Purification, refolding, and characterization of recombinant Pseudomonas fluorescens lipase.
Thermostable Pseudomonas fluorescens SIK W1 lipase (PFL), which is responsible for the spoilage of milk, was overexpressed as inclusion bodies in Escherichia coli. Renaturation of solubilized PFL was achieved by using size-exclusion protein refolding chromatography. The renatured enzyme was purified homogeneously using a combination of gel filtration and ion-exchange FPLC. Its specific activity was found to be enhanced in the presence of Ca2+. Secondary structural changes induced by Ca2+ were monitored by circular dichroism, which demonstrated that the activity increase of PFL in the presence of Ca2+ is strongly correlated with significant increases in alpha-helix and beta-sheet content. In the presence of Ca2+, the PFL structure was found resistant to denaturation by guanidine hydrochloride and to enzyme activity loss due to cosolvents like DMSO and trifluoroethanol, suggesting that Ca2+ plays an important role in inducing conformational changes and consequently in maintaining enzyme structural stability.
Kim
Kyu Rae
KR
Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Kwon
Dae Young
DY
Yoon
Suk Hoo
SH
Kim
Woo Yeon
WY
Kim
Kyung Hyun
KH
D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D002479:Inclusion Bodies / Q000378:metabolism; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D008049:Lipase; D002118:Calcium