A STUDY OF PHYTASE SYSTEM IN CUCURBITA MAXIMA (PUMPKIN) COTYLEDONS DURING GERMINATION

Abstract

The present study concerns with the isolation, charac terization, development and mode of action of multiple phytase species from Cucurbita maxima germinating cotyledons. Six molecular forms of phytase were separated and purified to homogeneity by SDS polyaery1amide gel electro phoresis (SDS PAGE) from the germinating Oucurhits, maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadcx gel (G-150) filtration and ion exchange chromato graphy on DBAS- and CM-cellulose. Gel filtration produced two peaks of phytase activity representing the high molecular weight (phytase I) and the low molecular weight (phytase II) species. The phytase I was further resolved into 5 distinct species on GM-cellulose. These were designated as phytase IA, IB, IC, ID and IB according to their elution order. The phy tase II emerged as a single pea,k both from DBAB and CMcellulose columns. The molecular weights of phytase IA, IB, IC, ID, IE and II as determined by SDS PAGE were found to be 6Q?56| 63,096; 43,657? 77,625; 33,905 and 29,5l2 Daltons res pectively. Some other properties characterising the multiple forms of phytase (IA, IB, IC, ID, IE and II) were determined using phytic acid as substrate. The PH optima curves for all the enzymes were sharply peaked at 5.0, 4.8, 5.0, 5.2, 4.4 and 5.6. The temperature optima of phytase I isoenzymes (IA, IB, IC, ID and IE) lie between 45-50°C but for phytase II it was

found to be sharp at 55°C. The values of temperature coef ficient Q10, between 30° and 40°C for phytase isoenzymes were found to be ranging from 1.3 to 2.3. All the phytase I isoenzymes were thermally stable upto 40 C, but phytase II was stable upto 50 C. The effect of temperature on the kinetic parameters of different phytase species are reported in terms of energy of activation (B)9 enthalpy change (aH), free energy change(AG) and entropy change (aS). Both Km and V^ values increase with increasing temperature. Ea of phvtase isoenzymes are different ranging from 3685 to 6909 Kcal/mole. aH values are positive indicating that in all cases the reaction is endothermic. AG values for phytase isoenzymes were found to be varying from 923 to 1845 Kcal/mole. aS values were found to be very low suggesting that phytase3 undergo little con formational change during enzyme substrate complex formation. The Arrhenius plots of log ?m(lv versus l/T in the temperature range 30°-50°C were found linear indicating that B& remains constant during reactions. The substrate affinity of different phytase species were determined towards different myoinositol polyphosphates on the basis of V^ /IC values. The data indicated that the Iuc—jC 111 myoinositol hexaphosphate is the most suitable substrate for enzyme II followed by IE and IB. Similarly myoinositol pentaphosphate is preferably hydrolysed by enzyme IB followed by enzyme II. The myoinositol tetraphosphate is a preferred substrate for both enzyme II and ID but the myoinositol iii triphosphate is the most suitable substrate for enzyme II. Diphosphates and monophosphates show preference for enzyme IB smd II. In addition, the phytase II exhibits high substrate affinity for all inositol polyphosphates which may be of physiological significance, as this enzyme makes its appea rance at a very late stage of germination when both the level of phytase I activity and the concentration of phytase are very low. The mode of dephosphorylation of myoinositol hexaphosphate (MlPg) by individual phytase isoenzyme was studied in vitro. The phosphorus-containing intermediates formed during hydrolysis of MlPg were isolated by ion-exchange and paper chromatography and identified by pcriodate oxidation and acid catalysed phosphate migration across cis OH groups and by IR spectroscopy of sugar alcohols formed on periodate cleavage of myoinositol phosphates followed by reduction and dephosphorylation with NaBH4 and HC1, respectively. These results showed that the dephosphorylation of phytic acid by all the molecular species of phytase, proceeds in a stepwise manner. On the basis of kinetic data and the pattern of deve lopment of different phytase isoenzymes end assuming that the relative concentrations of different phytases together with substrate affinity, nature and relative substrate concentra tions should determine the course of phytate dephosphoryla tion in vivo, the possible role of each phytase isoenzyme has been suggested (Fig. l). For example, phytase IB seems to be Phy. ID _ Phy IB Phy. I E MIP, MIPC MIP, (P-1.2.3,4,5.SJ (P-1.2,3,4,5) ( P-1,2, 3, C) Phy II ( M[p .. Myoinositol phosphate ester) ( P Phosphate group) MIP3 P-1,2 3) MIP3 [ P-2. 3.4 ) Fig 1 - Dephosphorylation Pathway of Phytic Acid Phy. IE Phy. IE MI R, —^ MI p! Phy II -> Inositol 2 7^ ' s~ A P-2, 3 ) Phy II ( P-2) Phy II involved to the selective dephosphorylation of MIP6 at position No. 6to yield corresponding MIP5 and the lower molecul*r for™, of phytase heing more specific for the lower inositol phosphates. The developmental pattern of the multimolecular spe cies to germinating Pipkin cotyledons was investigated hy separattog the different molecular forms and analysing for their relative levels of activities at different stages of germination. So phytase activity was present to the unimhihed intact cotyledons tat the early phase of germination perxed, hetween first and seventh day, was mart* hy the high r<*e of synthesis of the high molecular weight enzyme, phytase I. species with very little synthesis of low molecular weight enzyme, phytase II. In the later stages of germination, hetween 9th and 15th day, there was a sharp decline to the level of Phytase I activity with simultaneous rise in phy tase II activity level. It is remarkable to note that to the very onset of germination (first 12 hours of germination), the isoenzyme IB was found to account for more the* half of the total phytase I activity followed hy phytase IA (25 V. ). However, the activity level of IB enzyme declined rapidly and heoame nearly insignificant to ahout 48 hrs of germination. -,*= lndioate that to the very early period of ger- These results rnuice-Tie suw ** tb <• rasoonsihlo for dephosphorylation mination, isoenzyme IB is respond l of myoinositol hexaphosp»h,„a+t„e, tthhee oprriimma.rivj form of reserve phosphate. The enzyme IA shows somewhat a different pattern of

development. Its activity level increases upto 48 hrs contri buting about 50 V. of the total phytase I activity. This state is maintained upto 180 hrs. After this peak period the level of its activity starts declining slowly indicating that the enzyme IA plays a major role in the phytate metabolism during 43 to 130 hours germination period when the activity of all other phytase I isoenzymes was low. At this moment it is not certain if the enzyme IA and IB are structurally related. The molecular weight data and developmental pattern, however, indicate such relationship, i.e. IB may be the precursor of IA. Enzyme IC and ID showed minor activities. Their acti vities were found to be maximum at 84 and 48 hours of germi nation respectively. After that they started declining and became insignificant by the end of germination (15 days). The enzyme IE was found to follow yet another develop mental pattern as its activity increases progressively from merely 10 -/.after 12 hrs, upto 70 -/.of phytase I activity at the end of 372 hours of the germination. So it seems that enzyme IE plays rather a supplementary role for the enzyme IA especially in the later stages of germination. The synthesis of phytase II enzyme (low molecular weight) starts only after 36 hours of germination and unlike phytase I its activity goes on increasing through the total germination period of 15 days. In fact, the activity level of phytase II clearly becomes dominant between l2th and l5th day of germination. Like phytase IE, the role of this enzyme also Vll seems to supplement the phytase IA in the later period of germination when only lower inositol phosphates such as tri-, di- and monophosphates would be available in relatively higher concentrations than phytate as substrate. Thus, it seems that the relative concentrations of different isoenzymes are related to the stage of germination and may be responsible for the regulation of the overall phytate metabolism in germinating cotyledons of pumpkin seeds„