Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Characterization of Calcium- and Zinc-Tolerant Xylose Isomerase from Anoxybacillus kamchatkensis G10

  • Park, Yeong-Jun (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Jung, Byung Kwon (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Hong, Sung-Jun (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Park, Gun-Seok (Department of Biomedical Engineering, The University of Texas at Austin) ;
  • Ibal, Jerald Conrad (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Pham, Huy Quang (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Shin, Jae-Ho (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University)
  • Received : 2017.12.11
  • Accepted : 2018.02.06
  • Published : 2018.04.28
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Abstract

The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-${\beta}$-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was $80^{\circ}C$ and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at $60^{\circ}C$. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of $Ca^{2+}$ and $Zn^{2+}$ ions. The kinetic properties, $K_m$ and $V_{max}$, were calculated as 81.44 mM and $2.237{\mu}mol/min/mg$, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.

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