A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 106 Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ± 3.5 g/L of pullulan with a Mw of 0.35 × 106 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.
Keywords: Aureobasidium spp.; High Mw pullulan; Isopullulanase; α-Amylase, glucoamylase.
Copyright © 2018. Published by Elsevier B.V.