Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli.
It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure.
Tang
S J
SJ
Institute of Marine Biotechnology, Keelung, 20224, Taiwan. tsj@mail.ntou.edu.tw
Sun
K H
KH
Sun
G H
GH
Chang
T Y
TY
Lee
G C
GC
D002175:Candida / Q000201:enzymology*; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D013879:Thioredoxins / Q000235:genetics; D014280:Triglycerides / Q000378:metabolism
D003062:Codon; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D012694:Serine; D013879:Thioredoxins; D008049:Lipase