Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-Sepharose

The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.