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


ABSTRACT: Thermostable ?-amylases are widely used in industry. The ?-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 ?-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 ?-amylase BLA.

SUBMITTER: Hu X 

PROVIDER: S-EPMC6820627 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Hu Xinlin X   Yuan Xin X   He Nisha N   Zhuang Tony Z TZ   Wu Pan P   Zhang Guimin G  

3 Biotech 20191029 11


Thermostable α-amylases are widely used in industry. The α-amylase from <i>Bacillus licheniformis</i> (BLA) with six potential glycosylation sites possessed excellent thermal and pH stability and high activity. Here, it was expressed in <i>Pichia pastoris</i>. 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 <i>P. pastoris</i> was deeply  ...[more]

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