Cloning and expression of the mutated Exo-Inulinase gene from Aspergillus niger ۵۰۱۲ in E. Coli

Background and Aim: Inulinases are classified as hydrolases and widely used in the food and medical industries. Inulinases are mostly used for the production of ultra-high-fructose syrup, oligofructans, bioethanol, citric acid and single cell protein from inulin. Many yeasts, filamentous fungi and bacteria such as Pichia guilliermondii and Aspergillus niger can produce a large amount of inulinases. In this study, the expression of the mutated Exo-Inulinase gene derived from Aspergillus niger in E. coli strain BL۲۱(DE۳) has been studied.Methods: Exo-Inulinase gene from Aspergillus niger ۵۰۱۲ was mutated by SOEing PCR method using designed primers. Using this procedure, sixty nucleotides were deleted from chosen site of the gene which was found to be encoding ۲۰ hydrophobic amino acids. The new mutated gene, named Inu∆H, was cloned into the expression vector pET۲۶b through NdeI and NotI restriction endonuclease sites. E.coli DH۵α and E. coli BL۲۱(DE۳) were used as cloning and expression hosts, respectively. Finally, the protein expression was analyzed by SDS-PAGE. Heat shock protocol, described previously in our lab, was used to increase solubility of active enzyme. DNS method was applied to study enzyme activity.Results: Cloning of the mutated Exo-Inulinase gene in E. coli was studied on ۱% agarose gel and confirmed by sequencing. Expression of the recombinant protein was observed on ۱۰% SDS-PAGE. The protein size was about ۵۵kDa, approximately ۳ kDa smaller in comparison with its native form, as we expected. The recombinant Exo-Inulinase, Inu/\H, was highly expressed as inclusion bodies. Therefore, heat shock method was applied to increase solubility of properly folded enzyme. Preliminary experiment showed that the enzyme has a measurable activity.Conclusion: According to the wide industrial applications of Exo-Inulinase enzyme and due to the many benefits of protein expression in prokaryotic systems, we cloned the mutated Exo-Inulinase gene from Aspergillus niger ۵۰۱۲ into E. coli. We expressed the recombinant inulinase in E. coli BL۲۱(DE۳). The recombinant exo-inulinase showed measurable activity toward inulin as a substrate. The profile of enzyme activity and comparison with native enzyme is in progress.

Keywords:

Exo-Inulinase , Aspergillus niger ۵۰۱۲ , Fructose syrup , Inu∆H