Production of D-P-HYDROXYPHENYLGLYCINE BY N-CARBAMOYL-D-amino Acid Amidohydrolase-Overproducing Escherichia coli Strains

1999 ◽  
Vol 15 (4) ◽  
pp. 603-607 ◽  
Author(s):  
Y.-P. Chao ◽  
T.-Y. Juang ◽  
J.-T. Chern ◽  
C.-K. Lee
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acid Amidohydrolase

scholarly journals Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

2007 ◽  
Vol 402 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Shimin Jiang ◽  
Chunhong Li ◽  
Weiwen Zhang ◽  
Yuanheng Cai ◽  
Yunliu Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Solubility ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Triple Mutant ◽  
E Coli ◽  
Acid Amidohydrolase

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Production of thermotolerant N-carbamyl-d-amino acid amidohydrolase by recombinant Escherichia coli

1999 ◽  
Vol 87 (2) ◽  
pp. 149-154 ◽  
Author(s):  
Hirokazu Nanba ◽  
Yasuhiro Ikenaka ◽  
Yukio Yamada ◽  
Kazuyoshi Yajima ◽  
Masayuki Takano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Escherichia Coli ◽  
Acid Amidohydrolase

scholarly journals Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli.

1988 ◽  
Vol 263 (28) ◽  
pp. 14276-14280 ◽  
Author(s):  
T Kawakami ◽  
Y Akizawa ◽  
T Ishikawa ◽  
T Shimamoto ◽  
M Tsuda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitutions
Sign in / Sign up

Export Citation Format

Share Document