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We developed anin vitro enzyme system to producemyo -inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM),myo -inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeonThermococcus kodakarensis , MIPS from the hyperthermophilic archaeonArchaeoglobus fulgidus , and IMPase from the hyperthermophilic bacteriumThermotoga maritima . The enzymes were individually produced inEscherichia coli 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 ofmyo -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 gmyo -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 fromT. maritima into the system and observedmyo -inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively.IMPORTANCE myo -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 producemyo -inositol from starch with anin vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM),myo -inositol-3-phosphate synthase, andmyo -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 ofmyo -inositol.myo -Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g ofmyo -inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we producedmyo -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 ofmyo -inositol.

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