Economic Improvement of Myo-inositol Phosphate Generation by Enzyme Immobilization and Directed Enzyme Evolution

The potential health values of certain mo-inositol phosphates are well known. However, attempts to produce defined isomers of the different partially phosphorylated myo-inositol phosphates by non-enzymatic phytate hydrolysis or chemical synthesis is often associated with high expense and is not very efficient. Enzymatic phytate dephosphorylation was recognized as a cost effective strategy to make pure breakdown products of phytate available in high quantities. In order to improve enzyme stability and facilitate down-stream processing, the phytases were immobilized. Besides enzyme immobilization, directed enzyme evolution was identified as a strategy for an economic improvement of enzymatic myo-inositol phosphate production. Introduction Much scientific information has been reported in the last few years linking diet, specific foods, or individual food components with the maintenance of human health and the prevention of chronic diseases such as coronary heart disease, cancer or osteoporosis. Individual myo-inositol phosphate esters were shown to have important physiological functions in man (Shears, 1998). Some of the partially phosphorylated myo-inositol phosphates are involved in the phosphatidylinositol cycle, especially Dmyo-inositol(1,3,4)trisphosphate and D-myo-inositol(1,3,4,5)tetrakisphosphate play important roles as intracellular secondary messengers (Shears, 1998), and some are considered to be pharmaco-active (Carrington et al., 1993; Claxon et al., 1990; Maffucci et al., 2005; Phillippy and Graf, 1997). In addition, dietary myo-inositol phosphates have been suggested to bring about benefits for human health, such as amelioration of heart disease conditions by controlling hypercholesterolemia and atheriosclerosis (Jariwalla et al., 1990), protection against diabetes mellitus (Thompson, 1993) and caries (Kaufman and Kleinberg, 1971), prevention of renal stone formation (Grases et al., 2000), and protection against a variety of cancers, in particular colon cancer (Vucenik and Shamsuddin, 2003). So far, the diversity and practical unavailability of individual myo-inositol phosphates preclude their being used. Access to Individual Partially Phosphorylated myo-Inositol Phosphates The use of non-enzymatic phytate hydrolysis or chemical synthesis to provide access to individual myo-inositol phosphate isomers is often associated with high expense and is not very efficient. Chemical synthesis is a low yield multi-step process (Plettenburg et al., 2000) and attempts to produce certain partially myo-inositol phosphates non-enzymatically from phytate have resulted in mixtures of myoinositolpentakis(InsP5), -tetrakis(InsP4), -tris(InsP3), -bis(InsP2) and – monophosphate (InsP) isomers. Purification of these isomers from the mixture is arduous and uneconomical. All in all, 6 different InsP5, 15 different InsP4, 20 different InsP3, 15 different InsP2 and 6 different InsP exist. The direct production of the desired isomer using enzymes proves far more effective. Phytate was reported to be dephosphorylated by phytases regioand stereo-selective in a stepwise manner by producing, in general, only one myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphate isomer (Konietzny and Greiner, 2002). Phytases (myo-inositol