## 7 Methods of Increasing the Productivity and Activity of Laccases
### Improvement of Lacase Productivity  
Laccases are produced by various groups of organisms from bacteria to plants. The production by bacteria and fungi is biotechnologically important. The physiological requirements of microorganisms capable of producing lacases are different. To obtain the maximum amount of enzymes, the influence of many factors, such as the carbon source, concentration and type of nitrogen source, pH, temperature, inducer and other culture conditions on lacase production is studied [106].  
The synthesis and secretion of lacase are strictly dependent on the level of nutrients (bioavailability of mainly C and N), culture conditions (temperature, aeration, reaction and light) and the development stage of the cultured microorganism, as well as the addition of inducers to the media (e.g., metal ions). Most of these factors have been shown to operate at the transcriptional level, producing different enzyme isoforms within the same strain and between different fungal species [329]. An inducer of lacase production in fungal cultures, in addition to the high nitrogen content in the medium _Basidiomycte_ I-62 (CECT 2019) and _Lentitus sajor-caju_, is the presence of copper and other metal ion or aromatic compounds structurally similar to lignin precursors [106,194].  
The cheapest way to overproduce enzymes is to optimize the fermentation conditions, and a spectacular increase in the amount of enzyme produced by a given microorganism can be achieved by changing just a few parameters. A modern method more and more willingly used to optimize the composition of media is the methodology of the response surface (RSM) [316]. With good results, RSM was used to optimize the production of lacase by _A. bisporus_ CU13 [331]; _Pseudolagarobasidium acacciola_ AGST3 [332]; and _Penicillium chrysogenum_[225].  
[PERSON] et al. (2020) optimized lacase production by T. versicolor using RSM in batch-submerged fermenters. Lignocellulosic residues were used in the culture [333]. The optimization of the culture conditions with a temperature of 35 \({}^{\circ}\)C and 5 g/L of wheat bran in the medium allowed about 200 U/mL of lacase to be obtained in 11 days in a batch reactor. The postculture fluid was used for the enzymatic degradation of wastewater, with a two-fold increase in the biodegradability index. In conclusion, as noted by the authors, lacase-mediated biodegradation may become a feasible method for the pretreatment of real wastewater.  
Wild-type hosts can only produce small amounts of enzymes and are unable to meet industrial demands. Therefore, _P. pastoris_, _Aspergillus flavus_, _A. niger_, _A. nidulans_, _Trichoderma resei_ and _Yarrowia lipolytica_ are often used for the heterologous expression of lacases [329].  
More recently, [PERSON] et al. (2021) used the insect _Drosophila melanogaster_ to functionally express a lacase from the fungus _Trametes trogii_[334]. The short tubulin promoter was used for moderate expression in many tissues of the insect, and native fungal signal peptides were replaced with the signal peptide of the D. melanogaster larval epidermal protein to facilitate extracellular secretion. lyophilized powder from processed adult flies degraded over 90% of the blue synthetic dye Acid Blue 74 in aqueous solutions. The obtained results showed that transgenic animals can be used for the bioremediation of environmental pollutants in vivo and serve as a new way of producing industrial enzymes.  
In summary, the aim of traditional methods of optimizing the production of lacases, as well as methods of genetic engineering in the heterologous expression of enzymes, is to produce as many lacases as possible with appropriate biochemical properties at very low costs [106; 329].