3 research outputs found
Evolution of Chymotrypsin-Like Enzymes for Specific Hydrolytic Bioconversions of Industrial Interest
Expression of rat diamine oxidase in Escherichia coli
In this work, a reliable protocol was designed to rapidly express and purify a rat diamine oxidase in
Escherichia coli as a useful alternative to enzyme isolated from animal sources. The cDNA encoding for
rat diamine oxidase was overexpressed in an Origami2(DE3) E. coli strain and, by employing a rapid
purification protocol in which the hexahistidine tag was added at the C-terminal end of the enzyme,
the recombinant oxidase could be purified in a single step on a Ni-NTA column at >95% purity. The
enzyme was active but was largely produced in an immature quinone form: Cu2+ ions stimulated further
activation/maturation. This expression and purification procedure offers an easy and rapid means of
producing recombinant rat diamine oxidase from an animal-free source and represents a useful tool to
boost biotechnological application of this enzyme