Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli.
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.
Chao
Y P
YP
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan. ypchao@fcu.edu.tw
Chiang
C J
CJ
Lo
T E
TE
Fu
H
H
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics
D018835:Chaperonin 10; D018834:Chaperonin 60; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C020047:dihydropyrimidinase; C063842:dnaK protein, E coli