title: Novel cutinase from Pseudomonas cepacia NRRL B 2320: purification, characterization and identification of cutinase encoding genes.
abstract: An extracellular cutinase from Pseudomonas cepacia NRRL B 2320 was purified to apparent homogeneity. Upon biochemical characterization, the purified cutinase was found to be tolerant to organic solvents and surfactants under assay conditions. The molecular mass of cutinase was found to be 26.25 kDa by MALDI-TOF-MS analysis. The enzyme was able to show activity towards synthetic esters of chain length C4-C16. The activity of cutinase was enhanced by mono cations and various effectors, whereas it was moderately inhibited by various divalent cations and serine blocking reagent, phenyl methyl sulphonyl fluoride (PMSF). The optimal pH and temperature for highest activity were found to be 7.9 and 36.5°C, respectively. An overall 1.42-fold increase in activity was observed after optimization of both assay and process conditions. The exposure of hydrophobic amino acid to an aqueous environment and change in secondary structure of cutinase was observed from thermodynamic parameters (ΔH(*), ΔS(*)), fluorescence and circular dichorism spectra during the deactivation process. Two cutinase encoding genes were identified in P. cepacia, cloned and expressed in E. coli BL21 (DE3).
authors.affiliation: Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology (IIT), Guwahati, India. veeranki@iitg.ernet.in
authors.forename: Kasturi
authors.initials: K
authors.lastname: Dutta
authors.forename: Hegde
authors.initials: H
authors.lastname: Krishnamoorthy
authors.forename: Veeranki
authors.initials: V
authors.lastname: Venkata Dasu
mesh_terms: D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012997:Solvents / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002412:Cations; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D012997:Solvents; D002265:Carboxylic Ester Hydrolases; C019952:cutinase