Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability.
Although Candida rugosa utilizes a nonuniversal serine codon (CUG) for leucine, it is possible to express lipase genes (LIP) in heterologous systems. After replacing the 19 CUG codons in LIP4 with serine codons by site-directed mutagenesis, a recombinant LIP4 was functionally overexpressed in Pichia pastoris in this study. This recombinant glycosylated lipase was secreted into the culture medium with a high purity of 100 mg/liter in a culture broth. Purified recombinant LIP4 had a molecular mass of 60 kDa, showing a range similar to that of lipase in a commercial preparation. Since LIP4 has only a glycosylation site at position Asn-351, this position may also be the major glycosylation site in C. rugosa lipases. Although the thermal stability of recombinant LIP4 significantly increased from 52 to 58 degrees C after glycosylation, there were no significant differences in the catalytic properties of recombinant glycosylated lipase from P. pastoris and the unglycosylated one from Escherichia coil. These two recombinant LIP4s showed higher esterase activities toward long-chain ester (C16 and C18) and exhibited higher lipase activities toward unsaturated and long-chain lipids. In addition, LIP4 does not show interfacial activation as compared with LIP1 toward lipid substrates of tributyrin and triolein. These observations demonstrated that LIP4 shows distinguished catalytic activities with LIP1 in spite of their high sequence homology.
Tang
S J
SJ
Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, Republic of China. tsj@mail.ntou.edu.tw
Shaw
J F
JF
Sun
K H
KH
Sun
G H
GH
Chang
T Y
TY
Lin
C K
CK
Lo
Y C
YC
Lee
G C
GC
D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006358:Hot Temperature; D008049:Lipase; D010843:Pichia / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D003062:Codon; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C428110:Lip4 protein, Candida rugosa; D008049:Lipase