title: Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli.
abstract: BACKGROUND
  Degradation of phytic acid to inorganic phosphate in domestic animals' diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible for industrial application.
  
  
  METHODS
  The phytase gene from Bacillus subtilis DR8886 was isolated from Dig Rostam hot mineral spring in Iran and cloned into pET21(+) and pET32(+). Expression was induced with 1.5 mM IPTG and the proteins were purified.
  
  
  RESULTS
  The recombinant protein affected by thioredoxin (Trx) from pET32a-PhyC was estimated to constitute about 31% of the total soluble protein in the cells; its concentration was 3.5 µg/ml, and its maximal phytase activity was 15.9 U/ml, whereas the recombinant phytase from pET21a-PhyC was estimated to comprise about 19% of the total soluble protein; its concentration was 2.2 µg/ml, and its maximal phytase activity was 69 U/ml. The molecular masses of recombinant phytase with and without Trx were about 60 kDa and 42 kDa, respectively. Zymography confirmed that the recombinant enzymes were active. Although the concentration of the alkaline phytase expressed by pET32a was approximately 59% greater than that expressed by pET21, its phytase activity was approximately 77% less.
  
  
  CONCLUSIONS
  This study showed that the peripheral gene (Trx) encoded by the pET32a (+) vector are the principal reason for the decrease in recombinant phytase enzyme activity.
authors.affiliation: Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran ; Department of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
authors.forename: Mohammadreza
authors.initials: M
authors.lastname: Nassiri
authors.affiliation: Department of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Ariannejad
keywords: Bacillus subtilis DR8806; PhyC Gene; Thioredoxin