I= ISOLATION, PURIFICATION AND CHARACTERIZATION OF INVERTASES FROM SUGARCANE GROWN UNDER SALINE CONDITION
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Title of Thesis
ISOLATION, PURIFICATION AND CHARACTERIZATION OF INVERTASES FROM SUGARCANE GROWN UNDER SALINE CONDITION

Author(s)
Altaf Hussain
Institute/University/Department Details
University Of Agriculture,/ Botany
Session
2003
Subject
Botany
Number of Pages
120
Keywords (Extracted from title, table of contents and abstract of thesis)
sugarcane, abiotic stresses, chilling stress, salinity, invrtases, cp-77-400, coj-84

Abstract
The research project was carried out to assess the effect of salt stress on the kinetics of invertases in sugarcane . Two sugarcane cultivars, CP-77-400 and COJ-84 ser raise in filed plots at 50,100,150 and 200 mM salinity levels.

The salinity was started at late grand growth stage when the stalk formation was just to complete. Both the cultivars were grown for a period of three moths under saline conditions, after which time they were harvested. Effect of salinity on growth of canes was assessed by measuring the culm length leaf length, internode length and culm diameter. On the other hand, effect of salinity on biochemical characteristics of invertases was studied b y extracting the sugarcane juice. Sugarcane juice was dialyzed against distilled water to remove free sugars. Invertases were purified applying conventional purification methods, i.e., ammonium sulphate precipitation, anion exchange hydrophobic interaction and gel-filtration chromatography. Invertases were characterized for activity and stability. Kinetics and thermodynamics of sucrose hydrolysis and stability was determined to investigate the salinity-stability-functions relationship of invertases.

The results for different growth parameters showed that there was a significant effect of salt stress on culm length, leaf length and internode length, whereas the salt stress effect was non-significant on culm diameter. Both the cultivars differed significantly for culm length , leaf length and internode length. However, they did not differ significantly for culm diameter. The effect of salinity on biochemical parameters was very promising and like growth parameters, both varieties differed significantly. Sugar cont4ent of COJ-84 reflects that this was highly productive as compared to CP-77-400.

Purification of invertases was carried out on Fast Protein Liquid Chromatography(F.P.L.C) unit. Purification of invertases was up to homogeneity and was confirmed on 10% SDS-PAGE which displayed a single band indicating monomeric nature of enzymes. Molecular weight was determined to be 28kDa in both the cultivars. Soluble acid invertases (SAI) of sugarcane Cultivar CP-77-400 were highly acidic and their pH optima was lower than 2 while pH optima of SAI in COJ-84 was 2.65 which was higher than that of CP-77-400. However, salinity did not affect the pH optima in both the cultivars.

Optimum temperature of native SAI of CP-77-400 and COJ-84 was 55 and 50 C, respectively. Salinity did not affect temperature optima of COJ-84 invertases. In contrast, SAI from CP-77-400 showed a 5-degree at all salinity treatments. Kinetic and thermodynamic studies of SAI from both the cultivars shoed that they were afffect4ed by salinity. SAI of CP-77-400 were less stable to temperature, urea and proteases, but were very active in minute quantities. In contrast, SAI of COJ-84 were stable towards temperature and urea, but labile to proteolytic attack. However, these were less active, although their concentration was higher per ml juice as compared to those of Cp-77-400.

In the light of the present findings, it was concluded that the soluble acid invertases produced by sugarcane cultivar CP-77-400 were more active, but less stable than those of COJ-84. On the basis of catalytic efficiency ,ph and temperature optima, stability to temperature, urea and proteases, it was concluded that the soluble acid invertases produced by sugarcane cultivars under saline growth conditions were with improved characteristics, and hence showed that the expression of genes for invertases was affected by soil salinity.

Download Full Thesis
1311.83 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
114.8 KB
2 1 Introduction 1
43.17 KB
3 2 Review Of Literature 2
212.69 KB
  2.1 Sugarcane 3
  2.2 Specific Abiotic Stresses 12
  2.3 Chilling Stress 15
  2.4 High Temperature Stress 16
  2.5 Water Deficit Stress 16
  2.6 Salinity And Ionic Stress 18
4 3 Materials And Methods 24
143.86 KB
  3.1 Sugarcane Varieties 24
  3.2 Cultivation Of Canes 24
  3.3 Salinity Application 24
  3.4 Harvesting Of Canes 25
  3.5 Effect Of Salinity On Growth Of Sugarcanes 25
  3.6 Effect O Salinity On Biochemical Characteristics Of Sugarcane 25
  3.8 Effect Salinity On Invertases 27
  3.8 Purification Of Invrtases 27
  3.9 Molecular Weight Determination 29
  3.10 Characterization Of Invertases 33
  3.11 Statistical Analysis 37
5 4 RESULTS AND DISCUSSION 38
562.94 KB
  4.1 Influence Of Salinity On Growth Of Two Sugarcane Cultivars-Result 38
  4.2 Biochemical Attributes Of Two Sugarcane Cultivars Grown Under Salinity 40
  4.3 Effect Of Metal Ions On Activity Of Invertases 43
  4.4 Purification Of Sugarcane Invertases-Results 48
  4.5 Characterization Of Invertases –Results 59
  4.6 Effect Of Saline Growth Medium On Temperature Optima Of Invertases-Results 63
  4.7 Effect Of Saline Growth Medium On Michaelis Constants Of Sugarcane Invertases-Results 65
  4.8 Effect Of Salinity On Thermodynamics Of Sucrose Hydrolysis By Invertases –Results 68
  4.9 Kinetics And Thermodynamics Of Irreversible Thermal Stability Of Invertases Of Sugarcane Cultivars Grown Under Salinity-Results 71
  4.10 Total Thermal Denaturation Of Soluble Acid Invertases Of Sugarcane-Results 80
  4.11 Stability Of Soluble Acid Invertases Of Sugarcane To 8 M Urea –Results 83
  4.12 Stability Of Sugarcane Soluble Acid Invertases To Proteolyic Degradation-Results 85
6 5 General Discussion and Summary 88
157.5 KB
7 6 Literature Cited 102
226.62 KB
8 7 Appendix
226.62 KB