Construction of a Fusion Protein to Improve the Catalytic Performance of Recombinant Glucose Isomerase
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Abstract:
To study the effect of the N-terminal protein sequence of type II xylose/glucose on type I xylose/glucose, the N-terminal 31 amino acid sequence of type II glucose isomerase (GIase) from the hyperthermophilic bacterium, Thermoanaerobacter thermohydrosulfuricus (TTGIase) was fused to the N-terminus of the type I glucose isomerase from Thermobifida fusca (TFGIase), to construct the fusion protein N-TFGIase. The experimental results showed that under the same culture and induction conditions, total yield of N-TFGIase produced by the bacteria was approximately 40% higher than that of TFGIase. The specific enzyme activity of N-TFGIase was 26% higher than that of TFGIase and the optimum temperature of N-TFGIase was 5 ℃ higher than that of TFGIase. The half-life of N-TFGIase at 75 ℃ was extended by 30% compared to that of TFGIase, and the optimum pH of N-TFGIase was reduced by 1.0 compared to that of TFGIase. Sequence analysis showed that the mRNA secondary structure of the N-terminal sequence of TTGIase did not form a blocked cervical-loop structure, thereby improving the efficiency of expression of the fusion protein. The hydrophobicity indices of 31 amino acid residues of the N-terminal sequence were all less than zero, favoring the initial folding and packaging of the fusion protein. The proportion of acidic amino acid residues was approximately twice that of the basic amino acid residues of the N-terminal sequence, thereby reducing the impact of the acidic medium environment on the surface of the fusion protein molecules.
