An In Vitro Enzyme System for the Production of myo -Inositol from Starch

• Published in: Applied and environmental microbiology 2017-08, Vol.83 (16)
• Main Authors: Fujisawa, Tomoko, Fujinaga, Shohei, Atomi, Haruyuki
• Format: Article
• Language: English
• Subjects: Amylose; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Archaeoglobus fulgidus - enzymology; Biotechnology; Cells; E coli; Effects; Enzyme Stability; Enzymes; Heat treatment; Inositol - chemistry; Inositol - metabolism; Inositol-3-phosphate synthase; Intramolecular Lyases - chemistry; Intramolecular Lyases - genetics; Intramolecular Lyases - metabolism; Maltodextrin; Maltodextrin phosphorylase; Myo-Inositol-1 (or 4)-monophosphatase; NAD; NAD - metabolism; Phosphates; Phosphoglucomutase; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Phosphorylase; Potatoes; Proteins; Pullulanase; Starch; Starch - chemistry; Starch - metabolism; Thermococcus - enzymology
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00550-17

We developed an enzyme system to produce -inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), -inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon , MIPS from the hyperthermophilic archaeon , and IMPase from the hyperthermophilic bacterium The enzymes were individually produced in and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD , resulting in the production of -inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD was added every 2 h, we observed the production of 2.9 g -inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from into the system and observed -inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. -Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce -inositol from starch with an enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), -inositol-3-phosphate synthase, and -inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of -inositol. -Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of -inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced -inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of -inositol.