title: Production optimization and characterization of recombinant cutinases from Thermobifida fusca sp. NRRL B-8184.
abstract: Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318±0.73 and 316±0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40-80 °C) and pH (6.8-9) with optimum activity at pH 8.0 and 55 °C.
authors.affiliation: Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology, Guwahati 781039 Assam, India.
authors.forename: Krishnamoorthy
authors.initials: K
authors.lastname: Hegde
authors.forename: Venkata Dasu
authors.initials: VD
authors.lastname: Veeranki
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology; D013696:Temperature
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D012997:Solvents; D002265:Carboxylic Ester Hydrolases; C019952:cutinase