Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

Abstract

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Biochemical Characterization of Heat-Tolerant Recombinant l-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in d-Tagatose Production

“…The rL-AI isolated here presented the same optimum pH and temperature as shown by de Sousa et al [31]. The observed temperature curve (Fig.…”

“…In a previous report, Enterococcus faecium DBFIQ E36 L-AI was purified to homogeneity and primary characterization (monomer molecular weight, native mass, isoelectric point) was performed [30]. Furthermore, L-AI was immobilized onto several chitosan and agarose supports and then tested for the stability and activity of these derivatives [31,32]. Herein, we report gene virulence analysis of the thermotolerant L-AI producer Enterococcus faecium DBFIQ E36 and sequencing, primer design, cloning, and heterologous expression of L-AI in E. coli BL21 cells as well as further biochemical characterization of rL-AI such as ion metal requirement and thermal stability at different pHs and temperatures.…”

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From Bifidobacterium adolescentis for D-Tagatose Production

“…It is also known for its prebiotic action, capability of reducing cholesterol, preventing colon cancer, treatment of type II diabetes, and so on (Lu et al, 2008). It is widely used in many food and pharmaceuticals preparations, including drinks, yogurt, diabetes-specific foods, diet foods, cereals, meat products, candies, cough syrups, anti-adhesives for fixed dentures and oral disinfectants (Kim, 2004;Marylane et al, 2017).…”

“…In this context, milk industry wastes, particularly whey powder rich in lactose, would be an attractive source of substrates, which provides galactose upon hydrolysis of lactose (Zhang et al, 2020). As attempts to obtain potential source of L-AIs with a high efficiency, more than 30 L-AIs have been reported thus far from various natural or engineered microorganisms, such as Escherichia coli (Wu et al, 2020), Clostridium hylemonae (Nguyen et al, 2018), Bacillus subtilis (Kim et al, 2010), Bacillus coagulans (Mei et al, 2016), Lactobacillus plantarum (Chouayekh et al, 2007), Lactobacillus brevis (Guo et al, 2018), Geobacillus thermodenitrificans (Kim and Oh, 2005), Enterococcus faecium (Marylane et al, 2017) and Bifidobacterium longum (Salonen et al, 2012). However, almost all of these L-AIs showed higher specificity to L-arabinose than the specificity to D-Galactose (Xu et al, 2018).…”

Characterization of a d-lyxose isomerase from Bacillus velezensis and its application for the production of d-mannose and l-ribose

“…In order to meet the demand for production of high-value sugars in industry, sugar isomerases need to have good biochemical properties and thermal stability (De et al 2017). The isomerization reaction catalyzed by sugar isomerases should be performed in slightly acidic to reduce unwanted by-products (Rhimi et al 2009; Nguyen et al 2018).…”