Purification of Ectoine-Biosynthetic Enzymes

Ectoine was a kind gift from bitop AG (Witten, Germany). 5-Hydroxyectoine and glycine betaine were purchased from Merck KGaA (Darmstadt, Germany). Homoectoine was synthesized according to previously reported procedures (Koichi et al., 1991 ; Schnoor et al., 2004 (link)). Dithiothreitol (DTT) was purchased from AppliChem (Darmstadt, Germany) and phenylmethylsulfonyl fluoride (PMSF) from Roche (Basel, Switzerland). Desthiobiotin, Strep-TactinSuperflow chromatography material for the purification of proteins fused to a Strep-tag II affinity peptide and anhydrotetracycline hydrochloride (AHT) for the induction of the transcriptional activity of the TetR-regulated tet promoter present on the ectD expression plasmids pMP40 (ectD from Sphingopyxis alaskensis) and pMP41 (ectD from Pseudomonas stutzeri) (Widderich et al., 2014a (link)) were purchased from IBA GmbH (Göttingen, Germany). All other chemicals were purchased from Karl Roth GmbH (Karlsruhe, Germany), Merck KGaA (Darmstadt, Germany), and Sigma-Aldrich (Steinheim, Germany).