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Title of Thesis

Tehseen Aman
Institute/University/Department Details
Institute of Chemistry/University of the Punjab, Lahore
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
clycerolipids, seed maturation, germination, zea mays, neelum, lipase, plant lipids, plant phospholipases, maize seeds, thin layer chromatography, gas chromatography

An investigation was conducted to study the variation in the lipid class and fatty acid composition of the individual lipid fractions in Zea mays (Neelum) resting seeds, cotyledons and primary roots during germination to various root length (i.e. 5, 7, 10, 15, 20, 25, 30 mm root length). Lipolytic enzymes were subsequently studied regarding their isolation and characterization. Lipase, however, was further purified and its molecular weight determined.

During germination there was loss in both the dry weight and lipid content in the cotyledons, while in the roots the dry weight increased and lipids decreased. However, there was higher amount of lipid: in the cotyledons as compared to the primary roots which showed the rapid utilization of the lipids in the primary roots during germination.

Changes in the composition of lipids in the cotyledons during germination from that of the resting seeds was remarkable because the .triglycerides, decreased from 70.72% at the resting stage to 10.2% at 30 mm root length, while the free fatty acids increased to 31% at 30 mm root length from 7.2% at resting stage. 6-0-acyl monogalactosyl diglyceride and 6-0-acyl steryl glucoside were absent in the resting seeds but appeared upon germination and remained nearly constant at all stages of germination studied. In the phosphoglycerides, phosphatirdyl choline was the major fraction in both the resting seeds and cotyledons, however, phosphntidyl ethanolamine and its lyso compound which were absent in the resting seeds appeared upon germination. On the whole the phospholipids increased during germination.

Changes in the percentages of the different fractions in lipid extracts of primary roots during germination showed triglyceride to be still the major lipid fraction as in the cotyledons, but contrary to the cotyledons the free fatty acids decreased drastically with germination from 7.2% at resting stage to 0.02% at 30 mm root length. Sterol esters increased in the primary roots while free sterol was lesser in quantity than the sterol esters. This was vice versa in the cotyledons. Percentages of all the polar lipids increased with increase in the root length. However, the relative amounts of the neutral lipids were metabolized at a faster rate than polar lipids in both the cotyledons and primary roots, as is shown by the fact that neutral lipids decreased and the polar lipids increased in both the cotyledons and the primary roots.

In the fatty acid composition of the lipids of the resting seeds C18:2 was the most abundant fatty acid except in the triglycerides and polar lipid fractions where C18:0 dominated. C18:1 was the next major fraction while C18:0 was considerably reduced in the neutral lipids. C16:0 fatty acid composition did not show much variation in the lipid fractions. C14:0 was present in lesser quantities while C20:2 and present in the polar lipids in larger percent tages as compared to neutral lipids. C20:2 and C18:3 fatty ac ids were absent in the po1ar 1ipids. C24:0 was present in the polar lipids at 5% as compared to 2% of C22:0, C10:0 and C12:0 were present in fractional amounts only.

Fatty acid composition of cotyledons at various root lengths (5, 7, 10, 15, 20, 25, and 30 mm root length) showed that at all stages of germination there was not much perceptible change except that the only addition, to the existing lipid fractions was that of 6-0-acyl steryl-glucoside, 6-0-acyl monogalactosyl diglyceride, phosphatidyl ethanolamine and lyso phosphatidyl ethanolamine. These lipid fractions had the same fatty acid pattern except that in 6-0-acyl monogalactosyl diglyceride C16:2 and C16:3 appeared which increased with germination. As in the resting seeds C18:2 dominated in all the lipid fractions except in. the triglycerides and polar lipids and it increased with germination. C18:0 as the major fatty acid in the triglycerides and the polar lipids. with the increase in the germination the quantities of C18:0, C14:0, C18:0, C18:2, C20:0 and C20:4 increased while C10:0, C12:0, C18:1, C18:3, and C20:0 decreased. However, in C22:2 and C24:0 to much change was observed. Nevertheless there were higher quantities of saturated fatty acids in the polar lipids as compared to the neutral lipids C10: 0 was .absent in the mone-glyceride while C12:0 was absent in 6-0-acylmonogalactosyl diglyceride. C18:3 appeared in the sterol ester but was absent in diglycerides and monoglycerides. In both the glycolipids C18:1 was absent.

Fatty acid composition of the primary root lipid fraction at various root length showed that these quantities were lower than those of the cotyledons. The most abundant fatty acid was found to be C18:2 in all lipid fractions even in triglycerides except in polar lipids. It gradually decreased in all the lipid fractions with increase in root length. C18:0 till dominated in all fractions and also increased with germination, except for C16:0 which was higher in the neutral lipids than C18:0 which was contrary to that of cotyledons C16:0 too increased with germination in natural lipids. As in the cotyledons there were higher quantities of saturated fatty acids in the polar lipid fractions than in the neutral lipids. C18:3 appeared in the sterol ester as in the cotyledons and remained low. C10:0 and C12:0 decreased with germination. C20:4 disappeared in the polar lipids of the primary roots contrary to the cotyledons where it was present in only phosphatidyl choline and prosphatidyl ethanolamine lipid fractions. The rest of the fatty acid composition remained almost the same as those of cotyledons except for lesser percentages.

As the lipid mobilization is specifically based upon lipolytic enzymes, so studies upon maize lipase and phospho1ipase was also conducted.

While studying the effect of pH upon Zea mays (Nee1um) root lipase, two pH optimum peaks were obtained, showing an acidic lipase at pH 6 and the other alkaline lipase at pH 8.6. As at alkaline pH (8.6) lipase activity was found to be maximum, under the assay conditions and as this lipase was present in the soluble form, a condition which is required for the purification of the enzyme, so further studies were carried out upon the alkaline lipase. on germination, however, both these pH optima were maintained.

Studies upon germination of the resting seeds and cotyledons obtained from the root length of 5,7,10,15,20,25, and 30 mm showed maximum activity when the root length had reached 30 mm at pH 8.6, whereas at pH 6.0 maximum activity of the cotyledons at 30 mm root length was lesser. Similarly studies upon the primary roots of the same root lengths as those of the cotyledons showed that maximum lipase activity, at both the pH i.e. 6.0 and 8.6, was obtained when the root length had reached 10 mm root length. However, at pH 6.0 there was less lipase activity as compared to activity at pH 8.6 This partial inhibition of lipase activity at 20 mm root length showed that the fatty acid utilization during germination was faster as compared to its synthesis, due to presence absisic acid, which is a constituent of growth inhibiter. Thus further studies upon lipase were carried out upon 10 mm root length.

An optimum temperature of 37oC was observed at pH 8.6 under the assay conditions, while the lipase was stable from 50 to 200 minutes during incubation at 37oC. Both the Na+ and Ca2+ ions showed activating influence on the lipase activity. Since the crude lipase was extracted in 0.1M NaCl, so in order to study the effect of Na+, the enzyme employed was first salted out by ammonium sulfate at 50-70% concentration, then chromatographed on Sepliadex-25 column and lyophilized.

The enzyme was found to hydrolyze the triglycerides of almost all the oils subjected to study to a considerable extent. Olive oil, however, gave the highest activity so the enzyme implicated in the reaction analyzed was a lipase and not an esterase. with the increase of the substrate (10% olive oil emulsion in gum acacia) the lipase activity increased. Thus the Km value 62.5 mM, was also obtained for the system of zea mays crude alkaline root lipase and olive oil emulsion

Phospholipase studies of zea mays (N) on the other hand, showed only one optimum pH at 7.0 In contrast to lipase the phospholipase activity ,decreased with the progress of germination, 29.7 μM fatty acid were released per root at 5 mm root length while only 10.54 μM were released per root when the length had reached 30 mm under the assay conditions. Contrary to lipase the phospholipase showed an optimum activity between temperatures 30oC to 40oC and it was stable from 20 to 120 minutes when incubated at 37oC. Na+ and Ca2+ also had an activating effect upon the phospholipase with the increase of the substrate (10% lecithin in ethyl methyl ketone) the .lipase activity increased and the Km value of G 6.66 mM was obtained between the crude phospholipase extract of Zea mays and substrate.

There seemed to be a relationship between the distribution of lipids, lipase and phospholipase activities in the. roots, cotyledons and the resting seeds of the Zea mays (N). At the start .of germination the natural lipids present were in larger amount but as germination progressed the lipase enzyme became active and helped in the mobilization of the lipid fractions while phospholipase which was found to be in greater amount. as germination started showed a fall with the increase of the phosphoglyceride quantities.

Purification of talkative lipase was carried out by ammonium sulfate precipitation, where 50-70% saturation gave maximum activity. It was followed by gel filtration (employing Sephacryl S300 Column) where 14.3 times purification of zea mays (N) lipase was obtained. Further purification by ion exchange chromatography with sephadex A-50 gave 33.33 times purification showing a single band when subjected to SDS disc electrophoresis which proved homogeneity of the purified enzyme. The molecular weight of lipase was found, to be 55,000 daltons.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
139.94 KB
2 1 General 9
493.08 KB
  1.1 Plant lipids 9
  1.2 Gerimnation 29
  1.3 Distribution of plant lipases 35
  1.4 Distribution of plant phospholipases 39
  1.5 Characteristics of plant lipases 41
  1.6 Extraction and purification of plant lipases 54
3 2 Materials and methods 59
134.36 KB
  2.1 Germination of maize seeds 59
  2.2 Lipid extraction 60
  2.3 Thin layer chromatography and location of individual lipids( Lipid Class analysis) 60
  2.4 Preparative TLC 61
  2.5 Gas chromatography (fatty acid composition ) 61
  2.6 Isolation of lipase and phospholipase in roots and seeds of maize 62
  2.7 Lipase assay 62
  2.8 Phospholipase assay and characterization 63
  2.9 Characterization of the lipase 65
  2.10 Purification of zea mays (N) lipase 69
  2.11 Ion exchange chromatography (DEAE sephadex A 50) 69
  2.12 Electrophoresis 70
  2.13 Molecular weight determination 70
4 3 Results 90
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  3.1 Fatty acid composition 90
  3.2 Zea mays (N) Lipase 127
  3.3 Zea mays (N) Phospholipase 147
5 4 Discussion 175
189.14 KB
6 5 Bibliography 194
160.18 KB