Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

J Ind Microbiol Biotechnol. 2010 Apr;37(4):401-6. doi: 10.1007/s10295-009-0686-9. Epub 2009 Dec 29.

Abstract

A gene encoding the thermostable raw starch digesting alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZalphaA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-beta-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.

MeSH terms

  • Actinomycetales / enzymology*
  • Actinomycetales / genetics
  • Cloning, Molecular
  • Cycas / chemistry
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • Enzyme Stability
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Gene Expression*
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Molecular Sequence Data
  • Molecular Weight
  • Pichia / genetics*
  • Protein Stability
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Analysis, DNA
  • Starch / metabolism*
  • alpha-Amylases / biosynthesis*
  • alpha-Amylases / chemistry
  • alpha-Amylases / genetics

Substances

  • DNA, Fungal
  • Fungal Proteins
  • Recombinant Proteins
  • Starch
  • alpha-Amylases

Associated data

  • GENBANK/DQ473479