title: Novel S-enantioselective lipase TALipB from Trichosporon asahii MSR54: Heterologous expression, characterization, conformational stability and homology modeling.
abstract: A novel lipase encoding gene, TALipB from Trichosporon asahii MSR54 was heterologously expressed in Escherichia coli using three vectors, pET22b, pET28a & pEZZ18. The three recombinant proteins, viz. C-hexahistidine fused HLipB, N and C-hexahistidine fused HLipBH and ZZ-fused ZZLipB were purified using affinity chromatography. All the three enzymes were mid to long fatty acyl chain selective on p-NP esters and S-enantioselective irrespective of tags. HLipB had lowest activation energy (3.5 Kcal mol(-1)) and highest catalytic efficiency (254 mM(-1) min(-1)) on p-NP caprate followed by HLipBH and ZZLipB. However, ZZLipB demonstrated best pH stability (pH 6-10), thermostability (t1/2 of 50 min at 70 °C) and stability toward the denaturant Guanidium chloride (300 mM). Far-UV CD and fluorescence studies confirmed the role of N-terminal ZZ-tag in stabilizing the protein by altering its secondary and tertiary structures. All the three proteins were thiol activated. ZZLipB required higher concentration of β-mercaptoethanol as compared to the other two proteins to attain similar velocity. This indicated the involvement of additional disulfide bonds in its conformational stability. In silico analysis suggested low sequence identity of the enzyme with the available database but a close structural homology with Candida antarctica lipase B (CALB) was revealed by PHYRE(2). MULTALIN with CALB predicted the active site residues (Ser137-Asp228-His261) which were confirmed by superimposition and site directed mutagenesis.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Singh
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: ranigupta15@rediffmail.com.
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature; D014250:Trichosporon / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica
keywords: Enantioselective; In-silico; Lipase; Thiol; Trichosporon asahii MSR54