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2 protocols using d galactose

1

Enzymatic Characterization of Carbohydrate-Active Enzymes

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Cel2-5 and Lam2-5 were purchased from Megazyme. Sucrose, maltose, l-arabinose, d-fructose, d-galactose, d-xylose, d-mannose, l-rhamnose, and d-gluconate were purchased from Nacalai Tesque. Sop2-5 were prepared using SOGP from L. innocua and SGL from Chitinophaga pinensis (23 , 28 (link), 29 (link)). Lactose, α,α-trehalose, melibiose, esculin, d-amygdalin, methyl-α-Glc, ethyl-α-Glc, benzyl-α-Glc, pNP-α-Glc, and pNP-β-Glc were purchased from FUJIFILM Wako Chemical Corporation. Gentiobiose, d-talose, and gastrodin were purchased from Tokyo Chemical Industry. d-Gulose and 2-naphthyl-α-Glc were purchased from Toronto Research Chemicals. α-Arbutin was purchased from Ezaki Glico. n-Octyl-β-Glc and methyl-β-Glc were purchased from DOJINDO LABORATORIES and Merck, respectively. Salicin and β-arbutin were purchased from Combi Blocks.
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2

Enzymatic Characterization of Sucrose Phosphorylase

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d-Allose, d-glucose, α-Glc1P, d-gluconic acid, lactose, d-mannose, and d-xylose were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan); N-acetyl-d-glucosamine, d-galactose, and sucrose were purchased from Nacalai Tesque (Kyoto, Japan); d-glucosamine was purchased from Tokyo Chemical Industry (Tokyo, Japan); cellobiose, d-galacturonic acid, and d-glucuronic acid were purchased from Sigma (St. Louis, MO, USA). β-(1 → 4)-Mannobiose was prepared as previously described29 (link). Lactoless L3 (β-galactosidase) was provided by Daiwa Kasei (Shiga, Japan). SP from Bifidobacterium longum was prepared according to the method described by Nishimoto and Kitaoka5 (link). One unit of SP was defined as the amount of enzyme required to phosphorolyze 1 μmol of sucrose in 1 min. SP activity was measured as follows: a reaction mixture (50 μL) containing appropriate concentration of enzyme, 20 mM sucrose, 40 mM sodium phosphate, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer (pH 7.0), and 0.2 mg/mL bovine serum albumin (BSA, Nacalai Tesque) was incubated at 37 °C for 10 min. The enzymatic reaction was terminated by incubating the sample at 80 °C for 3 min, and the liberated d-fructose was measured using a d-Fructose/d-Glucose Assay Kit (Megazyme, Brey, Ireland).
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