Expression and purification of HLADH-EE and its application in L-fructose synthesis

• 1、Key Laboratory of Carbohydrate Chemistry and Biotechnology, Jiangnan University,Wuxi 214122

Abstract：To perform the enzymatic synthesis of L-fructose, E-type horse liver alcohol dehydrogenase (HLADH-EE) was heterologously expressed and purified in E. coli. The gene sequence ADH1E encoding HLADH-EE was obtained by gene synthesis and ligated into vector pET-28a. The recombinant plasmid was expressed in BL21 (DE3) by induction and the protein was purified based on His-tag. The optimal reaction temperature, pH condition, metal ion preference and substrate specificity were determined. Then, the enzymatic synthesis of L-fructose was performed and the final product L-fructose was isolated, purified and identified. The results showed that HLADH-EE was successfully expressed in BL21(DE3). SDS-PAGE verified that the band was about 40 kDa, which was consistent with the expectation. The maximum specific activity was 32.2 U/mg at pH 9.0 and 35 C, which was almost identical with HLADH-EE isolated from horse liver tissue. Under the optimal conditions, the yield of L-fructose was 8.1 g/L, which greatly reduce the cost for the enzymatic synthesis of L-fructose. This study provides a theoretical basis for biotransformation of L-fructose in industrialization.

Keywords： enzyme catalysis horse liver alcohol dehydrogenase (HLADH) heterologous expression L-fructose