Introduction

In-silico directed evolution of Laccase enzyme to enhance its stability and catalytic efficiency in the degradation of Aflatoxin B1 and Aflatoxin G1

Mycotoxins are secondary metabolites of fungi that cause hepatotoxicity, teratogenicity, and immunotoxicity, and are classified as group I carcinogens. Aflatoxins (AFs) are a type of mycotoxin produced by fungal species such as Aspergillus flavus, and A. parasiticus. These commonly infect cereal crops, including wheat, tree nuts, maize, cotton, and peanuts, and can pose serious threats to humans and animals by causing various complications such as hepatotoxicity, teratogenicity and immunotoxicity. The main aflatoxins are B1, B2, G1 and G2, and can be toxic to the body through inhalation, mucous membranes or skin,leading to an overactive inflammatory response. Various methods such as physical strategies, chemical methods and biodegradation are applied to detoxify aflatoxin. However, many of the existing physical and chemical methods for the detoxification of aflatoxin from the contaminated food and feed are unprofitable for practical application due to loss of nutritional quality, safety concerns, environment pollution, limited efficiency and high marketing costs. In contrast, biological processes involving biodegradation and biosorption are more effective and promising strategies. Microbial and enzymatic applications are moderate, inexpensive and effective with little to no harmful intermediates for humans and animals consumption. Globally, fungal laccase have better pH/temperature stability, greater tolerance to metals, and can oxidize a wide variety of substrates of different origins. The use of laccase can lead to rapid and significant degradation of various substrates.

Laccases belong to the family of multiple copper (Cu) containing enzymes, which are able to perform the four-electron reduction of dioxygen to water and subsequent oxidation of organic and inorganic compounds by the mechanism of transferring one-electron. The laccase derived from Myceliophthora thermophilus has a monomeric active site and differences in the T1-Cu active site topology and polar motifs, amongst different sources of laccases within the taxonomic subgroup, each employ different molecular evolution to serve different functional roles such as in degradation of environmental pollutants, wastewater treatment, and endocrine disruptor chemicals under varying conditions. The conserved histidine residues are found to interact with the copper ions found in the structure which might play a role in degrading aflatoxin through accelerated Redox reactions with the help of copper ions.

Molecular docking is an effective analytical tool to assess the enzyme–substrate interaction. With the development of high technology, the use of computer simulation, and the deep understanding of the three-dimensional structure of laccase, it becomes easy to study the relationship between laccase and its interaction with the substrate. easier and faster.

In-silico Directed evolution of enzymes studies hold the potential to develop preventative strategies to reduce AFB1 and AFG1 contamination of food . Our present study has aimed to analyze the interaction of AFB1 and AFG1 with the site-directed mutated laccase to find possible differences in enzyme and ligand pocket recognition towards various catalytic sites using different docking simulations and procedures