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Background Catalase (EC 1.11.1.6) is a heme-containing tetrameric enzyme that plays a critical role in signaling and hydrogen peroxide metabolism. It was the first enzyme to be crystallized and isolated. Catalase is a well-known industrial enzyme used in diagnostic and analytical methods in the form of biomarkers and biosensors, as well as in the textile, paper, food, and pharmaceutical industries. In silico analysis of CAT genes and proteins has gained increased interest, emphasizing the development of biomarkers and drug designs. The present work aims to understand the catalase evolutionary relationship of plant species and analyze its physicochemical characteristics, homology, phylogenetic tree construction, secondary structure prediction, and 3D modeling of protein sequences and its validation using a variety of conventional computational methods to assist researchers in better understanding the structure of proteins. Results Around 65 plant catalase sequences were computationally evaluated and subjected to bioinformatics assessment for physicochemical characterization, multiple sequence alignment, phylogenetic construction, motif and domain identification, and secondary and tertiary structure prediction. The phylogenetic tree revealed six unique clusters where diversity of plant catalases was found to be the largest for Oryza sativa. The thermostability and hydrophilic nature of these proteins were primarily observed, as evidenced by a relatively high aliphatic index and negative GRAVY value. The distribution of 5 sequence motifs was uniformly distributed with a width length of 50 with the best possible amino residue sequences that resemble the plant catalase PLN02609 superfamily. Using SOPMA, the predicted secondary structure of the protein sequences revealed the predominance of the random coil. The predicted 3D CAT model from Arabidopsis thaliana was a homotetramer, thermostable protein with 59-KDa weight, and its structural validation was confirmed by PROCHECK, ERRAT, Verify3D, and Ramachandran plot. The functional relationships of our query sequence revealed the glutathione reductase as the closest interacting protein of query protein. Conclusions This theoretical plant catalases in silico analysis provide insight into its physiochemical characteristics and functional and structural understanding and its evolutionary behavior and exploring protein structure-function relationships when crystal structures are unavailable.

A catalase from a novel source Sechium edule (squash) has been purified to homogeneity using ammonium sulfate fractional precipitation, dialysis, and anion exchange chromatography on Diethylaminoethyl (DEAE) cellulose in sodium acetate buffer pH 6.0. The purity of the enzyme was analyzed by SDS-PAGE. The molecular weight of the enzyme using the SDS-PAGE method was found to be 55 kDa with specific activity 6.35 U/mg. The molecular weight of the enzyme was further confirmed by the INTACT mass spectrometric technique and found to be 52.5 kDa. The Reinheitzahl (Rz) value of the enzyme was 1.5. The MALDI-TOF analysis of the purified enzyme showed that it contains 529 amino acid residues. The K m , V max, optimum pH, and optimum temperature of the free enzyme using H 2 O 2 as the substrate were found to be 0.03 mM, 200 μmol/min, 7.6, and 24 • C, respectively. The purified enzyme was immobilized on chitosan beads which was prepared by extracting the chitosan from Cornu aspersum (garden snail) having highest degree of deacetylation%. The kinetic characteristics, K m , V max, optimum pH, and optimum temperature of the immobilized catalase were found to be 0.065 mM, 250 μmol/min, 7.6, and 41 • C, respectively. The immobilized catalase is more thermostable in comparison to free catalase.

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