Bacterial expression and characterization of an active recombinant lipase A from Serratia marcescens with truncated C-terminal region

Mohammadi, Mohsen and Sepehrizadeh, Zargham and Ebrahim-Habibi, Azadeh and Shahverdi, Ahmad Reza and Faramarzi, Mohammad Ali and Setayesh, Neda Bacterial expression and characterization of an active recombinant lipase A from Serratia marcescens with truncated C-terminal region. Journal of Molecular Catalysis B: Enzymatic.

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Abstract

Abstract: The lipase of Serratia marcescens (SML) with 614 amino acid residues belongs to the lipase family I.3 has an important pharmaceutical application in production of chiral precursors. Like other members of this family, the SML consists of the N-catalytic domain (/) that contains the active-site residues and the C-terminal domain including the two parallel -roll domains, the first and second -roll. The repetitive sequences, a nine-residue sequence motif (GGXGXDXUX), in SML are somewhat degenerated as well as are not consecutive. The parallel -roll domains separated by a 72 residues spacer from each other. The importance of these repetitive sequences is not yet fully understood. In the present investigation, as an approach, a C-terminally truncated (~13kD) SML was generated using PCR-based site-directed mutagenesis method (designated SML-128). Both wild-type and truncated forms of SML were constructed , overexpressed in E. coli without Lip-system and purified by affinity chromatography on the Ni-NTA system. Kinetic parameters and circular dichroism (CD) spectra were determined and compared. The SML-128 showed an approximately 3.7-fold increase in the turnover rate (kcat), relative to that of the full-length enzyme. In conclusion, this report demonstrates that the SML could tolerate extensive modification in the C-terminal extreme of the protein, as deletion of the C-terminal region (~13 kDa), consisting of several tandem repeats of glycine-rich and 49 residues including signal peptide, significantly increases the catalytic efficiency of the enzyme. Keywords: Serratia marcescens, Family I.3, lipase, -roll, Expression, CD spectra

Item Type: Article
Subjects: R Medicine > R Medicine (General)
Depositing User: sobhan rezaiian
Date Deposited: 28 Sep 2016 05:53
Last Modified: 28 Sep 2016 05:53
URI: http://eprints.lums.ac.ir/id/eprint/213

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