Immobilization of the recombinant (His)6-tagged l-arabinose isomerase from Thermotoga maritima on epoxy and cupper-chelate epoxy supports

Abstract

l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was overexpressed in Escherichia coli as a (His)6-tagged protein and purified by heat treatment followed by immobilized Ni2+ affinity chromatography. TMAI from a heat-treated preparation was immobilized on the epoxy support Eupergit C250L (Eu) and on its copper–chelate form (Eu–Cu). The immobilization yield and the specific activity at 80°C and pH 7.5 for the former or the latter derivatives were, respectively, 7.2 or 25mg BSAE per gds (grams of dry solid) and 0.44±0.04 or 3.1±0.4IU per gds. TMAI immobilized on Eu–Cu incubated in absence of substrate exhibited an improved thermostability compared to its soluble counterpart, the half-life time at 80 or 90°C being not detectable for 5h or equal to 2h for Eu–Cu and to 1.0h or 0.3h for the free enzyme, respectively. However, in bioconversions in three repeated cycles at 80°C with 18gL−1 of initial d-galactose, the immobilized biocatalyst activity declined rapidly. At 60°C, not only Eu–Cu derivatives used in three repeated bioconversions with initial d-galactose concentration of 18gL−1 were stable for at least 242h, they also yielded a d-galactose conversion and an average productivity of 29.1% and 0.06gL−1h−1, respectively, exhibiting better performances compared to the results at 80°C.