Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymic thermostability.

ABSTRACT

Thermostable α-amylases are widely used in industry. The α-amylase from Bacillus licheniformis (BLA) with six potential glycosylation sites possessed excellent thermal and pH stability and high activity. Here, it was expressed in Pichia pastoris. The Pic-BLA-producing yeast without any antibiotics-resistant gene was cultivated in flasks and the amylase activity in fermentation supernatant reached 900 U/mL. The recombinant α-amylase Pic-BLA produced in P. pastoris was deeply glycosylated with 30% increase in molecular mass (MM). The deglycosylation treatment by Endoglycosidase H (Endo H) reduced the MM of Pic-BLA. Thermostability analysis showed that Pic-BLA and deglycosylated Pic-BLA were similar in heat tolerance. In order to eliminate the extra impact of Endo H, the BLA was also expressed in Escherichia coli to get non-glycosylated Eco-BLA. A comparative study between non-glycosylated Eco-BLA and glycosylated Pic-BLA showed no obvious difference in thermostability. It is speculated that the glycosylation has little effect on the thermostability of α-amylase BLA.