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Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter
Authors:Alberto Buson  Alessandro Negro  Luigi Grassato  Massimo Tagliaro  Marina Basaglia  Claudio Grandi  Angelo Fontana  Marco P. Nuti
Affiliation:CRIBI Biotechnology Centre, University of Padua, Via Trieste 75, 35121 Padua, Italy
Abstract:Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro14→Leu14 exchange leads to an inactive enzyme species, while a Cys279→Ser279 exchange did not impair the functional properties of the enxyme.
Keywords:d-Carbamoylase    d-Amino acid    Agrobacterium radiobacter    Escherichia coli expression    cauA
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