Efficient solubilization, purification of recombinant extracellular alpha-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli

J Ind Microbiol Biotechnol. 2007 Mar;34(3):187-92. doi: 10.1007/s10295-006-0185-1. Epub 2006 Nov 22.

Abstract

The gene encoding the Pyrococcus furiosus extracellular alpha-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant alpha-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90 degrees C treatment for 3 min in Britton-Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher than previously reported.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enzyme Stability
  • Escherichia coli / genetics*
  • Escherichia coli / ultrastructure
  • Hydrogen-Ion Concentration
  • Inclusion Bodies / enzymology*
  • Pyrococcus furiosus / enzymology*
  • Recombinant Proteins / isolation & purification
  • Temperature
  • alpha-Amylases / genetics
  • alpha-Amylases / isolation & purification*
  • alpha-Amylases / metabolism

Substances

  • Recombinant Proteins
  • alpha-Amylases