A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

Abstract

A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 degrees C, respectively. The enzyme retained a high activity at 45-60 degrees C, while it could be quickly inactivated by a heat treatment at 80 degrees C for 15 min, exhibiting a half-life of 8 min at 70 degrees C. The K(m) and V (max) for pNPP were determined to be 31.5 muM and 430 muM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.

A thermo‐alkaline lipase from a new thermophile Geobacillus thermodenitrificans AV‐5 with potential application in biodiesel production

“…Here we describe a thermostable lipase from a newly isolated Geobacillus thermodenitrificans AV5 found in Yellowstone National Park (YNP), WY. Geobacillus thermodinitrificans has been researched for its long‐chain alkane‐degrading capabilities, as a metal‐binding sorbent for bioremediation of industrial waste waters, as source of extracellular polysaccharides with antiviral activities, of bacteriocins of medical importance, of thermostable carboxylesterase, α‐glucosidase, arabinose isomerase, phosphatase, and xylanases . Although lipase production and purification from G .…”