Overproduction of D-hydantoinase and Carbamoylase in a Soluble Form in Escherichia Coli

Appl Microbiol Biotechnol. 2000 Sep;54(3):348-53. doi: 10.1007/s002530000385.

Abstract

The production of D-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of D-hydantoinase in an active form can be achieved at low temperature (25 degrees C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, D-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing D-hydantoinase and carbamoylase in a soluble form in E. coli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / biosynthesis*
  • Amidohydrolases / chemistry
  • Amidohydrolases / genetics
  • Amidohydrolases / metabolism
  • Chaperonin 10 / genetics
  • Chaperonin 10 / metabolism
  • Chaperonin 60 / genetics
  • Chaperonin 60 / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins*
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Plasmids
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Solubility
  • Temperature
  • Thioredoxins / biosynthesis
  • Thioredoxins / genetics

Substances

  • Chaperonin 10
  • Chaperonin 60
  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Recombinant Fusion Proteins
  • Thioredoxins
  • Amidohydrolases
  • N-carbamoyl-D-amino acid amidohydrolase
  • dihydropyrimidinase
  • dnaK protein, E coli