Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253

Abstract

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and stability. Cell envelope components such as mycolic acids are also influenced by sugars and inducers such as cobalt and urea. This is information that can be useful when implementing rhodococcal catalysts in industrial applications.

Lawsonella clevelandensis gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from human abscesses

“…The positive control was Rhodococcus rhodochrous strain DAP 96253 grown on yeast extract (10 g) malt extract (10 g) agar (20 g) (YEMEA) supplemented with cobalt (100 p.p.m. ), urea (7.5 g), and glucose (4 g) for 7 days at 30 C (Tucker et al, 2012;Dietz & Thayer, 1980). Cells were combined with 2 ml of saponification reagent (200 g KOH, 400 ml deionized water and 400 ml methanol) and mixed for 30 s, then autoclaved for 80 min at 121 C. The cooled mixture was combined with 1.5 ml of acidification reagent (6 M HCl) and shaken for 30 s. Mycolates were extracted twice with 2 ml dichloromethane (DCM).…”