Pakistan Research Repository Home

Title of Thesis

Muhammad Saleem Akhtar
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)
bioconversion, cellulosic materials, microbial cellulases, thermophilic cellulolytic bacterium, b. subtilis, waste paper, cellobiose, xylose, arabinose, cmc, avicel, cellulase genes

A thermophilic cellulolytic bacterium identified as B. subtilis was isolated from waste paper compost by enrichment culture and serial dilution methods. The organism produced higher levels of CM Case and avidcelase activities in the presence of cellobiose as compared to those produced in the presence of other carbon sources including xylose, arabinose, CMC and Avicel, when cultivated in the medium of initial pH 7.0 at 50oC for ten hours. When the medium was supplement with 0.2% Avicel in addition to 0.5% cellobiose, 1.5 fold increase in cellulase activity was observed when medium containing cellobiose and 0.2% Avicel as substrate was supplemented with .02% Tween 80. the Maximal CMCase activity after optimizing fermentation conditions was 16.8 U/ml of the culture supernatant. Production of extracellular cellulases was also studied in the presence of 2.0% lignocellulosic materials including wheat straw, rice straw, bagasse, kallar grass and cotton stalks when used as powder of 20 mesh size. Wheat straw produced higher cellulase activities in the culture supernatant after 12 hours of fermentation as compared to the other lignocellulosic substrates. CM Case and avicelase activities were most active at pH 7.0 and 60 oC. Analysis of the enzyme on native-PAGE followed by zymogram technique showed at least three CMC Case activity bands.

DEAE-Sepharose chromatography of the crude enzyme followed by chromoatofocusing produced three CM Case fractions, named as E1, E2, and E3. Each of these fractions showed a single band on native-PAGE. The molecular weight of the CM Case E1, E2, and E3. were found to be 37, 38.5 and 40.5 kDa, respectively. Isoelectric points were found to be 6., 6.5 and 5.6 for E1, E2, and E3. respectively. All the CM Case showed maximum activity in the temperature range of 55-60 oC under the assay conditions used. pH optima of these enzymes ranged between 6.5 to 7.5. Endoglucanase activities were stable in the pH range of 5.5 to 8.5 and up to 65oC as there were little losses of activity when the enzymes were incubated for two hours under these conditions. E1 and E2 showed only CM Case activity while Ca+2 , Mg+2,, Z+2 Fe+2 Ni+2 activated the cellulase were strongly inhibited by Hg+2 and C2+ at 2m M concentration, while Ca+2 , mg+2 , zn+2, Fe+2 and Ni+2 activated the cellulases. Ca+2 when used in the assay mixture, enhanced activity by about 100%. Each of the enzyme was inhibited in the presence of cellobiose, glucose and β-mercaptoethanol when added to the assay mixture. The major hydrolytic products of all the endoglucanases were oligosaccharides along with small amounts of cellobiose and glucose. Km values of the purified endoglucanases (E1, E2 and E3)were found to be 8.33 10 and 12.5 mg CMC/ml, respectively. 58.1% synergism was observed for the hydrolysis of CMC when E1, E2 and E3 were used in the ratio 2.3:1.67:1, respectively.

Cellulases produced by B. subtilis were used for the saccharification of wheat straw, rice straw and bagasse. 33.0, 25.5 and 35.5 % saccharification was obtained with 20U of enzyme when 2% NaOH pretreated wheat straw, rice straw and bagasse were used at a concentration of 4%, respectively. When the concentration of these substrates were increased to 10%, the percent saccharification decreased to 16.9,17.4 and 18.7%, respectively. Percent saccharification of these pretreated substrates increased with the increase of enzyme concentration.

Preliminary investigation on the application of cellulases produced by B. subtilis has shown promising results in removing fuzziness and pilling from the samples of untreated cotton fabrics like, kora latha (fine thread raw cotton fabric), khaddar (coarse thread raw cotton fabric) and denim. The percent loss in weight of the fabric increased with increase in incubation period. When kora latha was treated with 20U of cellulases the percent loss in fabric weight was 2.8 after 3hours of incubation. However the percent loss in weight for denim cloth was 2.1. The weight loss of the fabrics increased with the increase of enzyme concentration up to 40U. The lint build up on the surface of the fabrics was reduced and pilling tendency of the fabric was lower.

Download Full Thesis
2043.46 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
114.22 KB
2 1 Introduction 1
393.21 KB
  1.1 Chemistry and structure of lignocellulosic materials 2
  1.2 Celluloytic enzymes 5
  1.3 Mechanism of enzymatic hydrolysis 8
  1.4 Lignocellulosic reserves 10
  1.5 Microbial cellulases 12
  1.6 Purification of cellulases 16
  1.7 19
  1.8 Pretreatment of lignocellulosic substrates 23
  1.9 Saccharification of lignocellulosic materials 27
  1.10 Cloning of cellulase genes 31
3 2 Materials and Methods 35
290.89 KB
  2.1 Isolation and identification of the organism 35
  2.2 Fermentation conditions 35
  2.3 Protein estimation 37
  2.4 Enzyme assay 39
  2.5 Ultrafiltration 40
  2.6 DEAE-Sepharose chromatography 43
  2.7 Chromatofocusing 44
  2.8 Native polyacrylamide gel electrophoresis 44
  2.9 SDS-Polyacrylamide gel electrophoresis 45
  2.10 Molecular weight determination 46
  2.11 Isoelectric point 46
  2.12 Identification of hydrolytic products 47
  2.13 Saccharification of lignocellulosic materials 47
  2.14 Cellulase treatment of cotton fabrics 48
  2.15 Cloning of cellulase genes 49
4 3 Results 60
817.85 KB
  3.1 Screening for celluloytic thermophiles 60
  3.2 Growth conditions and cellulase production 62
  3.3 Purification of cellulases 83
  3.4 Characteristics of purified cellulases 85
  3.5 Saccharification of lignocellulosic materials 101
  3.6 Cellulase treatment of cotton fabrics 109
  3.7 Cloning of cellulase genes 115
5 4 Discussion 123
219.58 KB
6 5 References 140
184.77 KB