Abstract Laccases are members of the multicopper oxidases, having the ability to catalyze the oxidation of a wide spectrum of phenolic compounds. These enzymes are capable of oxidizing lignin-related compounds and highly resistant environmental pollutants, and hence can be used in wastewater treatment and detoxification. Laccases are mostly derived from fungi, bacteria, and plants. In general, fungal laccases are known for their high redox potential, while bacterial laccases have a better tolerance to temperature. A combination of these properties is ideal for industrial processes. Directed evolution and the consensus method are often used to engineer protein stability. However, they are time-consuming and expensive. Ancestral sequence reconstruction (ASR), an approach whereby probable ancestral sequences are obtained, is known to provide thermostable variants of modern proteins as the output. In this study, ASR was used to design a laccase with high thermostability and high redox potential. ASR was performed through the web tool Phylobot, where fungal laccases were used as the input to obtain a phylogenetic tree with ancestral variants of the fungal laccases. For rooting the phylogenetic tree, multiple outgroups were tested, and the ‘Three-outgroups’ scenario proved to be the most efficient. In all cases, the phylogenetic tree was branched into major clusters of thermostable clades and non-thermostable clades. Nodes representing ancestral sequences were selected based on their sequence length and proximity to the thermostable clades. Sequence alignment of the selected nodes to the fungal laccases showed that the selected nodes have a high percentage of identity to well-known, highly thermostable laccases like Trametes pubescens and Basidiomycete PM1. All selected nodes have some common conserved motifs in the vicinity of the copper ligands. It also shows that the number of prolines in nodes 229 and 345 is more than in other selected nodes. The gene for the most promising candidate, Node 345, was synthesized, cloned into the pPICZ A vector, and transformed into Pichia pastoris cells. In the future, the protein will be expressed and characterized. Furthermore, the engineered laccases from the directed evolution method and the ancestral sequence reconstruction method will be compared for their activity and thermostability. This work would pave the way for using ASR as a less resource-intensive and time-consuming method for protein engineering. 2.12.0.0 / <p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>2.12.0.0</p>
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-474854 |
| Date | January 2019 |
| Creators | Najafi Abedi, Akram |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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