I= ISOLATION, PURIFICATION AND CHARACTERIZATION OF GULCANSUCRASE FROM LOCALLY ISOLATED STRAIN OF LEUCONOSTOC MESENTEROIDES
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Title of Thesis
ISOLATION, PURIFICATION AND CHARACTERIZATION OF GULCANSUCRASE FROM LOCALLY ISOLATED STRAIN OF LEUCONOSTOC MESENTEROIDES

Author(s)
Afsheen Aman
Institute/University/Department Details
Department of Biochemistry/ University of Karachi
Session
2007
Subject
Biochemistry
Number of Pages
164
Keywords (Extracted from title, table of contents and abstract of thesis)
gulcansucrase, leuconostoc mesenteroides, streptococcus, acetobacter, glucan, dextran

Abstract
Glucansucrase is an industrially important enzyme that is produced by different species of bacteria like Leuconostoc, Streptococcus and Acetobacter. Among them Leuconostoc mesenteroides is used for the production of glucansucrase and glucan on industrial scale. Due to its increasing demand, isolation of new strains of Leuconostoc mesenteroides for glucansucrase production is of valuable importance. In this work a new strain of Leuconostoc mesenteroides AA1 is isolated and various special conditions and aspects regarding glucansucrase production and extracellular activity have been studied. This newly isolated strain was found to be a producer of a novel type of glucansucrase that catalyzes the synthesis of high molecular weight glucan from sucrose. This strain was selected among eleven strains isolated from various vegetables and fruits on the basis of high enzyme production.

Different media composition and factors, which play a vital role during fermentation, were optimized for the better production of extracellular glucansucrase. Leuconostoc mesenteroides AA 1 showed maximum glucansucrase production in 8 hours in medium containing 2.5 % of sucrose. Addition of calcium chloride resulted in very high expression of extracellular glucansucrase and the enzyme production was enhanced up to three times when medium was supplemented with 0.01 % calcium chloride. Maximum enzyme production of 53.0 DSU/ml/hr of glucansucrase was achieved when the initial pH for the fermentation medium was kept at 7.5 at 25°C.

Biochemical characteristics of extracellular glucansucrase showed that when sucrose is used as a substrate, the enzyme had a Km value of 69.88mM and a Vmax of 61.75 DSU/ml/hr. The optimum activity was observed at pH 5.0 in 0.lM citrate phosphate buffer at 35°C. Studies on the effect of various meta1 ions on the glucansucrasc activity showed that metal ions like A12+ and Mg2+ stimulated the activity, whereas Hg2+, Ba2+, K2+ and Na2+ showed a potent inhibitory effect on the enzyme activity in various concentrations. However, Cd2+, Mn2+, Ca2+ and Ni2+ showed a stabilizing effect on the enzyme activity. Effect of different organic solvents on enzyme activity revealed that extracellular glucansucrase showed 50 % of its relative activity with different organic solvents i.e. DMSO, 2-propanol, ethanol and acetone, while activity was completely inhibited by ethanolamine. It was also observed that glucansucrase from Leuconostoc mesenteroides AA 1 is more thermostable at -18°C as compared to when it is stored at 4°C and 30°C. Thermal stability revealed that when the enzyme was kept at 30°C and 35°C for one hour, the loss in the activity was less as compared to 40°C and 45°C.

The partial purification of extracellular glucansucrase was carried out using ethanol precipitation and PEG 4000 precipitation. It was observed that a higher specific activity of glucansucrase was found when partial purification was carried out with PEG 4000 as compared to ethanolic precipitation. Glucansucrase was further purified to homogeneity using affinity chromatography with DEAE Sephadex A-50 followed by gel permeation chromatography using Sepharose CL6B. The purified sample showed an enzyme activity of 1050 DSU/ml/hr with a specific activity of 2692 DSU/mg. These procedures yeilded a pure glucansucrase with 162.5 fold purification of. This purified enzyme had a molecular mass of 177,000 Daltons as determined by SDS PAGE and in situ electrophoresis. Homogeneity of the purified enzyme was confirmed by HPLC analysis. The amino acid composition of the enzyme showed that the enzyme is rich in both the basic and polar/hydrophilic amino acid and is less rich in acidic amino acids. The N-terminal amino acid analysis showed that the first six amino acid residues at the N-terminal end are: D-S-T-N-T-V, which is a unique sequence among different glucansucrases, characterized previously.

Download Full Thesis
3207.74 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
186.4 KB
2 1 Acknowledgement 1
69.5 KB
  1.1 Abbreviations 3
3 2 Abstract 4-6
121.99 KB
4 3 Introduction 7-34
463.11 KB
  3.1 Lactic Acid Bacteria 7
  3.2 Characterization Of Gulcasucrase 13
  3.3 Glucan ( Dextran ) Synthesis 28
5 4 Plan Of Work 32-34
51.29 KB
6 5 Materials & Methods 35-81
712.45 KB
  5.1 General Section 35
  5.2 Experiment Section 44
  5.3 Analytical Section 71
7 6 Results And Discussion 82-143
1190.03 KB
  6.1 Bacterial Taxonomy 82
  6.2 Selection Of Strain 86
  6.3 Glucansucrase 87
  6.4 Partial Purification Of Glucansucrase 121
  6.5 Purification Of Glucansucrase By Gel Permeation Chromatography 127
  6.6 Assessment Of Homogeneity Of Gulcansurase By HPLC 130
  6.7 Characterization Of Glucansucrase 133
8 7 Conclusion 144-146
60.28 KB
9 8 Bibliography 147-163
574.59 KB
  8.1 Publications 164