Alcohol dehydrogenase from Saccharomyces cerevisiae (yeast) was purchased from Sigma-Aldrich as a lyophilized powder and used as received. ADH (~50 units) was mixed with 200 μM NADH and 400 μL of CYP121 reaction flow-through from the previous section. The absorbance was monitored at 339 nm using a UV–vis spectrophotometer (Agilent 8453) during the reaction.
Formaldehyde dehydrogenase from Pseudomonas was purchased from Sigma-Aldrich as a lyophilized powder and used as received. FDH (~0.5 units) was mixed with 200 μM NAD+ and CYP121 reaction flow-through, and the reaction was monitored as described above.
Chemical method of formaldehyde formation quantitation was also attempted. 2,4-Dinitrophenylhydrazine (2,4-DNP) was purchased from Sigma-Aldrich and prepared using previously published methods.27 The reaction of CYP121 with cYF-4-OMe was completed as described above and heated to precipitate the enzyme, and then the solids were pelleted. The supernatant was collected, and 2, 4-DNP was added to make up 1.5% of the mixture. The resulting mixture was then analyzed via HPLC, utilizing a gradient of 5–95% acetonitrile in H2O with 0.1% formic acid.
Formaldehyde dehydrogenase from Pseudomonas was purchased from Sigma-Aldrich as a lyophilized powder and used as received. FDH (~0.5 units) was mixed with 200 μM NAD+ and CYP121 reaction flow-through, and the reaction was monitored as described above.
Chemical method of formaldehyde formation quantitation was also attempted. 2,4-Dinitrophenylhydrazine (2,4-DNP) was purchased from Sigma-Aldrich and prepared using previously published methods.27 The reaction of CYP121 with cYF-4-OMe was completed as described above and heated to precipitate the enzyme, and then the solids were pelleted. The supernatant was collected, and 2, 4-DNP was added to make up 1.5% of the mixture. The resulting mixture was then analyzed via HPLC, utilizing a gradient of 5–95% acetonitrile in H2O with 0.1% formic acid.