Bioprospecting for enzymes in bryophytes: Extraction of L-Myo-inositol-1-phosphate synthase from Sphagnum junghuhnianum Doz. et Molk. and its characterization

Abstract

L-myo-inositol-1-phosphate synthase (MIPS) and its product, free myo-inositol have been isolated from different bryophytes. MIPS was purified for the first time from the moss, Sphagnum junghuhnianum to 58.67 fold over its homogenate fraction with about 32.86 % recovery. D-glucose-6-phosphate was the exclusive substrate of the enzyme without which there was no enzyme activity and the deduction of NH4Cl, ME and NAD+ substantially reduced the activity of the enzyme. The Km for D-glucose-6-phosphate and NAD+were 1.81 mM and 0.25 mM, while the Vmax for the same were 1.42 mM and 1.12 mM respectively. The molecular weight of the enzyme was assessed to be approximately 174 kDa. The activity of the enzyme increased with the increase in the duration of incubation time for up to 90 min and with the increase in protein concentration for up to 300 µg. The pH and temperature maxima were pH 7.0 and at 30 °C, respectively, but a significant activity was observed at 10 °C also. NH4Cl substantially stimulated the enzyme activity while K+ and Ca2+ also raised the activity slightly . Li+ greatly inhibited the activity. Inhibitory activity was also shown by Cd2+, Mn2+, Zn2+and Hg2+ of which Hg2+ showed the maximum inhibition. Interestingly, the Sphagnum junghuhnianum MIPS showed the characteristics of both Class-II and Class-III aldolase.