Gene expression, enzyme purification and physicochemical characterization of Weissella confusa P2 glucansucrase.
TL;DR
These findings enrich the genetic resources of glucansucrase, provide a promising biocatalyst for efficient EPS production, and establish a foundation for the application of glucansucrase and its polysaccharides products in the food and biotechnology industries.
Abstract
In this study, the glucansucrase-encoding gene (dsr) from Weissella confusa P2 was cloned and heterologously expressed in Escherichia coli BL21(DE3). A 4542 bp fragment encoding 1510 amino acids was obtained, and the recombinant enzyme exhibited a molecular weight of approximately 161 kDa with a theoretical pI of 4.74. After Ni-IDA affinity chromatography, the specific activity reached 300.19 U/mg with a purification fold of 18.43. Enzymatic characterization revealed that the recombinant glucansucrase displayed optimal activity at pH 5.5 and 30 °C, and retained over 70% relative activity after 48 h incubation at 10-30 °C. The enzyme was significantly activated by Ca2+, while strongly inhibited by Ni2+, Cd2+, SDS, EDTA, DTT, and high concentrations of organic solvents. Kinetic analysis using sucrose as substrate yielded Km of 0.56 mM and Vmax of 4.57 U/mg, indicating exceptionally high substrate affinity. Using this recombinant enzyme, exopolysaccharide (EPS) was synthesized in vitro, achieving a yield of 59.23 ± 0.21 g/L, representing an 18.79% improvement over EPS synthesis by the wild-type strain. These findings enrich the genetic resources of glucansucrase, provide a promising biocatalyst for efficient EPS production, and establish a foundation for the application of glucansucrase and its polysaccharides products in the food and biotechnology industries.