High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris.
High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZalphaA with the Saccharomyces cerevisiae alpha-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)6-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.
Kwon
Min-A
MA
Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology, Yuseong, Daejeon 305-600, Republic of Korea.
Kim
Hyun Suk
HS
Yang
Taek Ho
TH
Song
Bong Keun
BK
Song
Jae Kwang
JK
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase