Abstract
Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.
Keywords: Laccase, Lignin, Cell wall, Phenylpropanoid pathway, Rice, Plants
Background
Laccases are oxidases ubiquitously present in bacteria, fungi, animals, and plants. They are some of the oldest enzymes identified. Laccase is involved in diverse functions such as pigmentation of fungal spores, regeneration of plants, as fungal virulence factors, and in lignification of cell walls and delignification during wood rotting. They oxidize the biosynthesis of secondary metabolite called lignin in vascular tissues of plants. Purification of laccases is very challenging as laccases are expressed in very low amounts. Existing protocols to purify laccases are from either microorganism or softer tissues of plants such as leaves. This protocol provides an efficient approach to extract and purify laccases from harder tissues of plants such as rice stems.
Materials and Reagents
Equipment
Note: The materials, reagents and equipment not provided with company and catalog number can be ordered from any qualified company for using in this experiment.
Procedure
Data analysis
Data analysis could refer to Figures 3 and 4, and also Supplementary figures S7 and S8 from Swetha et al., 2018.
Notes
Recipes
Acknowledgments
This protocol was developed by modifying the method from Jaiswal et al. (2015). Thanks to Prof. K. Veluthambi for rice seeds. The authors acknowledge financial support from Ramanujan Fellowship (SR/S2/RJN-109/2012; Department of Science and Technology, Government of India) to PVS. PVS lab is supported by NCBS-TIFR core funding and a grant (BT/PR12394/AGIII/103/891/2014) from Department of Biotechnology, Government of India. SC acknowledges a fellowship from DBT, India.
Competing interests
The authors declare no competing interests.
References
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