The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Abstract

BackgroundL-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values.ResultsThe gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C.ConclusionsHere we reported the purification and the biochemical characterization of the novel Shewanella sp. ANA-3 L-arabinose isomerase. Determination of the biochemical properties demonstrated that this enzyme was highly active at low temperatures. The generated T268K mutant displays an increase of the enzyme stability essentially at low pH. These features seem to be very attractive for the bioconversion of D-galactose into D-tagatose at low temperature which is very interesting from industrial point of view.

Characterization of an <small>L</small>-Arabinose Isomerase from <i>Bacillus thermoglucosidasius</i> for <small>D</small>-Tagatose Production

“…ANA-3-derived L-AI after 6 h of incubation. 9,20) A recent study describes an enhanced conversion ratio to L-ribulose from L-arabinose upon the addition of a borate, possibly due to alteration of the reaction equilibrium by the formation of a complex of borate with carbohydrate substrates. 21,22) In our study, an investigation of the borate effect showed that the conversion ratio by BTAI to D-tagatose increased slightly, to 55.6%, in borate buffer after 30 h of incubation (Fig.…”

A new l-arabinose isomerase with copper ion tolerance is suitable for creating protein–inorganic hybrid nanoflowers with enhanced enzyme activity and stability

“…[9][10][11][12] Thus, it is importance to develop novel immobilization techniques using various materials. [17][18][19][20][21][22][23] Thus, it is not suitable to prepare nanoowers using AI as the component enzyme. Feasibility and price are the basic criterion for the immobilization material selection.…”

Novel D-Galactose Isomerases from Lactobacillus Strains Isolated from the Sweet Sap of Agave atrovirens

“…The same enzyme catalyzes the isomerization of D-galactose into D-tagatose (Zhang et al, 2007;Cheng et al, 2009;Mei et al, 2016). L-arabinose has been previously reported as the main inducer for the expression of the araA gene (Yanjun et al, 2011;Rhimi et al, 2011;Xu et al, 2013) and thus, the preferred carbon source for enrichment and selective media, to be used in a large scale screening for the detection and selection of novel bacterial isolates producers of D-GI. Due to the high cost of pure Larabinose, in the present study a process to obtain Larabinose from arabic gum was adapted and improved.…”

“…Protein concentration was determined by the Bradford method using bovine serum albumin as the as the standard (Protein standard II lyophilized bovine serum albumin) (Bradford, 1976;Rhimi et al, 2011). Dtagatose was determined by the cysteine-carbazole sulphuric-acid method and the absorbance was measured at 560 nm, using a spectrophotometer (Hach, model DR 5000) (Dische and Borenfreund, 1951).…”