Publications
ChemCatChemAug 2020 |
12
(
16
),
4044-4051
DOI:
10.1002/cctc.202000561

Extending the Library of Light‐Dependent Protochlorophyllide Oxidoreductases and their Solvent Tolerance, Stability in Light and Cofactor Flexibility

Schmermund, Luca; Bierbaumer, Sarah; Schein, Viktor K.; Winkler, Christoph K.; Kara, Selin; Kroutil, Wolfgang
Product Used
Genes
Abstract
SYNTHESIS AND PURIFICATION OF PCHLIDE: The protochlorophyllide was produced and purified form a _Rhodobacter capsulatus_ ZY5 culture. _Rhodobacter capsulatus_ ZY5 cells from a glycerol stock were plated on agar plates with VN medium (10 g/L yeast extract, 1 g/L MgSO4, pH 7.0) containing rifampicin (rif) (25 μg/mL). The plates were incubated at 34 °C for 48 hours until greenish‐red colonies were formed. Then VN medium (100 ml) containing rif (25 μg/mL) were inoculated with several colonies and the preculture was incubated at 34 °C and 120 rpm. After 31 h the preculture (75 mL) was transferred to a 2 L flask with fresh VN medium (1 L) containing rif (25 μg/mL). In addition, two white FisherbrandTM polyurethane foam stopper (height/diameter 50/35 mm, Fisher Scientific) were added to the culture and the culture was incubated for 72 h at 34 °C and 130 rpm. The foam stopper turned dark green and were replaced every 24 h (Figure SI5). The dark green foam stopper were dried overnight in the dark. Pchlide was washed from foam bungs with methanol (800 mL) and the solvent was removed under reduced pressure. Then the crude pchlide was resuspended in acetone (800 mL). For better resuspension methanol (1.5 % v/v) was added. Subsequently, pchlide was purified by a CM Sepharose Fast Flow column (SIGMA). For this, 50 mL CM Sepharose were poured onto a Buchner funnel and washed with ddH2O (500 mL). Then resin was resuspended in acetone (200 mL) and was dried. The acetone treatment was repeated twice before the resin was finally resuspended in acetone (100 mL) and poured into a glass column (5 cm wide). Then, the green pchlide suspension was loaded onto the column. The column was washed with acetone (10 cv) in order to remove all carotenoids and with acetone (containing 5 % v/v methanol, 6 cv) in order to remove phytol or pheophorbide.22 Finally, the pchlide was eluted with acetone (25 % v/v methanol, 10 cv). The elution fractions were concentrated under reduced pressure to a volume of 50 mL. The final pchlide solution was aliquoted (1 mL, 1 mg/mL), the solvent was removed completely under reduced pressure and the aliquots were stored at −21 °C in the dark.
Product Used
Genes

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