Laccases

Laccases (EC 1.10.3.2; benzenediol: oxygen oxidoreductases) are a class of enzymes known as oxidoreductases that catalyze the oxidation of various substrates. They belong to the multicopper oxidase family and are widely distributed in nature, including bacteria, fungi, plants, and insects. Laccases play essential roles in many biological processes and have significant biotechnological applications. Most of them are found in fungi on decaying wood samples.

The name “laccase” originates from the first known source of this enzyme, the sap of the lacquer tree (Rhus vernicifera). However, laccases have since been identified in numerous organisms, including fungi such as the basidiomycete Trametes versicolor, Pleurotus ostreatus, and Aspergillus species (Baldrian, 2006). A number of white rot fungi such as Pycnoporus also produce highly active laccases because they are needed to catalyse hydrolysis of wood materials such as lignin. They are sometimes referred to as lignin-modifying enzyme (LME) (Pointing et al., 2000).

Actinomycetes such as Streptomyces sp. have been collected from decayed wood (Gogotya et al., 2021). Typical bacteria include Bacillus spp..

Fermentation

 Trametes can be grown on cellubiose-asparagine liquid medium. Induction of laccase production is achieved using kraft lignin. Two laccases were produced but the main one was laccase A that was 85% of the total activity (Xiao et al., 2003).

Purification

Downstream processing is best achieved using column chromatography. The main steps to achieve electrophoretic purity are: DEAE-Sepharose FF, Superdex-200 and Mono-Q (Xiao et al., 2003).

The laccase A from Trametes species is between 57 and 62 kDa based on the type of method used (Xiao et al., 2003). The protein is a monomeric glycoprotein where the carbohydrate content is between 11 and 12% content and the isoelectric point is 4.2. The substrate used for testing activity is to oxidise guaiacol. The pH optimum of activity is 4.5 and is highest around 50ºC.

Thermostable Laccases

Laccases tend to be thermostable which is a benefit in biotechnology but not all. One thermostable laccase was produced by a Bacillus species (Sharma et al., 2019).

Laccases are notable for their ability to oxidize a wide range of substrates by utilizing molecular oxygen as a co-substrate. They typically contain four copper ions distributed among three copper centers: Type 1, Type 2, and Type 3. These copper ions are crucial for their catalytic activity. Solutions of this enzyme are blue because of the presence of copper.

The oxidation reactions catalyzed by laccases involve the transfer of electrons from the substrate to molecular oxygen. As a result, laccases can oxidize a variety of phenolic compounds, aromatic amines, and even non-phenolic compounds. The reaction leads to the formation of free radicals or quinones, which have applications in various fields, including biodegradation, bioconversion, and biosensing.

Laccases have gained considerable attention in environmental and industrial applications. They are capable of degrading pollutants and xenobiotic compounds, making them useful in bioremediation processes. Laccases can break down lignin, a complex polymer found in plant cell walls, which has potential applications in biofuel production, paper and pulp industry, and textile processing.

In addition to their role in environmental remediation, laccases are utilized in the food industry, where they can improve food quality, enhance flavors, and participate in processes such as wine clarification. They are also used in the production of bio-based materials, including adhesives, coatings, and dyes.

Overall, laccases are versatile enzymes with broad substrate specificity and significant potential in various fields. Their ability to catalyze oxidative reactions and their applications in biotechnology and environmental sciences make them valuable tools for sustainable and eco-friendly processes.

References

Baldrian, P. (2006). Fungal laccases–occurrence and properties. FEMS Microbiology Reviews, 30(2), pp. 215-242

Farnet, A.M., Criquet S, Tagger S, Gil G, Le Petit J (2000) Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17. Can. J. Microbiol. 46 pp. 189–194
Farnet, A.M., Criquet S, Pocachard E, Gil G, Ferre E (2002) Purification of a novel isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L.). Mycologia 94 pp. 735–740
Farnet, A.M., Criquet S, Cigna M, Gil G, Ferre´ E. (2004) Purification of a laccase from Marasmius quercophilus induced with ferrulic acid: reactivity towards natural and xenobiotic aromatic compounds. Enzyme Microb. Technol. 34 pp. 549–554 

Gogotya, A., Nnolim, N.E., Digban, T.O. et al. (2021) Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO. Biotechnol Lett 43, 1429–1442 (Article). 

Hildén, K., Hakala, T. K., & Lundell, T. (2009). Thermotolerant and thermostable laccases. Biotechnology Letters, 31, pp. 1117-1128

Mun˜oz C, Guillen F, Martınez AT, Martı´nez MJ (1997) Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl Environ Microbiol. 63 pp. 2166–2174

Pawlik A, Wójcik M, Rułka K, Motyl-Gorzel K, Osińska-Jaroszuk M, Wielbo J, Marek-Kozaczuk M, Skorupska A, Rogalski J, Janusz G (2016) Purification and characterization of laccase from Sinorhizobium meliloti and analysis of the lacc gene. Int. J. Biol. Macromol. 92 pp. 138–147 (Article)

Pointing SB, Jones EBG, Vrijmoed LLP (2000) Optimization
of laccase production by Pycnoporus sanguineus in submerged liquid culture. Mycologia 92 pp. 139–144

Sharma, V., Ayothiraman S, Dhakshinamoorthy V (2019) Production of highly thermo-tolerant laccase from novel thermophilic bacterium Bacillus sp. PC-3 and its application in functionalization of chitosan film. J Biosci. Bioeng. 127 pp. 672–678 (Article)

Thurston, C.F. (1994) The structure and function of fungal laccases. Microbiology 140 pp. 19–26

Tong, P., Hong, Y, Xiao Y, Zhang M, Tu X, Cui T (2007) High production of laccase by a new basidiomycete, Trametes sp. Biotechnol. Lett 29 pp. 295–301

Xiao, .Y., Tu, .X., Wang, .J. et al. (2003) Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. Appl Microbiol Biotechnol 60, pp. 700–707 (Article).

Yang, J., Li, W., Ng, T. B., Deng, X., Lin, J., & Ye, X. (2017). Laccases: production, expression regulation, and applications in pharmaceutical biodegradation. Frontiers in Microbiology, 8, 832