Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis

Appl Microbiol Biotechnol. 2002 Jul;59(2-3):190-7. doi: 10.1007/s00253-002-1033-5. Epub 2002 Jun 8.

Abstract

A novel phytase gene ( phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene ( 168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 6-Phytase / chemistry
  • 6-Phytase / genetics*
  • 6-Phytase / metabolism
  • Amino Acid Sequence
  • Bacillus / enzymology*
  • Bacillus subtilis / enzymology*
  • Base Sequence
  • Cloning, Molecular
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data

Substances

  • 6-Phytase