Substrate Specificity of the Bacillus Licheniformis Lyxose Isomerase YdaE and Its Application in in Vitro Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization

Appl Environ Microbiol. 2011 May;77(10):3343-50. doi: 10.1128/AEM.02693-10. Epub 2011 Mar 18.

Abstract

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on D-lyxose, suggesting that the enzyme is D-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²+. The apparent K(m) values for D-lyxose and D-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k(cat)/K(m)) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for D-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of D-lyxose and D-glucose from d-xylose and D-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM D-lyxose and D-mannose, 3.7 mM and 3.8 mM D-lyxose and D-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus / enzymology*
  • Bacillus / genetics
  • Coenzymes / metabolism
  • Enzyme Stability
  • Escherichia coli / genetics
  • Gene Expression
  • Glucose / metabolism*
  • Hydrogen-Ion Concentration
  • Isomerases / chemistry
  • Isomerases / genetics
  • Isomerases / metabolism*
  • Kinetics
  • Manganese / metabolism
  • Mannose / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Pentoses / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Temperature
  • Thermus thermophilus / enzymology
  • Xylose / metabolism

Substances

  • Coenzymes
  • Pentoses
  • Recombinant Proteins
  • Manganese
  • lyxose
  • Xylose
  • Isomerases
  • Glucose
  • Mannose