Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications

A comparison between engineered and commercially available L-lactate oxidases from Aerococcus viridans was conducted for biosensing applications. Enzymes were adsorbed onto the surfaces of graphite electrodes modified with multi-walled carbon nanotubes. Thermostable L-lactate oxidases were cloned with a (i) N-, (ii) a C-terminal His-tag and (iii) a wild-type enzyme. Subsequently to the heterologous expression in Escherichia coil and purification, we determined the kinetic parameters of these enzymes in solution. The kinetics of the wild-type, of the N-terminally His-tagged enzyme and of the commercial L-lactate oxidase from A. viridans were studied with a classical Michaelis-Menten as well as with a substrate inhibition model, while the enzyme carrying a C-terminal His-tag showed no activity. The active enzymes were used to fabricate and comparatively investigate multi-walled carbon nanotubes-based biosensors. The enzyme kinetic results were compared with electrochemical studies. By using both spectrophotometric and amperometric techniques, the inhibition phenomenon fits better to the data especially those data related with Lox-His-N. The electrochemical data of the fabricated enzymatic biosensors showed that the N-terminally His-tagged L-lactate oxidase performed best on carboxyl-modified carbon nanotubes. The sensor based on this engineered enzyme showed the highest sensitivity and lowest detection limit in the range of L-lactate concentration 0-1 mM as well as long term stability over one month. (C) 2013 Elsevier Ltd. All rights reserved

Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

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.ISSN:2045-232