A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched ß-cyclodextrin (Glcn-ß-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.

Novel formulation of low-fat spread using rice starch modified by 4-α-glucanotransferase.

Low-fat spreads were developed using a thermoreversible gelling agent, the 4-α-glucanotransferase (4αGT)-modified rice starch. The low-fat spreads consisted of the modified starch paste (or rice starch or maltodextrin), olive oil (0-30% w/w), egg yolk, salt, xanthan gum, and butter flavor, and were produced by homogenization, ultrasonic processing at 50% amplitude for 2min, and cold-gel setting at 4°C for 24h. Formulations with 15% and 20% of the modified starch paste resulted in highly stable oil-in-water low-fat spreads having varied textural properties and acceptable spreadability, whereas formulations with rice starch and maltodextrin did not yield enough stability and consistency. Moreover, the modified starch-based low-fat spreads showed high thermoreversibility. These results indicated that 4αGT-modified starch could be used in the preparation of low-fat spreads, allowing the formulation of functional products for healthy diets.