title: Phenylalanine to leucine point mutation in oxyanion hole improved catalytic efficiency of Lip12 from Yarrowia lipolytica.
abstract: In lipases, oxyanion hole has crucial role in the stabilisation of enzyme-substrate complex. Majority of lipases from Yarrowia lipolytica consist of two oxyanion hole residues viz.; Thr and Leu. However, Lip12 has Phe instead of Leu at second oxyanion hole residue. It was observed that Lip12 has lower specific activity and catalytic efficiency than other lipases of Yarrowia. In silico analysis of Phe to Leu mutation revealed improved binding energy of Lip12 for p-np palmitate. This was validated by Phe148 to Leu point mutation where, specific activity of mutant was 401U/mg on olive oil, which was two fold higher in comparison to wild-type. Kcat, remained unaltered, while decrease in Km was predominant for all the substrates used in the study. Improved catalytic efficiency of mutant was a function of chain length in case of p-np esters, with 73% improvement for p-np stearate. However, hydrolysis of triacylglycerides improved by 20%, irrespective of chain length. Decrease in activation energy for all the substrates, was observed in mutant in comparison to wild-type, indicating better stabilisation of transition state complex. Further, unaltered differential activation energy for mutant depicts that substrate specificity of enzyme remained same after mutation.
authors: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors: Arti
authors: A
authors: Kumari
authors: Rani
authors: R
authors: Gupta
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D010938:Plant Oils / Q000378:metabolism; D017354:Point Mutation; D013379:Substrate Specificity; D013816:Thermodynamics; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; D010938:Plant Oils; D010649:Phenylalanine; D008049:Lipase; D007930:Leucine
keywords: Catalytic efficiency; Differential activation energy; Lipase; Oxyanion hole; Yarrowia lipolytica