Engineering of l arabinose isomeraseenzyme for the production of tagatose

Abstract

The L-arabinose isomerase (LAI) gene from Shewanella sp. ANA3 newline(ShLAI) was engineered to enhance the bioconversion of D-galactose. The newlineShLAI protein is a homohexamer with six identical subunits, each made up of newlinea single polypeptide chain of 500aa. The ShLAI protein is structurally similar newlineto fucose isomerase and functionally similar to other sugar isomerases such as newlineXylose isomerase and L-rhamnose isomerase. There are only two highresolution newlinecrystal structures of L-arabinose isomerase are available. Based on newlinemultiple sequence alignment, the ShLAI - Geobacillus kaustophilus LAI newline(GKLAI) sequence identity was found to be 54.95% and ShLAI - E.coli LAI newline(ECLAI) sequence identity was 51.61%. Although ShLAI shows marginally newlinehigh sequence similarity with GKLAI, phylogenetic analysis revealed that newlineShLAI and ECLAI are evolutionarily more conserved than GKLAI. newlineTo study the effect of mutation on the substrate affinity, a molecular newlinemodel of ShLAI dimer was constructed. The predicted model was refined and newlinethen validated with a Ramachandran plot generated using the PROCHECK newlinewebserver. The active site residues essential for the enzyme s catalytic newlineactivity were determined by the combination of metaPocket prediction and newlinecomparative analysis of amino acid sequences of 22 L-arabinose isomerase newlineproteins. The amino acid residues L18, Y19, M185, F279, E306, E333, H349, newlineM350, I371 and H447 were highly conserved in the L-arabinose isomerase of newlinebacteria. Glutamate residues at 306 and 333 positions were determined to be newlinecatalytic residues based on COFACTOR analysis and literature survey. newlineSuperimposition of ShLAI active site with E. coli active site also confirmed newlinethe significance of E306 and E333.Based on the Docking results, it was deduced that the presence of amino acids F279 and I371 might sterically hinder the O6 of D-galactose. The newlineeffect of active site amino acid substitutions on substrate affinity was newlinepredicted by in silico mutagenesis followed by in silico docking. The active newlinesite variants of F279 and I371 were created by in silico mutagen