Effect of Molecular Chaperone Co-Expression on Heterologous Soluble Expression of Recombinant Sucrose Isomerase

Abstract:

Sucrose isomerase (SIase) plays an important role in the enzymatic preparation of isomaltulose. In this study, a SIase gene from Klebsiella sp. LX3 was expressed in E. coli by using the molecular chaperone co-expression system, and then purified and characterized. The SIase gene was cloned into pET-24b to yield the construct pET-24b-SIase, which was introduced into E. coli BL21(DE3) for expression. Unfortunately, the enzyme was expressed mainly as inclusion bodies. In order to enhance the soluble form of the enzyme, SIase was coexpressed with a molecular chaperone. Four plasmids (pKJE7, pGro7, pG-Tf2, pTf16), each carrying a molecular chaperone gene, were separately introduced into E. coli BL21(DE3) carrying pET-24b-SIase and the expression of SIase was evaluated. The plasmid pGro7 was the most effective at improving the expression of recombinant SIase as a soluble enzyme, yielding a specific activity of 14.8 U/mL for the crude cell extract compared with a mere 3.5 U/mL for the crude cell extract derived from cells harbouring only pET-24b-SIase. The expressed SIase was purified by affinity chromatography using Ni-NTA. The purified enzyme exhibited the maximum activity at 40 ℃ and pH 6.0. Its K_m for sucrose was(179.10±20.65) mmol/L and the k_cat/K_m was (5.44±0.72) L/(mmol·s). The results of the product specificity study showed that the proportion of isomaltulose and trehalulose in the products decreased and the proportion of monosaccharides increased with increasing reaction temperature. The results of this study showed that the level of soluble SIase expressed in E. coli could be enhanced by coexpressing the enzyme with a molecular chaperone, suggesting a potential strategy for the large-scale production of SIase and its applications.