I= MOLECULAR CLONING, OVEREXPRESSION AND CHARACTERIZATION OF ENDOGLUCANASE FROM CHAETOMINUM THERMOPHILE
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Title of Thesis
MOLECULAR CLONING, OVEREXPRESSION AND CHARACTERIZATION OF ENDOGLUCANASE FROM CHAETOMINUM THERMOPHILE

Author(s)
Saima Naim
Institute/University/Department Details
Department of Chemistry / University of Agriculture Faisalabad
Session
2005
Subject
Biochemistry
Number of Pages
92
Keywords (Extracted from title, table of contents and abstract of thesis)
molecular cloning, overexpression, endoglucanase, chaetominum thermophile, thermostable fungus, egl gene

Abstract
Cellulases provide a key opportunity for achieving the tremendous benefits of biomass utilization in the long term because of high glucose yields possible, and the opportunity to apply the modern tolls of biotechnology to reduce costs. Endoglucanase( endo-1, 4-D-glucanase, EC 3.2.1.4 enzyme of ecelluase complex was produced by many fungi. The enzyme was produced from a thermostable fungus Chaetomium thermophile. It was grown on Vogelā€™s medium with different carbon sources like xylan, CMC, corncobs and glucose for 5-days at 180 rpm at 28oC in orbital shaker. Glucose repressed the synthesis of endoglucanase whereas xylan and CMC produced the enzymes in appreciable amount. Growth conditions of Chaetomium thermophile were optimized for maximal production of endoglucanse (EG) : pH 5.0 oC, incubation period 120 h, substrate 1% CMC.

The enzyme was produced and isolated from the culture filtrate through centrifugation. The crude enzyme extract had 0.064 IU/ML endoglucanse activity. Total protein in the crude extract was 0.103 mg/ mL and the specific activity was 0.615 IU/mg. The crude extract was subjected to various purification procedures. The enzyme was precipitated at 20-40 % ammonium sulphate saturation and had activity and specific activity 0.15 IU/mL and 0.76 IU/mg, respectively. Gel filtration chromatography was performed on sephadex G200 . The enzyme activity and specific activity of the gel filtration fraction was found to be 0.02 IU/mL and 2.19 IU/mg, respectively . The enzyme was, therefore purified to 3.57 fold. SDS-PAGE revealed single band showing that the enzyme was purified to homogeneity. The enzyme was characterized. Optimal pH and temperature of the enzyme was marked at 6.0 and 50 oC respectively . KM Vmax of the enzyme was also determined by applying various transformations of Michaelis-Menton equation i.e. Line-Weaver Burk Plot and Hans-Woolf Plot. KM Vmax of enzyme were found to be 0.4 % and 0.75 Āµmol/min, respectively. The egl gene was successfully isolated with the help of RT-PCR from C. Thermophile. Overespression (9.8 fold) of EG was obtained using inducible FLAG- expression vector. Optimal pH and temperature of recombinant and wild type EG were 6.0 and 50 oC, respectively. Molecular weight of the enzyme was 41 kD. Both enzymes were 70 % stable at 80 oC for 30 minutes. Recombinant enzyme had higher KM Vmax lower Km values as compared to the wild type EG.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
74.48 KB
2 1 Introduction 1
59.38 KB
3 2 Review Of Literature 6
428.68 KB
4 3 Material And Methods 29
234.95 KB
5 4 Results 46
332.63 KB
6 5 Discussion 65
171.62 KB
7 6 Summary 78
180.13 KB
8 7 Literature Cited 80
180.11 KB