D-allulose, which is one of the important rare sugars, has gained significant attention in the food and pharmaceutical industries as a potential alternative to sucrose and fructose. Enzymes belonging to the D-tagatose 3-epimerase (DTEase) family can reversibly catalyze the epimerization of D-fructose at the C3 position and convert it into D-allulose by a good number of naturally occurring microorganisms. However, microbial synthesis of D-allulose is still at its immature stage in the industrial arena, mostly due to the preference of slightly acidic conditions for Izumoring reactions. Discovery of novel DTEase that works at acidic conditions is highly preferred for industrial applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on D-fructose at pH 6.0 and 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on -fructose at pH 6.0 and 50°C with a Kcat/Km value of 45 mM−1min−1. The 500 g/L D-fructose, which corresponded to 30% conversion rate. With these interesting catalytic properties, this enzyme could be a promising candidate for industrial biocatalytic applications.
Mycobacterial pathogenesis is hallmarked by lipidic polyketides that decorate the cell envelope and mediate infection. However, factors mediating persistence remain largely unknown. Dynamic cell wall remodeling could facilitate the different pathogenic phases. Recent studies have implicated type III polyketide synthases (PKSs) in cell wall alterations in several bacteria. Comparative genome analysis revealed several type III pks gene clusters in mycobacteria. In this study, we report the functional characterization of two novel type III PKSs, MMAR_2470 and MMAR_2474, in Mycobacterium marinum. These type III pkss belong to a unique pks genomic cluster conserved exclusively in pathogenic mycobacteria. Cell-free reconstitution assays and high-resolution mass spectrometric analyses revealed methylated polyketide products in independent reactions of both proteins. MMAR_2474 protein exceptionally biosynthesized methylated alkyl-resorcinol and methylated acyl-phloroglucinol products from the same catalytic core. Structure-based homology modeling, product docking, and mutational studies identified residues that could facilitate the distinctive catalysis of these proteins. Functional investigations in heterologous mycobacterial strain implicated MMAR_2474 protein to be vital for mycobacterial survival in stationary biofilms. Our investigations provide new insights into type III PKSs conserved in pathogenic mycobacterial species.
D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from Bifidobacterium adolescentis (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those found in L-arabinose-BAAI bonding site. The activity of BAAI was 24.47 U/mg, and it showed good stability at temperatures up to 65 • C and a pH range 6.0-7.5. The K m , V max , and K cat /K m of BAAI were found to be 22.4 mM, 489 U/mg and 9.3 mM −1 min −1 , respectively for D-Galactose, while the respective values for L-arabinose were 40.2 mM, 275.1 U/mg, and 8.6 mM −1 min −1 . Enzymatic conversion of D-Galactose into D-tagatose by BAAI showed 56.7% conversion efficiency at 55 • C and pH 6.5 after 10 h.
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