장 효과적이었다. 기질 특이성에 관한 연구에서는 D-xylose를 사용했을 때 높은 활성을 가짐을 알 수 있었다. 이 효소의 pI 값은 4.8이었고, D-xylose에 대한 Km 값은 5.9 mM이었다.

• 한국미생물·생명공학회지
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• 제8권3호
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• pp.173-180
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• 1980

Streptomyces sp. strain K-17의 포도당 이성화효소의 강력한 분비를 위해서는 inducer로서 D-xylose를 필요로 하고 있다. 그런데 D-xylose를 가하지 않고 1.0% glucose를 가한 배지에서 배양한 이성화효소 역가가 낮은 균체를 모아서 이것을 다시 0.05M인 산 완충액 (PH7.0)에 현탁시켜 0.5 % xylose를 가하여 호기적으로 해주었을 때 효소의 induction pattern을 조사한 결과 효소활성이 10시간까지는 처리 시간에 따라 직선상으로 증가하고 이에 비례해서 D-xylose의 양이 감소했으나 cell mass에 있어서는 거의 변동이 없었다. 이때 효소단백의 합성이 일어나고 있지만, 전 RNA함량에 있어서는 오히려 감소하였다. 이와 같이 질소원을 가하지 않는 non-growth phase에서도 효소단백의 합성이 일어나는 것은 세포내에 축적되어 있는 단백질의 turn-over에 의한다는 것을 starvation 실험에서 알 수 있었다. D-xylose 이외에 D-ribose, L-arabinose, D-glucose, D-mannose, citrate, succinate 및 tartrate는 inducer로서의 효과가 없었다. 효소의 induction시, D-glucose를 가했을 경우catabolite repression 이 일어났으며 succinate 나 citrate 에 의해서도 강하게 효소생성이 억제되었다. 이와 같은 현상은 growth phase에서도 마찬가지 결과를 나타내었다. Induction시, $Ba^{2+}$, $Mg^{2+}$ 및 $Co^{2+}$가 효소생성을 발전시켰으며, C $u^{2+}$, C$d^{2+}$, A $g^{+}$ 및 H $g^{2+}$ 와 같은 중금속이 효소생성을 저해하였고, mitomycin C 몇 penicillin G는 효소생성에 영향을 주지 못하였으나, actinomycin D, streptomycin, chlora-mphenicol 및 tetra cycline등에 의해 강하게 저해되었다. 또 p-CMB 및 uncoupler 인 arsenate와 2.4-DNP에 의해서도 효소생성이 저해되었다.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.571-578
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• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• 한국미생물·생명공학회지
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• 제40권2호
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• pp.163-167
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• 2012

D-Xylose는 Saccharomyces cerevisiae에서 기질로 이용될 수 없어 S. cerevisiae가 이용 가능한 D-xylulose로의 전환이 요구된다. 효소고정화 시스템을 통한 D-xylose로부터 D-xylulose로의 전환을 위해 대장균의 D-xylose 이성화효소(XI)의 카르복시 말단에 양이온 교환수지를 이용한 단순 정제 및 고정화가 가능하도록 10-arginine tag(R10)을 융합하였다. 융합단백질인 XIR10은 재조합 대장균에서 과잉발현되었고 단일 단계의 양이온 교환 크로마토그라피를 통하여 고순도로 정제되었다. 정제된 XIR10은 카르복시 말단의 10-arginine tag과의 정전기적 상호작용을 통하여 양이온 교환수지에 고정화되었다. 고정화 및 비고정화된 XIR10은 넓은 범위의 pH 및 온도에서 비슷한 D-xylose 이성화효소 활성을 보였다. 이 결과는 양이온 교환수지로의 고정화는 XIR10의 효소적 기능에 영향을 주지 않는다는 것을 나타낸다. 고정화된 XIR10의 최적화된 조건에서 D-xylose의 25%는 D-xylulose로 이성화되었다. 본 연구의 결과들은 10-arginine tag과 양이온 교환수지간의 상호작용을 통해 정전기적 고정화된 XIR10이 D-xylose로부터 D-xylulose로의 전환에 응용 가능하다는 것을 명확하게 보여주었다.

• 미생물학회지
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• 제15권1호
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• pp.9-19
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• 1977

Xylone isomerase (D-xylose ketol-isomerase, EC 5,3,1,5) have been demonstrated in the cell-free extracts of Stroptomuces canus and Streptomuces malachiticus grown in the presence of xylose. Xylose, glucose and ribose served as substrates for the enzymes of the two strains with respective $K_m$ values of 22, 130, 290 mM (S. canus) and 7,83,637 mM(S.malachiticus), and $V_max$ values of 1,000, 0.087, $\0.0222{\mu}moles/min/mg$ protein (S. canus) and 0.312, 0.083, 0.500.$\mu$moles/min/mg protein (S. malachiticus). L-Rhammose was also isomerized by the crude enzyme solutions of the two strains. The maximal activities of the two xylose-isomerases were observed at pH 7.5 and $75^{\circ}C$. The xylose isomerase activities of the two strains were activated two-three times by $Mg^{++}\;and\;Co^{++}$ as that of control, partially activated by $Ba^{++}$ and inhibited by $Ni^{++},\;Ca^{++}\;and\;Zn^{++}\$. Particulary, the addtion of $Mn^{++}$ stimulated xylose-isomerizing activities, but inhibited glucose-isomerizing activities in both strains. However, $Cu^{++}$ inhibited xylose-isomerizing activities, while stimulated glucose-isomerizing activities of the enzymes.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.757-763
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• 1999

The metal specificity of D-xylose isomerase from Streptomyces rubiginosus was examined by site-directed mutagenesis. The activation constants for metal ion ($Mg^{2+},{\;}Mn^{2+},{\;}or{\;}Co^{2+}$) of wild-type and mutant enzymes were determined by titrating the metal ion-free enzyme with $Mg^{2+},{\;}Mn^{2+},{\;}and{\;}Co^{2+}$, respectively. Substitutions of amino acids either on coordinated or around the M2 site (His-22O, Asn-185, Glu-186, and Glu-221) dramatically affected the activation constants as well as activity. A decrease of metal binding affinity was most significant in the presence of $Mg^{2+}$. When compared with the wild-type enzymes, the binding affinity of H220S and Nl85K for Mg^{2+} was decreased by 10-15-fold, while the affinity for $Mn^{2+}{\;}or{\;}Co^{2+}$ only decreased by 3-5-fold. All the mutations close to the M2 site changed their metal preference from $Mg^{2+}{\;}to{\;}Mn^{2+}{\;}or{\;}Co^{2+}$. These altered metal preferences may be caused by a relatively weak binding affinity of $Mg^{2+}$ to the enzyme. Thermal inactivation studies of mutants at the M2 site also support the importance of the M2 site geometry for metal specificity as well as the thermostability of the enzyme. Mutations of other important groups hardly affected the metal preference, although pronounced effects on the kinetic parameters were sometimes observed. This study proposes that the metal specificity of D-xylose isomerase can be altered by the perturbation of the M2 site geometry, and that the different metal preference of Group I and GroupII D-xylose isomerases may be caused by nonconserved amino acid residues around the M2 site.

• 동아시아식생활학회지
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• 제7권1호
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• pp.35-39
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• 1997

Studies on the glucose isomerase produced by alkalophilic Streptomyces sp. B-2. Glucose Isomerase (E. C. 5.3.1.5) which reversibly catalyzes reaction between D-glucose and D-fructose was demonstrated in cell free extracts of alkalophilic Streptomyces sp. B-2 isolated form soil. The maximum enzyme activity was found at glucose concentration 4(g/$\ell$) , xylose concentration 6(g/$\ell$), magnesium ion 1.0(g/$\ell$), yeast extract concentration 2.0(g/$\ell$), peptone concentration 3(g/$\ell$).

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.385-392
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• 1996

Bacillus stearotheymophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substrate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of $^{D}$-xylose isomerase, $^{D}$-xylulokinase and $^{D}$-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

• 미생물학회지
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• 제16권2호
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• pp.47-61
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• 1978

Strptomyces albus T-12 which ahd been isolated and identified in the laboratory, was selected for the studies on the cultural conditions on the production of D-xylose iosmerase and the enzymological characteristics using the partially purified enzyme. The best results in the enzyme production came from D-xylose medium than wheat bran. The divalent metla ions as $Co^{2+},\;Fe^{2+},\;Zn^{2+}\;and\;Cu^{2+}$ retard or inhibit the cell-growth at the early stages of mycelia propagations, and T-12 strain is especially sensitive to $Co^{2+}$. After 60 hours of shaking cultivation at $30^{\circ}C$ and 200 rpm, a maximum enzyme activitz, 0.49 enzyme units, was obtained. Cell-free enzyme obtained from mycelia heat-treated in the prescence of 0.5mM $Co^{2+}$, showed a 2.4-fold increase in specific than the enzyme from untreated mycelia. The specific activity of the purified enzyme through Sephadex G-150 columm showed 180 fold to the crude enzyme. The effective activators of the enzyme appeared to be $Mg^{2+}\;and\;Co^{2+}$ ions, and it exhibited the maximal enzyme activity showed at pH 7.0 and at tempersture around $80^{\circ}C$ when $Mg^{2+}\;and\;Co^{2+}$ ions were added. The enzyme isomerized D-glucose, D-xylose, D-ribose, L-arabinose, D-mannose, and L-rhamnose in the present of $Mg^{2+}\;and\;Co^{2+}$ ions as an activatiors. $Mg^{2+}\;and\;Co^{2+}$ ions were non-competitively bound at different allosterix sites of enzyme molecule. $Mg^{2+}(5mM)\;or\;Co^{2+}(1.0mM)$ protected against the thermal denaturations of the enzyme activities. The michelis constant(Km) and $V_{max}$ values of the emzyme for D-glucose and D-xylose were 0.52M, $2.12{\mu}moles/ml{\cdot}min.\;and\;0.28M,\;0.65moles/ml{\cdot}min.$, respectively.