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

摘要

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 degrees C and pH 7.5 for the former or the latter derivatives were, respectively, 7.2 or 25 mg BSAE per gds (grams of dry solid) and 0.44 +/- 0.04 or 3.1 +/- 0.4 IU 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 degrees C being not detectable for 5h or equal to 2h for Eu-Cu and to 1.0 h or 0.3 h for the free enzyme, respectively. However, in bioconversions in three repeated cycles at 80 degrees C with 18 g L-1 of initial D-galactose, the immobilized biocatalyst activity declined rapidly. At 60 degrees C, not only Eu-Cu derivatives used in three repeated bioconversions with initial D-galactose concentration of 18 g L-1 were stable for at least 242 h, they also yielded a D-galactose conversion and an average productivity of 29.1% and 0.06 g L-1 h(-1), respectively, exhibiting better performances compared to the results at 80 degrees C. (C) 2015 The Institution of Chemical Engineers. Published by Elsevier BM. All rights reserved.