Purification and Characterization of Recombinant Levansucrase From Bacillus lichniformans MJ8

Abstract

The ability to produce levansucrase was estimated in fifty Bacillus spp. isolates obtained from various sources. One isolate was found to be the higher producer of this enzyme through primary and secondary screening, which was performed by estimating levansucrase activity. It was found to be 6.05 U/ml. The results show, after identification by the Vitek 2 compact system with 92% probability and 16S ribosomal RNA gene sequencing, that this isolate is Bacillus licheniformis, designated in this study as MJ8, and registered in the Gene Bank under the accession number OM672244.1. Then, the gene of (SacB) was studied in detail. It contained 1449 nucleotides (accession number ON811641.1), expressed for 482 amino acids and 29 amino-acid as signal peptides. A recombinant plasmid, pET28a(+), with the levansucrase (SacB) gene, was over expressed in high efficiency E. coli BL21 (DE3). Then the enzyme was purified in three steps; firstly, it was precipitated with ammonium sulfate 40-80%. Secondly, DEAE cellulose ion exchange chromatography was applied then the gel filtration chromatography. The activity of levansucrase at the final purification steps reached 48.65 U/ml with a purification fold of 18.0 and a recovery of 48.9%. The levansucrase characterization was also studied. It was found that the molecular weight by SDS-PAGE and gel filtration methods was 43 kDa and 47 kDa, respectively, and the optimum temperature activity and stability were 45 ◦C, 30-35 ◦C, respectively while the pH activity and stability were 6.0, and 7.0, The levan was produced from cloned (SacB) levansucrase, separated, purified, and identified by NMR, AFM, and SEM analyses showed that its structure mostly consists of fructose residues.