Purification and characterization of recombinant Acetohydroxyacid synthase catalytic subunit in Haemophilus influenzae

Kyoung Mi Noh, Kyoung Jae Choi, Joon Shik Park, Moon-Young Yoon

Research output: Contribution to journalArticle

Abstract

Acetohydroxyacid synthase (E.C. 2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by Ni2+-charged HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, MgCl2 and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at 37°C. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained. Copyright

Original languageEnglish
Pages (from-to)19-22
Number of pages4
JournalKorean Journal of Microbiology
Volume43
Issue number1
StatePublished - 2007 Aug 7

Keywords

  • Acetohydroxyacid synthase
  • Characterization
  • Haemophilus influenzae
  • Purification

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