title: Enhanced archaeal laccase production in recombinant Escherichia coli by modification of N-terminal propeptide and twin arginine translocation motifs.
abstract: Laccases are multicopper oxidases that couple the oxidation of phenolic polymers to the reduction of molecular oxygen. While an archaeal laccase has only recently been described (LccA from the culture broth of Haloferax volcanii), this enzyme appears promising for biotechnology applications based on its robust bilirubin oxidase and laccase activities as well as its ability to withstand prolonged exposure to extreme conditions. To further optimize LccA productivity and develop an option for LccA purification from whole cells, the encoding gene was modified through deletion of the twin-arginine translocation motif and N-terminal propeptide, and the modified genes were expressed in Escherichia coli. With this approach, LccA was readily purified (overall yield up to 54 %) from the soluble fraction of E. coli as a 74-kDa monomer with syringaldazine oxidizing activity as high as 33 U mg(-1). LccA proteins prepared from H. volcanii culture broth and the soluble fraction of E. coli cells were compared by ICP-AES, EPR, DSC, CD, and UV-Vis spectroscopy and found to have a similar folding pattern with T (m) values and a rich β-sheet structure analogous to other multicopper oxidases. However, in contrast to the H. volcanii-purified LccA, which was loaded with copper, copper was not fully incorporated into the type-I Cu center of E. coli purified LccA, thus, providing insight into avenues for further optimization.
authors: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA. usivakumartnau@gmail.com
authors: Sivakumar
authors: S
authors: Uthandi
authors: Laurence
authors: L
authors: Prunetti
authors: Ian Mitchelle S
authors: IM
authors: De Vera
authors: Gail E
authors: GE
authors: Fanucci
authors: Alexander
authors: A
authors: Angerhofer
authors: Julie A
authors: JA
authors: Maupin-Furlow
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D019622:Haloferax volcanii / Q000201:enzymology* / Q000235:genetics; D042845:Laccase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D003300:Copper; D001120:Arginine; D042845:Laccase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase