|
|
|
| Cloning of glucoamylase gene from Thermomyces lanuginosus and its expression in Pichia Pastoris |
|
|
|
|
Abstract The gla gene of Thermomyces lanuginosus was cloned by PCR. Sequencing analysis revealed that gla has an open reading frame of 1854bp, which encodes a putative polypeptide of 617 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 64 kDa. The glucoamylase belongs to the glycoside hydrolase family 15. The gla sequence without signal sequences was obtained and cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. The recombinant enzymatic activity reached 11.6 U/mL after fermatation by Erlenmeyer flask. The recombinant glucoamylase was purified by using ammonium sulfate fraction、DEAE-sepharose fast flow chromatography. A molecular mass of the purified enzyme is 67 kDa determined by SDS-PAGE and a little bigger than the deduced molecular mass, possibly related to the glycosylation site. The recombinant glucoamylase exhibited optimum catalytic activity at pH5.0 and 60 ℃ respectively. It was thermostable at 60 ℃ and remained 54% of its original activity after 20 min at 70 ℃.
|
|
Received: 22 June 2009
|
|
|
|
|
|
|
