Abstract:
Produced by levansucrase that uses sucrose as a substrate, levan and levan oligosaccharides (GFn) have potential applications in food and pharmaceutical industries such as prebiotics, anti-obesity and anti-tumor agents. Previous study reported that N251A and N251Y mutants of levansucrase from Bacillus licheniformis RN-01 could effectively produce short-chain oligosaccharides upto GF3, instead of long-chain levan synthesis. This study hypothesized that these mutations probably reduced GF3 binding affinity in the active site of levansucrase that contains fructosyl-Asp93 intermediate and caused GF3 to be in an unfavorable orientation for transfructosylation; therefore, levansucrase could not effectively extend GF3 to GF4. However, these mutations probably did not significantly reduce the binding affinity or drastically change orientation of GF2; therefore, levansucrase could still extend GF2 to GF3. To test this hypothesis, this study employed molecular dynamics to investigate effects of these mutations on GF2/GF3 binding in the active site of levansucrase. The results reasonably support this hypothesis as N251A and N251Y mutations did not significantly reduce the GF2 binding affinity and did not drastically change orientation of GF2 in the active site of levansucrase. However, these mutations drastically decreased GF3 binding affinity and caused GF3 to be in an unfavorable orientation for transfructosylation. Furthermore, the free energy decomposition and hydrogen bond occupation results suggest the importance of Arg255 in GF2/GF3 binding in the active site of levansucrase. To elucidate the importance of Arg255 on the production of levan, R255A mutant of levansucrase was successfully cloned and expressed. The optimum pH and temperature of R255A mutant was at 6.0 and 50 oC, respectively. This study found that R255A mutant could not synthesize long-chain levan, but it synthesized a small amount of short-chain levan oligosaccharides with the chain length upto GF2, supporting the computational results. This knowledge could be beneficial in designing levansucrase to efficiently produce levan oligosaccharides with desired length.
