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

Abstract

ABSTRACT We developed an in vitro enzyme system to produce myo -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 archaeon Thermococcus kodakarensis , MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus , and IMPase from the hyperthermophilic bacterium Thermotoga maritima . The enzymes were individually produced in Escherichia 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 of myo -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 myo -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 T. maritima into the system and observed myo -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 produce myo -inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo -inositol-3-phosphate synthase, and myo -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 myo -inositol. myo -Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo -inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo -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 myo -inositol.