## 7 Methods of Increasing the Productivity and Activity of Laccases
### Improvement of Catalytic Activity of Lacases
Genetic engineering and protein engineering techniques are used to modify the thermostability, catalytic activity and substrate specificity of enzymes. Three types of enzyme modification strategies are used in this field, such as rational, semifational and directed evolution methods [87; 154]. The site-directed mutagenesis of [PERSON] et al. (2020) obtained mutant S208G/F227A Cota-Laccase _Bacillus pumilus_ W3, which showed more than five times higher catalytic efficiency than the laccase wild-type Cota and improved the methyl red decolorizing capacity [325]. Based on the method of site-directed mutagenesis, [PERSON] et al. (2019) also investigated the role of N-glycosylation in the specific laccase activity of _Coprinopsis cinerea_ Lec9 expressed in _Pichia pastoris_[326]. It turned out that the glycosylation at N313 affects the affinity of the enzyme for substrates, and the glycosylation at N45 affects the catalytic rate. Notably, N-glycosylation modifications in eukaryotic hosts can significantly improve the thermostability of lignocellulosic enzymes, as the glycan chains act as a protector by attaching to the protein surface via extensive hydrogen bonding [327]. Heterologically expressed fungal laccases exhibit different biochemical properties compared to native fungal laccases. The laccase of _Cyathus bulleri_ when expressed in _P. pastoris_, in which the glycosylation pattern was changed, increased thermal stability and salt tolerance, which is likely related to the change in protein structure and function. Rational engineering increased the substrate specificity of the laccase lccb of _T. versicolor_ for the oxidation of large polycyclic aromatic hydrocarbons without a mediator [184]. The authors used structure-based protein engineering to generate rationally modified laccases with an enhanced ability to process large PAHs without a mediator. Computational simulations were used to estimate the effect of mutations in the enzymatic binding pocket on the binding and oxidizing capacity of a selected set of organic compounds. The generated mutants with an enlarged laccase binding pocket showed increased activity without the mediator and functioned over a wider pH range compared to the wild-type enzyme. The enlargement of the binding pocket allowed macromolecular substrates to come into direct contact with the catalytic residues of the laccase, which eliminated the use of mediators for electron transport. The modified laccase degraded ethyl green, a synthetic triphenylmethane dye, by over 90% within 24 h without mediators. The researchers proposed the use of the M1 catalytic site mutant of the F162A/L164A laccase in mediator-free, difficult-to-degrade macromolecular compounds. Noncatalytic enzyme functional sites are modified according to the primary and tertiary structure of the protein, while catalytic functional sites are designed semirationally and rationally based on molecular docking [328; 329]. Using advanced molecular biology tools, i.e., next-generation sequencing, site-directed mutagenesis, fusion proteins, surface display, etc., scientists can now develop enzymes to improve activity, stability and substrate specificity to meet the stringent conditions of industrial processes and biodegradation of waste from their activities [315]. Currently, work is being undertaken to modify the enzymes, which are then immobilized. When a charged polypeptide tail was added at the C-terminus, the salt resistance of lacase placed in coacervate core micelles (C3M) by encapsulation was enhanced [330]. The use of CotA with a polyglutamic acid tail resulted in more micelles with fewer enzyme molecules per micelle, better stability and increased lacase activity. Enriching the charge of enzymes by genetic engineering appears to be a potential strategy to improve the practical application of C3 Ms as enzyme delivery systems in reaction environments.