Abstract
BackgroundLipoxygenase (LOX) from Anabaena sp. PCC 7120 (Ana-rLOX) offers important applications in the food industry, especially for improving aroma and dough rheological properties. However, industrial applications of LOXs have been limited by their poor thermostability. Herein, we report a bioinformatics-based consensus concept approach for the engineering of thermostable Ana-rLOX.ResultsA series of mutations (N130D, G260A, S437T, N130D/G260Q, N130D/S437Y) showed higher thermostability and activity than the wild-type enzyme. Thus, N130D/G260Q exhibited a 6.6-fold increase in half-life and 2.45 °C increase in unfolding temperature; N130D/S437Y showed a 10 °C increase in optimal temperature. The secondary structure did not change much that contributed to improved thermostability were investigated in detail using circular dichroism. Homology modeling suggested that enhanced thermostability and specific activity may result from favorable hydrophobic interactions.ConclusionsA series of mutations were achieved, showing higher thermostability and activity than the wild-type enzyme by semi-rational mutagenesis with limited structure information. Our findings provide important new insights into molecular modifications aimed at improving Ana-rLOX thermostability and activity.
Highlights
Mutation design guided by the consensus concept The consensus concept is a bioinformatics-based protein design method that replaces specific amino acids with the amino acid that most frequently occurs at that position in the protein family [32, 33]
Tm values determined by circular dichroism (CD) spectroscopy (Additional file 1: Figure S2), revealed a modest increase of 1.4 °C in mutant N130D compared to wild-type
We tried to combine this mutation with S437Y and G260Q and obtained increases of 2.1–2.5 °C
Summary
PCC 7120 (Ana-rLOX) offers important applications in the food industry, especially for improving aroma and dough rheological properties. Industrial applications of LOXs have been limited by their poor thermostability. LOX-catalyzed oxidative coupling reactions are of interest to the food industry. They are applied in bread-making to bleach carotenoids and to improve dough rheology [2, 3]. LOX played an important role in aroma compound formation. Because of their unique properties, LOXs have been widely utilized in the food, pharmaceutical [4, 5] and textile industries [6, 7].
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