This chapter discusses the experimental study, focusing on the determination of nicotinamide. Nicotinamide and nicotinate are generally determined, by some modification of the Koenig reaction— that is, the reaction with cyanogens bromide and an aromatic amine to give a color. In this study, first the nicotinamide deamidase is prepared. The yeast Saccharomyces cervisiae, obtained from a spent fermentation tank, is washed thrice with 0.1 M potassium phosphate buffer, pH 7.5. The washed cells are homogenized in a Waring Blender, with 10 volumes of cold (–10°) acetone and filtered on a Buchner funnel. The residue is homogenized again in cold ether, filtered, and dried in a vacuum. The acetone powder is autolyzed, by incubation in six volumes (10 g/60 ml) of 0.1 M KHCO3 for 4 h at 37° with shaking. The autolysate is centrifuged in cold at 20,000g for 15 min. The resulting supernatant fluid is brought to 35% saturation, by the addition of solid ammonium sulfate (24.7 g/100 ml). The mixture is allowed to stand for 1 hr and is centrifuged. The resulting nicotinamide deamidase preparation contains ca. 200 units/mg protein when assayed by the procedure of Joshi and Handler, employing [7‑14C]nicotinamide. Series: Methods in Enzymology (Book 66) Hardcover: 755 pages Publisher: Academic Press; Language: English ISBN-10: 0121819663 ISBN-13: 978-0121819668 Product Dimensions: 6 x 1.6 x 9 inches Link Download http://nitroflare.com/view/0E3C057FE437E07https://drive.google.com/drive/folders/1yLBzZ1rSQoNjmWeJTZ3WGQHg04L1
