A GENETICALLY MODIFIED E. COLI BL21(DE3) ROSETTA STRAIN/PET-28A(+): OPTIMIZED EXPRESSION OF RECOMBINANT TYPE I PULLULANASE FROM METABACILLUS INDICUS

Background: Pullulanase is commonly utilized when hydrolyzing the ?-1,6 glycosidic bonds in starch and pullulan. The production level of pullulanase type I is still not meeting the demand for industrial applications from wild-type microorganisms and heterologous expression systems. Aims: To clone and express the type I pullulanase gene from Metabacillus indicus LGM 22858 in E. coli BL21(DE3) Rosetta cells. Then, the expression conditions for recombinant pullulanase can be optimized by investigating the effects of different IPTG concentrations, various growth media, and different induction times. Finally, the optimal incubation time for the pullulanase enzyme assay must be determined. Methods: Different concentrations of the induction of the inducer IPTG were investigated:0.3, 0.5, 0.7, and 1 mM. The effect of using various production media (e.g., LB, TB, 2xTY, M9, 5xLB, and SB was studied. The influence of induction time was also tried 8, 20, and 36 h of induction. Moreover, the optimal incubation time for enzyme assay was tested: 5, 10, 15, 20, 25 and 30 min. Results: The ultimate level of Pull activity 2.55±0.18 U/mg was achieved at 0.3 mM IPTG. LB was the most appropriate growth medium, with 0.96 ± 0.11 U/mg. SDS-PAGE analysis revealed the same results. The best induction time was realized after 20 h of induction 1.08±0.02 U/mg. The optimal incubation time was 5 min to realize 11.57±0.37 U/mL. Discussion: The preliminary level of attained pullulanase activity was subjected to an optimization strategy to attain the optimal conditions encouraging the ultimate level of pullulanase activity. Conclusions: Results underpin the recombinant type I pullulanase of Metabacillus indicus, which was overexpressed in E.coli BL21(DE3) Rosetta cells.