Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6

J Biotechnol. 1996 Nov 15;51(3):259-64. doi: 10.1016/s0168-1656(96)01604-5.

Abstract

Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6. Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 degrees C and was used successfully on an industrial-scale mill trial. To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli. The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized. The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step. Over 2 g soluble and active enzyme per 1 culture were achieved.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Biotechnology
  • Cloning, Molecular
  • DNA Primers / genetics
  • Enzyme Stability
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Bacterial
  • Genetic Vectors
  • Geobacillus stearothermophilus / enzymology*
  • Geobacillus stearothermophilus / genetics*
  • Molecular Sequence Data
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Temperature
  • Xylan Endo-1,3-beta-Xylosidase
  • Xylosidases / genetics*
  • Xylosidases / isolation & purification*

Substances

  • DNA Primers
  • Recombinant Proteins
  • Xylosidases
  • Xylan Endo-1,3-beta-Xylosidase