title: Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.
abstract: Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development.
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstr.5, 9014 St. Gallen, Switzerland.
authors.forename: Julian
authors.initials: J
authors.lastname: Ihssen
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstr.5, 9014 St. Gallen, Switzerland.
authors.forename: Renate
authors.initials: R
authors.lastname: Reiss
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstr.5, 9014 St. Gallen, Switzerland.
authors.forename: Ronny
authors.initials: R
authors.lastname: Luchsinger
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstr.5, 9014 St. Gallen, Switzerland.
authors.forename: Linda
authors.initials: L
authors.lastname: Thöny-Meyer
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biointerfaces, Lerchenfeldstr.5, 9014 St. Gallen, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Richter
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D042845:Laccase