title: Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis.
abstract: Engineering of invertases has come to attention because of increasing demand for possible applications of invertases in various industrial processes. Due to the known physicochemical properties, invertases from micro-organisms such as Saccharomyces cerevisiae carrying SUC2 gene are considered as primary models. To improve thermostability and catalytic efficiency of SUC2 invertase (SInv), six influential residues with Relative Solvent Accessibility<5% were selected through multiple-sequence alignments, molecular modelling, structural and computational analyses. Consequently, SInv and 5 mutants including three mutants with single point substitution [Mut1=P152V, Mut2=S85V and Mut3=K153F)], one mutant with two points [Mut4=S305V-N463V] and one mutant with three points [Mut5=S85V-K153F-T271V] were developed via site-directed mutagenesis and produced using Pichia pastoris as the host. Physicochemical studies on these enzymes indicated that the selected amino acids which were located in the active site region mainly influenced catalytic efficiency. The best improvement belonged to Mut1 (54% increase in Kcat/Km) and Mut3 exhibited the worst effect (90% increase in Km). These results suggest that Pro152 and Lys153 play key role in preparation of the right substrate lodging in the active site of SInv. The best thermostability improvement (16%) was observed for Mut4 in which two hydrophilic residues located on the loops, far from the active site, were replaced by Valines. These results suggest that tactful simultaneous substitution of influential hydrophilic residues in both active site region and peripheral loops with hydrophobic amino acids could result in more thermostable invertases with enhanced catalytic efficiency.
authors.affiliation: Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology. P.O. Box: 149651/161, Tehran, Iran.
authors.forename: Nooshin
authors.initials: N
authors.lastname: Mohandesi
authors.affiliation: Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology. P.O. Box: 149651/161, Tehran, Iran. Electronic address: Kamahl@nigeb.ac.ir.
authors.forename: Kamahldin
authors.initials: K
authors.lastname: Haghbeen
authors.affiliation: Department of Biotechnology, Faculty of New Technologies Engineering, Shahid Beheshti University, Tehran, Iran.
authors.forename: Omid
authors.initials: O
authors.lastname: Ranaei
authors.affiliation: Faculty of Biological Science, Bioinformatics Department, Tarbiat Modares University, Tehran, Iran.
authors.forename: Seyed Shahriar
authors.initials: SS
authors.lastname: Arab
authors.affiliation: Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology. P.O. Box: 149651/161, Tehran, Iran.
authors.forename: Sorour
authors.initials: S
authors.lastname: Hassani
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D001709:Biotechnology; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase
keywords: Invertase engineering; Pichia pastoris; Saccharomyces cerevisiae; Site-direct mutagenesis