Gene cloning, expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus KP1289

Appl Microbiol Biotechnol. 2003 Aug;62(2-3):180-5. doi: 10.1007/s00253-003-1261-3. Epub 2003 Feb 22.

Abstract

The gene ( inuA) encoding exo-inulinase (EC 3.2.1.80) was cloned from the thermophilic Geobacillus stearothermophilus ( Bacillus stearothermophilus) KP 1289 growing at between 41 degrees C and 69 degrees C. The inuA gene consisted of 1,482 bp encoding a protein of 493 amino acids. The deduced polypeptide of molecular mass ( M) 56,744 Da showed strong sequence similarity to Pseudomonas mucidolens exo-inulinase, Bacillus subtilis levanase, Paenibacillus polymyxa ( Bacillus polymyxa) fructosyltransferase, and so on, indicating that the enzyme belonged to glycosyl hydrolase family 32. The M of the purified exo-inulinase, expressed in Escherichia coli HB101, was estimated as approximately 54,000 Da by both SDS-PAGE and gel filtration. These results suggested that the active form of the enzyme is a monomer. The enzyme was active between 30 and 75 degrees C with an optimum at 60 degrees C. The properties were identical to those of the native enzyme. Additionally, for the first time for a prokaryotic GH32 protein, crystals of the recombinant enzyme were obtained.

MeSH terms

  • Amino Acid Sequence
  • Bacillaceae / enzymology*
  • Bacillaceae / genetics*
  • Base Sequence
  • Cloning, Molecular
  • Crystallization
  • DNA Probes
  • DNA, Bacterial / genetics
  • Enzyme Stability
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Bacterial*
  • Hexosyltransferases / genetics*
  • Hexosyltransferases / isolation & purification*
  • Hexosyltransferases / metabolism
  • Hot Temperature
  • Molecular Sequence Data
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • DNA Probes
  • DNA, Bacterial
  • Recombinant Proteins
  • Hexosyltransferases
  • inulin fructotransferase (depolymerizing)

Associated data

  • GENBANK/AB086444