Expression in Aspergillus niger and characterization of β-mannanases from Stachybotrys chartarum

Abstract:

[Objective] Using Aspergillus niger as host to express β-mannanases from Stachybotrys chartarum.[Methods] Through sequence analysis of Stachybotrys chartarum genome, two β-mannanase genes (s16942 and s331) were identified. The primers were designed based on the DNA sequence and the β-mannanase genes (s16942 and s331) were obtained, and then inserted to the vector pGm. The expression plasmids were transferred into Aspergillus niger. β-mannanase producing strains (G1-pGm-s16942 and G1-pGm-s331) were isolated after screening several transformants using amdS selection plates and confirmed by PCR fragment sequencing.[Results] The molecular weight of the enzymes from G1-pGm-s16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis, and the recombinant proteins did not present in the negative control. Assays of enzymatic property using the crude enzyme preparations indicated that the enzyme from G1-pGm-s16942 exhibited maximum activity (521 U/mL) under the optimum conditions (pH 7 and 60℃); the enzyme from G1-pGm-s331 exhibited maximum activity (84 U/mL) under the optimum conditions (pH 7 and 50℃).[Conclusion] This was the first study of the heterologous expression of the β-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the β-mannanase genes could be expressed successfully with high activities and protein titers.

Key words:mannanase;Stachybotrys chartarum;Aspergillus niger;genetic transformation;recombinant protein expression