title: Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5(T).
abstract: We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation, lpetr65@yahoo.com.
authors.forename: L E
authors.initials: LE
authors.lastname: Petrovskaya
authors.forename: K A
authors.initials: KA
authors.lastname: Novototskaya-Vlasova
authors.forename: E A
authors.initials: EA
authors.lastname: Kryukova
authors.forename: E M
authors.initials: EM
authors.lastname: Rivkina
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D019295:Computational Biology; D004926:Escherichia coli; D004950:Esterases / Q000737:chemistry*; D006868:Hydrolysis; D007477:Ions; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry; D065946:Permafrost / Q000382:microbiology; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry
chemical_list: D007477:Ions; D011994:Recombinant Proteins; D012997:Solvents; D004950:Esterases; D010447:Peptide Hydrolases