TY - JOUR
T1 - Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli
AU - Tang, Shye Jye
AU - Sun, Kuang Hui
AU - Sun, Guang Huan
AU - Chang, Terng Yuan
AU - Lee, Guan Chiun
N1 - Funding Information:
We thank Dr. Fung-Fang Wang for fruitful discussions. This work was supported by Grants NSC-86-2313-B-019-002 and NSC-87-2313-B-019-001 from the National Science Council, Republic of China.
PY - 2000
Y1 - 2000
N2 - 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 RL21(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. (C) 2000 Academic Press.
AB - 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 RL21(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. (C) 2000 Academic Press.
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U2 - 10.1006/prep.2000.1304
DO - 10.1006/prep.2000.1304
M3 - Article
C2 - 11049754
AN - SCOPUS:0033777407
SN - 1046-5928
VL - 20
SP - 308
EP - 313
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -