It is difficult to express recombinant Candida rugosa lipases (CRLs) in het
erologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG
for leucine. In this study, recombinant LIP4 in which all 19 CUG codons ha
d been converted to a universal serine codon was overexpressed in Escherich
ia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion b
odies and showed a low catalytic activity. To increase the amount of solubl
e 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 a
ddition, recombinant LIP4 had higher esterase activities toward long-chain
ester and lower lipase activities toward tributyrin, triolein, and olive oi
l. 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. (C) 2000 Academic Press.