title: Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.
abstract: Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T1/2, respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations.
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Lorestan University of Medical Sciences, Khorramabad, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Mohammadi
authors.affiliation: Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Zargham
authors.initials: Z
authors.lastname: Sepehrizadeh
authors.affiliation: Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: aehabibi@sina.tums.ac.ir.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Ebrahim-Habibi
authors.affiliation: Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Shahverdi
authors.affiliation: Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Faramarzi
authors.affiliation: Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research Center, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: nsetayesh@tums.ac.ir.
authors.forename: Neda
authors.initials: N
authors.lastname: Setayesh
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008049:Lipase
keywords: CD spectra; Differential scanning calorimetry; Family I.3 lipase; Serratia marcescens; Site-directed mutagenesis