Enzymatic synthesis of c-di-GMP using inclusion bodies of Thermotoga maritima full-length diguanylate cyclase

• Published in: Journal of biotechnology 2013-03, Vol.164 (2), p.276-280
• Main Authors: Korovashkina, A.S., Rymko, A.N., Kvach, S.V., Zinchenko, А.I.
• Format: Article
• Language: English
• Subjects: Allosteric Site; Allosteric site mutation; Biotechnology; c-di-GMP; Cyclic GMP - analogs & derivatives; Cyclic GMP - analysis; Cyclic GMP - metabolism; Diguanylate cyclase; Electrophoresis, Polyacrylamide Gel; Enzymatic synthesis; enzymes; Escherichia coli; Escherichia coli Proteins - metabolism; gene overexpression; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Inclusion bodies; Inclusion Bodies - metabolism; Kinetics; mutants; Mutation; Phosphorus-Oxygen Lyases - metabolism; Recombinant Proteins - metabolism; Salts; Thermotoga maritima; Thermotoga maritima - enzymology
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2012.12.006

► E. coli strains producing diguanylate cyclase of Thermotoga maritima were constructed. ► The obtained recombinant enzymes are produced in the form of active inclusion bodies. ► Diguanylate cyclase allosteric site inactivation resulted in increase of its activity. ► c-di-GMP was originally synthesized with the diguanylate cyclase inclusion bodies. Recombinant full-length diguanylate cyclases (DGCs) of Thermotoga maritima with native and mutant allosteric sites were overexpressed in Escherichia coli cells and characterized. It has been shown that target enzymes are produced substantially in the form of active inclusion bodies. Introduction of the mutation in allosteric site resulted in 7-fold increase of the T. maritima DGC activity. Possibility of applying full-length DGC of T. maritima in the form of inclusion bodies for synthesis of c-di-GMP was originally demonstrated.