Vol.:(0123456789) 1 3 Waste Biomass Valor DOI 10.1007/s12649-016-9824-6 ORIGINAL PAPER Development of a Novel Micro-Aerobic Cultivation Strategy for High Potential CotA Laccase Production Nadia A. Samak 1,2  · Jianhua Hu 1,2  · Kefeng Wang 1,2  · Chen Guo 1  · Chunzhao Liu 1   Received: 13 October 2016 / Accepted: 27 December 2016 © Springer Science+Business Media Dordrecht 2017 Introduction Bacterial laccases have a great advantage over fungal lac- cases due to their activation and stabilization at alkaline conditions, elevated temperatures and mediums sup- plemented with high concentration of copper ions [1, 2]. A thermo stable CotA laccase in Bacillus subtilis, a 65-kDa protein, takes part in the production of the mela- nin like brown spore pigment in the endospore coat. It is also responsible for the defense provided by the spore coat against H 2 O 2 and UV light [3]. CotA-laccase is 513 amino acids [4] and it is reported for industrial textile waste water detoxifcation and decolorization [5], and for catalyzing phenolic acids dimerization [6]. Low production yields of the enzyme by native strains are considered as the main obstacle of bacterial laccases applications in industry [7]. Easily handling and cultivable organisms can be used to overcome this problem and facili- tate the higher production of these enzymes [8]. Escheri- chia coli is considered the most preferable recombinant protein expression system due to its rapid growth and easy genetic manipulation and considered as the most dynamic system for industrial biocatalysts [9–11]. Culture condi- tions also have a strong efect in controlling the formation of soluble proteins in vivo. The insufcient content of cop- per in the cytoplasm upon heterologous expression of CotA laccases in E. coli is disadvantageous due to production of an inactive enzyme. This problem can be resolved by add- ing copper to the medium and expressing CotA laccase under micro-aerobic conditions for obtaining fully cop- per-loaded enzyme [12]. E. coli has the ability to grow in oxygen availability and defciency in which, it can take the advantage of oxygen defciency which exists during micro- aerobic cultivation and preserve the redox balance [13–15]. Abstract Recently, bacterial laccases has drawn research- ers’ interest due to their ability to overcome high pH and salt concentration conditions compared to fungal laccases. Here we report a novel micro-aerobic cultivation strategy for enhancing CotA laccase expression and study its appli- cation for dye decolorization. Micro-aerobic cultivation of Escherichia coli BL21 (DE3) strain carrying pT7-FLAG- MAT-TAG-1-CotA had signifcantly enhanced CotA lac- case activity up to 13903 U/L. The most unique fndings of this investigation are that micro-aerobic cultivation strategy enhanced the reactive oxygen species production which consequently led to the over expression of CotA lac- case gene. Malachite green, Crystal violet, Congo red and Bromophenol blue were efciently decolorized by using purifed CotA laccase without presence of any mediators at pH 6 and 9. These results provide a great platform for the dynamic production and application of bacterial laccase in industry. Keywords Bacillus subtilis · Bacterial laccase · CotA · IPTG · Micro-aerobic condition · ROS * Chunzhao Liu czliu@ipe.ac.cn 1 State Key Laboratory of Biochemical Engineering & Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China 2 University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China