Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris.
The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6g/L, and the concentration of dextranase and enzyme activity in the supernatant were 0.46 g/L and 83900 U/L, respectively. The activity of dextranase was improved 17.56-fold by cation-exchange chromatography only with a final yield of 71.61% and the specific activity of the purified enzyme was 181.96 U/mg. The purified dextranase, analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. Then the factors affecting the dextranase activity were evaluated. The optimal temperature and pH was 30 degrees C and pH 4.5, respectively. Metal ions Al(3+), Cu(2+), Fe(3+), and SDS could completely inhibit the enzyme activity, whereas Mg(2+) enhanced 145% of the enzyme activity. These characters are much different from what was previously reported for the L. starkeyi dextranase that was either expressed in S. cerevisiae or purified from natural L. starkeyi.
Chen
Lin
L
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, P.O. Box 283, Meilong Road 130, Shanghai 200237, PR China.
Zhou
Xiangshan
X
Fan
Weimin
W
Zhang
Yuanxing
Y
D019149:Bioreactors; D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005800:Genes, Fungal; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D003910:Dextranase