Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

  • Kang, Han-Chul (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Yoon, Sang-Hong (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Chang-Muk (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Koo, Bon-Sung (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration)
  • Received : 2011.07.27
  • Accepted : 2011.10.14
  • Published : 2011.12.31
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Abstract

A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring $6{\times}histidine$ tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

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References

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