{Reference Type}: Journal Article
{Title}: Enhancement of L-ribulose Production from L-ribose Through Modification of Ochrobactrum sp. CSL1 Ribose-5-phosphate Isomerase A.
{Author}: Zheng Q;Wang R;Ju X;Gu X;Xu X;Chen Z;Li L;Zheng Q;Wang R;Ju X;Gu X;Xu X;Chen Z;Li L;
{Journal}: Appl Biochem Biotechnol
{Volume}: 194
{Issue}: 10
{Year}: Oct 2022
{Factor}: 3.094
{DOI}: 10.1007/s12010-022-04015-2
{Abstract}: L-ribulose, a kind of high-value rare sugar, could be utilized to manufacture L-form sugars and antiviral drugs, generally produced from L-arabinose as a substrate. However, the production of L-ribulose from L-arabinose is limited by the equilibrium ratio of the catalytic reaction, hence, it is necessary to explore a new biological enzymatic method to produce L-ribulose. Ribose-5-phosphate isomerase (Rpi) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose, which is of great significance for the preparation of L-ribulose. In order to obtain highly active ribose-5-phosphate isomerase to manufacture L-ribulose, ribose-5-phosphate isomerase A (OsRpiA) from Ochrobactrum sp. CSL1 was engineered based on structural and sequence analyses. Through a rational design strategy, a triple-mutant strain A10T/T32S/G101N with 160% activity was acquired. The enzymatic properties of the mutant were systematically investigated, and the optimum conditions were characterized to achieve the maximum yield of L-ribulose. Kinetic analysis clarified that the A10T/T32S/G101N mutant had a stronger affinity for the substrate and increased catalytic efficiency. Furthermore, molecular dynamics simulations indicated that the binding of the substrate to A10T/T32S/G101N was more stable than that of wild type. The shorter distance between the catalytic residues of A10T/T32S/G101N and L-ribose illuminated the increased activity. Overall, the present study provided a solid basis for demonstrating the complex functions of crucial residues in RpiAs as well as in rare sugar preparation.