Cloning, expression and characterisation of Erwinia carotovora L-asparaginase

dc.contributor.author	Kotzia, GA	en
dc.contributor.author	Labrou, NE	en
dc.date.accessioned	2014-06-06T06:46:27Z
dc.date.available	2014-06-06T06:46:27Z
dc.date.issued	2005	en
dc.identifier.issn	01681656	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.jbiotec.2005.04.016	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/3017
dc.subject	Enzyme therapy	en
dc.subject	Hydrolase	en
dc.subject	Kinetic analysis	en
dc.subject	L-Asparaginase	en
dc.subject	Leukaemia	en
dc.subject.other	Biotechnology	en
dc.subject.other	Catalysis	en
dc.subject.other	Cloning	en
dc.subject.other	Diseases	en
dc.subject.other	Escherichia coli	en
dc.subject.other	pH effects	en
dc.subject.other	Purification	en
dc.subject.other	Enzyme therapy	en
dc.subject.other	Hydrolase	en
dc.subject.other	Kinetic analysis	en
dc.subject.other	L-Asparaginase	en
dc.subject.other	Leukaemia	en
dc.subject.other	Leukemia	en
dc.subject.other	Enzymes	en
dc.subject.other	asparaginase	en
dc.subject.other	aspartic acid	en
dc.subject.other	bacterial enzyme	en
dc.subject.other	glutamine	en
dc.subject.other	recombinant enzyme	en
dc.subject.other	acidity	en
dc.subject.other	acute lymphoblastic leukemia	en
dc.subject.other	affinity chromatography	en
dc.subject.other	article	en
dc.subject.other	catalysis	en
dc.subject.other	childhood leukemia	en
dc.subject.other	entropy	en
dc.subject.other	enzyme active site	en
dc.subject.other	enzyme activity	en
dc.subject.other	enzyme analysis	en
dc.subject.other	enzyme mechanism	en
dc.subject.other	enzyme purification	en
dc.subject.other	enzyme specificity	en
dc.subject.other	enzyme structure	en
dc.subject.other	enzyme therapy	en
dc.subject.other	Escherichia coli	en
dc.subject.other	ion exchange chromatography	en
dc.subject.other	ionization	en
dc.subject.other	kinetics	en
dc.subject.other	molecular cloning	en
dc.subject.other	molecular model	en
dc.subject.other	nonhuman	en
dc.subject.other	nucleotide sequence	en
dc.subject.other	Pectobacterium carotovorum	en
dc.subject.other	Pectobacterium chrysanthemi	en
dc.subject.other	pH	en
dc.subject.other	priority journal	en
dc.subject.other	protein expression	en
dc.subject.other	protein structure	en
dc.subject.other	temperature	en
dc.subject.other	thermodynamics	en
dc.subject.other	viscosity	en
dc.subject.other	Amino Acid Sequence	en
dc.subject.other	Asparaginase	en
dc.subject.other	Binding Sites	en
dc.subject.other	Catalysis	en
dc.subject.other	Computer Simulation	en
dc.subject.other	Enzyme Activation	en
dc.subject.other	Enzyme Stability	en
dc.subject.other	Escherichia coli	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Models, Chemical	en
dc.subject.other	Models, Molecular	en
dc.subject.other	Molecular Sequence Data	en
dc.subject.other	Pectobacterium carotovorum	en
dc.subject.other	Protein Binding	en
dc.subject.other	Recombinant Proteins	en
dc.subject.other	Sequence Homology, Amino Acid	en
dc.subject.other	Substrate Specificity	en
dc.subject.other	Temperature	en
dc.subject.other	Bacteria (microorganisms)	en
dc.subject.other	Erwinia chrysanthemi	en
dc.subject.other	Pectobacterium carotovorum	en
dc.title	Cloning, expression and characterisation of Erwinia carotovora L-asparaginase	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.jbiotec.2005.04.016	en
heal.publicationDate	2005	en
heal.abstract	Bacterial l-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. l-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised l-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of Vmax and V max/Km were analyzed. The pH-dependence of Vmax shows one transition in the acidic pH range with pKa = 5.4, and the pH-dependence of Vmax/Km exhibits two transitions with pKa = 5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pKa at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pKa observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity. © 2005 Elsevier B.V. All rights reserved.	en
heal.journalName	Journal of Biotechnology	en
dc.identifier.issue	4	en
dc.identifier.volume	119	en
dc.identifier.doi	10.1016/j.jbiotec.2005.04.016	en
dc.identifier.spage	309	en
dc.identifier.epage	323	en