Experimental studies for production of cytoplasmic and periplasmic thermostable glucose isomerase in recombinant escherichia coli

Abstract

Glucose isomerase (GI), which catalyzes the reversible isomerization of Glucose toFructose, is of commercial importance in the production of high fructose corn syrup. Athigh temperature, equilibrium for the isomerisation reaction is shifted toward fructose. The low expression level of GI in the thermophilic microorganisms, the difficulties encounteredduring cultivation of the thermophilic microorganisms and purification of the enzyme fromits native host make obtaining large amounts of the thermostable enzymes from the naturalhost impractical. To overcome these problems, GI from thermophile Thermus thermophilus was cloned into two different pET vectors, and expressed in Escherichia coli.In the first part of this study, the GI gene was cloned into pET20b vector containing pelBsignal sequence, which enables the periplasmic localization of the recombinant protein.The ratio of the translocated enzyme to the total produced enzyme changed with time which could probably be varying the folding rate of GI in cytoplasm. The maximumvolumetric and specific activities of GI in periplasm and cytoplasm of the recombinantcells were calculated as 50.6 U/L culture and 42.8 U/mg protein, 36.6 U/L culture and 19.6U/mg protein, respectively. In the second part of this study, the same gene was cloned into pET28a vector for high level cytoplasmic expression of the thermostable GI enzyme. Themaximum volumetric and specific activities were calculated as 1474.9 U/L culture and366.0 U/mg protein, respectively.