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Title of Thesis

Naheed Kausar
Institute/University/Department Details
Institute of Chemistry/ University of the Punjab
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
lipase, lipolytic enzymes, cucumis melo, hibiscus cannabinus, c melo

The work presented here deals with studies on lipolytic enzymes and lipids of Cucumis melo seeds both in the resting and germinated states. Also some work was undertaken on the lipase activity and lipids of Hibiscus cannabinus seeds.

C melo seed lipase in the resting state showed two pH optima i.e. 4.0 and 6.0. On germination both these pH optima were maintained although level of activities at both the pH increased. Loss in activity of the lipase having optimum pH 4.0 was greater as compared-to the other when incubated at pH 6.0 for different periods. The enzyme extract showed two peaks on fractionating with Sephadex G-75. One fraction .was found to have optimum pH 4.0 and the other pH 6.0. C. melo seed lipase therefore contains two distinct lipases.

On germination of C. melo seeds upto a primary root length of 30 mm, activity of the lipase having pH optimum 6.0 increased more than three-fold. An optimum temperature of 31-40oC was observed at pH 6.0 under the assay conditions used. Both the Na+ and Ca2+ ions showed activating influence on the lipase activity. The lipase was shown to be more active against ricinoleic acid glycerides as compared to the others used, which seems due to a genuine affinity for the former. Using powder of C melo germinated seeds greatest lipase activity was observed in ethyl methyl ketone system and this was followed by activities in di-isopropyl ether, n-heptane, cyclohexane and cyclohexanol systems. The pattern of specific solvent effects on lipase activity of C. melo seems to be different from those of other lipases studied.

The primary root of 30mm length showed an activity of 49 μM fatty acids released per gram of the fresh roots at pH 6.0. Studies on distribution of lipase activity in different segments of primary root of 30mm length showed that the enzyme activity in apical portion of 6 mm length was most active releasing 91 μM fatty acids per gram of fresh sample.

Phospholipase activity showed only one optimum pH, i.e. 5.0, in the primary roots of the germinated seeds. Relative phospholipase activities in the ethyl methyl ketone, di-isopropyl ether and n-heptane systems were similar to those of lipase. In contrast to lipase, the phospholipase activity decreased with increase in the length of the primary root. 100 mg dry roots of 5mm released 210 μM fatty acids while only 80 pM were released when the length reached 30mm under the assay conditions used. The pattern of distribution of phospholipase activity in the various segments of the primary root of 30 mm length was reverse to. that of lipase. The portion attached to the cotyledon showed greatest activity and it reduced while moving towards the tip. On fractionating a concentrated extract of the primary root in a Sephadex G-75 column the phospholipase activity could be separated from the two lipases described above.

On germinating C melo seeds, lipid content in the cotyledons did not change much but in the primary roots there was a rapid decrease with increase in length. The growing tip of the primary roots contained greatest amount of lipids i.e. 17% on a dry weight basis which decreased to 2.7% in the segments attached to the cotyledons. Analysis of the lipids of the resting seeds and the cotyledons and the primary roots of the germinated seeds showed triglycerides as the major fraction. In the primary root quantities of triglycerides, partial glycerides and free fatty acids decreased whereas percentages of the various polar lipids and sterols and their esters increased alongwith increase in its length. Proportion between neutral and polar lipids in the resting seed was 24:1 whereas in the freshly germinated roots this proportion dropped to 4.92 am reduced further with increase in length of the primary root. Changes in the composition of lipids in the cotyledons during germination from that of the resting state were not as remarkable as in the case of primary roots. As compared to the lipids of the resting seeds percentages of 1-3 diglycerides and 2-monoglycerides decreased, whereas phosphatidyl ethanolamine was not detectable, in the cotyledons. Proportion between neutral and the polar lipids in the cotyledons was although reduced as compared to that of resting seeds but the change was not as remarkable as in the case of primary roots.

Studies on distribution of different lipid fractions in the various segments of the primary roots grown to )0 mm showed that there was a slight decrease in the percentages of free fatty acids, 1-3 diglycerides and phosphatidyl serine from tip towards the base of the root. Percentages of triglycerides, monogalactosyl diglycerides, phosphatidyl ethanolamine phosphatidyl choline and lysophospbatidyl choline increased slightly. However, quantities of sterol esters, 1-2 diglycerides, 6-O-acyl sterylglucoside, lysophosphatidyl ethanolamine and sterols were found to be nearly constant throughout the length of the root.

There seemed to be a relationship between the distribution of lipids and lipase and phospholipase activities in the various segments of the primary root. In the growing tip portion both the lipase activity and the total glycerides and free fatty acids existed in greater amount whereas phospholipase activity and the total phosphoglycerides were found to be present in greater amounts in the portions nearer to the cotyledons. lipids of the resting seeds contained C18:2 (48.5%) as the major fatty acid and this was followed by C18:1(20.3%), C16:0(9.8%), C22:1(6.1%), C18:0(6.2%), C20:1(5.4%) and small amounts of C12:0 and C14:0. C18:3 was not detectable. On germination C16:0 and C18:0 contents increased while that of C1S:2 decreased remarkably in the lipids of freshly germinated primary roots. Alongwith increase in the root length quantity of C16:0 decreased while those or C20:1 and C22:1 gradually increased. C18:3 which was not detectable in the lipids of primary root in initial stages of germination, also appeared in significant amounts later on. However, fatty acid composition in the cotyledons did not change much on germination although some amounts of C18:3 also appeared in the lipids of cotyledons. A study on the distribution of fatty acids in the various segments of the primary roots showed that amounts of C16:0 C18:0 and C20:1 deceased while moving from the portion attached to the cotyledons towards the growing tip while t his order was reverse in the case of C8:2 and C18:3 The results showed that the. dietr1bution pattern of fatty acids in various segments of the primary root did not change gradually from that of the cotyledons, rather there was an abrupt change in the fatty acid composition in the portion of the primary root immediately attached to the cotyledons.

Lipase activity of the H cannabinus seeds in the resting state showed only one pH optimum i.e. 5.0. The enzyme was found to be stable over a pH range 4.0 to 6.0 and lost the activity rapidly at pH values below 4.0 or above 6.0. An optimum temperature of 37°C was observed under the assay conditions employed. Above 40°C the enzyme was found to be rapidly inactivated. Mg2+and Ca2+ had an activating effect on tile lipase activity while EDTA. Ba2+ . Pb2+,Zn2+, K4Fe (CN)6 at concentration of 0.025 M inhibited activity. NaCl activated the enzyme at concentrations of 0.3-0.5 M. The enzyme was found to be specifically active against butyric acid glycerides. Amongst the solvent systems studied greatest lipase activity was found in the ethyl methyl ketone and n-heptane systems and this was followed by activities in cyclohexane. di-isopropyl ether and cyclohexanol systems.

Analysis of the lipids of g. cannabinus seeds showed that triglycerides was the major fraction. Some free fatty acids apart from sterols were also present in small amounts. Composition of the lipids of g. cannabinus seeds showed all those fractions which were found in cotton seed oil but the quantity of free fatty acid was greater in cotton seed oil as compared to H. cannabinus seed oil. Fatty acid composition of the lipids of g. cannabinus showed that C16:0, C18:1 and C18:2 were the major fatty acids and C14:0 C16:1, C18:0, C18:3 and C22:1 were present in small amounts in the oils of both H. cannabinus and cotton seed. However, H cannabinus contained cyclopropenoid and epoxy acids in amounts slightly higher than in cotton seed oil. Similarity of fie cannabinus oil with that of cotton seed warrant its possible use as an edible oil.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
111.98 KB
2 1 Introduction
392.23 KB
  1.1 General 8
  1.2 Distribution Of Plant Lipases 11
  1.3 Distribution Of Plant Phospholipases 13
  1.4 Germination 28
  1.5 Extraction And Purification Of Lipases 30
  1.6 Characteristics Of Plant Lipases 35
3 2 Materials And Methods 59
92.14 KB
  2.1 Seeds Of Cucumis Melo And Hibiscus 59
  2.2 Germination Of The Seeds 60
  2.3 Lipase Assay 61
  2.4 Phospholipase Assay 62
  2.5 Stability Of The Lipase 63
  2.6 Lipase Activity In Organic Solvent Systems 64
  2.7 Fractionation Of Lipolytic Enzymes 65
  2.8 Extraction Of Lipids 65
  2.9 Fatty Acid Composition 66
4 3 Results 70
492.62 KB
  3.1 Cucumis Melo Seed Lipase 70
  3.2 Cucumis Melo Seed Phospholipase 84
  3.3 Cucumis Melo Seed Lipids 93
  3.4 Hibiscus Cannabinus Seed Lipase 121
  3.5 Hibiscus Cannabinus Seed Lipids 137
5 4 Discussion 142
181.01 KB
  4.1 Cucumis Melo Seed Lipase 142
  4.2 Cucumis Melo Seed Phospholipase 147
  4.3 Cucumis Melo Seed Lipids 150
  4.4 Hibiscus Cannabinus Seed Lipase 156
  4.5 Hibiscus Cannabinus Seed Lipids 159
6 5 Bibliography 164
159.74 KB