• Current Research on Agriculture and Life Sciences
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• v.11
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• pp.81-89
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• 1993

In order to isolate a mutant which was constitutively expressed in xylA gene, Pxyl-cat-xylA fusion gene was constructed by the insertion of cat gene between xylA promoter and xylA structural gene in pEX13 contained xylA gene. The expression of cat and xylA gene from transformants of xylA mutant DH77 with plasmid pEXC131 containing Pxyl-cat-xylA fusion gene was induced by the addition of 0.4% xylose to media. This results indicated that cat and xylA gene were expressed under control of xylA promoter the presence of xylR gene. We have also isolated constitutive mutant plasmid pEXC131-39 from pEXC131 by trementment with N-methyl-N'-nitro-N-nitrosoguanidine(NTG). cat and xylA gene from pEXC131-39 were constitutively expressed without induction of xylose regardless of xylR gene. Transformants of xylR mutant DH60 with pEXC131-39 also expressed chloramphenicol resistances and xylose isomerase without induction of xylose. This result shows that mutation in region of xylA promoter might make it possible to be constitutively expressed.

• Current Research on Agriculture and Life Sciences
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• v.10
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• pp.125-135
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• 1992

Nine strains of xyl mutants that could not utilize xylose as a carbon source were isolated from E. coli JM109 by the treatment of NTG in order to investigate the regulation of xylose operon and to use recipient cells for the cloning of xylR gene. For the characterization of all isolated mutants, colony colors of all mutants on MacConkey-xylose and MacConkey-xylulose agar plate were observed for the utilization of xylose and xylulose, and the growth level and the activity of xylose isomerase and xylulokinase were determined in need. The isolated xylR/T mutants formed the white colony on MacConkey-xy-lose and MacConkey-xylulose agar plate. They did not detect the activity of xylose isomerase, and the activity of xylose isomerase was not restored in transformants of xylR/T mutant with pEX13 which contained xylA gene. xylR and xylT mutants were classified from xylR/T mutants depending upon the growth level in minimal medium. xylT mutants; DH13, DH121 and DH125 could grow a little in that medium, but xylR mutants; DH10, DH53, and DH60 could not grow that medium.

• BMB Reports
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• v.31 no.6
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• pp.600-603
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• 1998

A highly thermostable xylose isomerase from Thermus thermophilus has been expressed in Escherichia coli and crystallized. The purified enzyme shows its optimum temperature at $90^{\circ}C$. It has been crystallized at room temperature using polyethylene glycol 4000 as the precipitant. The crystal belongs to the orthorhombic space group $P2_12_12_1$, with unit cell parameters of a = 73.34 ${\AA}$, b = 144.05 ${\AA}$, c = 155.07 ${\AA}$. The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass ($V_m$) of 2.32 ${\AA}^3/Da$ and the solvent content of 47.0% by volume. The diffraction pattern extends to 1.9 ${\AA}$ Bragg spacing with synchrotron radiation and a set of native data has been collected to 2.3 ${\AA}$.

• Journal of Microbiology
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• v.43 no.1
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• pp.34-37
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• 2005

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was $85^{\circ}C$ and the enzyme exhibited a high level of heat stability.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.15-22
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• 1997

A bacterial strain J-59 was isolated from a humus soil, which produced simultaneously a thermostable glucose isomerase as well as xylanase. The morphological, cultural and physiological characteristics of the isoisomerase strain J-59 were detemined by the use of the media and methods described in International Streptomyces Project. The chemotaxonomic characteristics of the isolated strain J-59 were determined by the analysis of G+C molar % of DNA, diaminipimelic acid, composition of fatty acid and menaquinone. As the results of various examinations, the strain J-59 was identified to be Streptomyces chibaensis. This strain produced glucose isomerase intracellularly and xylanase extracellularly when grown in a medium containing xylan, but it was not able to utilize the xylose or xylan as a carbon source. The glucose isomerase of S. chibaensis J59 was highly thermostable, which retained more than 75% activity in the presence of Co$^{2+}$ at 80$\circ $C for 72 h.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.163-167
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• 2012

Since D-xylose is not fermentable in Saccharomyces cerevisiae, its conversion to D-xylulose is required for its application in biotechnological industries using S. cerevisiae. In order to convert D-xylose to D-xylulose by way of an enzyme immobilized system, D-xylose isomerase (XI) of Escherichia coli was fused with 10-arginine tag (R10) at its C-terminus for the simple purification and immobilization process using a cation exchanger. The fusion protein XIR10 was overexpressed in recombinant E. coli and purified to a high purity by a single step of cation exchange chromatography. The purified XIR10 was immobilized to a cation exchanger via the electrostatic interaction with the C-terminal 10-arginine tag. Both the free and immobilized XIR10 exhibited similar XI activities at various pH values and temperatures, indicating that the immobilization to the cation exchanger has a small effect on the enzymatic function of XIR10. Under optimized conditions for the immobilized XIR10, D-xylose was isomerized to D-xylulose with a conversion yield of 25%. Therefore, the results of this study clearly demonstrate that the electrostatic immobilization of XIR10 via the interaction between the 10-arginine tag and a cation exchanger is an applicable form of the conversion of D-xylose to D-xylulose.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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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.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1975.12a
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• pp.181.1-181
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• 1975

토양에서 분리한 glucose isomerase를 생성하는 Streptomyces속 균주중에서 xylanase 활성이 가장 높은 균주 Streptomyces sp. K-53을 xylan을 함유한 영양배지에서 배양하여 xylan에 의한 xylanase의 유도 과정과 xylan의 분해산물이 xylose를 이용하여 glucose isomerase를 생성하는 과정의 일연의 관계를 알아보기 위해서 몇가지 실험한 결과는 다음과 같다.(중략)

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1976.10a
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• pp.190.3-190
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• 1976

본 균주가 생성하는 glucose isomerase에 관한 제성질에 대해서는 전보에 이미 발표한 바 있다. 금반에는 isomerase 생성에 있어서 제요인의 검토의 일환으로 비증식상에서 inducer로서 0.5％ Xylose를 첨가해 주었을 때의 효소생성능에 관하여 검토하였다.(중략)

• Journal of the East Asian Society of Dietary Life
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• v.7 no.1
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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/$\ell$).