Bacterial Expression and Characterization of an Active Recombinant Lipase a From Serratia Marcescens With Truncated C-Terminal Region Publisher



Mohammadi M^{1, 2} ; Sepehrizadeh Z¹ ; Ebrahimhabibi A^{3, 4} ; Reza Shahverdi A¹ ; Ali Faramarzi M¹ ; Setayesh N¹ Show Affiliations
Source: Journal of Molecular Catalysis B: Enzymatic Published:2015

Abstract

The lipase of Serratia marcescens (SML), containing 614 amino acid residues and belonging to the lipase family I.3, has an important pharmaceutical application in production of chiral precursors. Similar to other members of this family, the SML consists of the N-catalytic domain (α/β) that contains the active-site residues and the C-terminal domain that includes the two parallel β-roll domains, the first and second β-roll. The repetitive sequences, a nine-residue sequence motif (GGXGXDXUX), in SML are somewhat degenerated as well as not consecutive. The parallel β-roll domains are separated from each other by a 72-residue spacer. The importance of these repetitive sequences has not been fully understood yet. In the present investigation, as an approach, a C-terminally truncated (∼13 kD) SML was generated using a polymerase chain reaction (PCR)-based site-directed mutagenesis method (designated SML-Δ128). Both wild-type and truncated forms of SML were constructed, overexpressed in Escherichia coli without the Lip system and purified by affinity chromatography on the Ni-NTA system. The kinetic parameters and circular dichroism (CD) spectra were determined and compared. The SML-Δ128 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), which consists of several tandem repeats of glycine-rich and 49 residues including the signal peptide, significantly increases the catalytic efficiency of the enzyme. © 2015 Elsevier B.V. All rights reserved.