Investigation on the expression of a Fusarium oxysporum Endoglucanase in Kluyveromyces lactis /
Cellulose is the main component in plant cell wall and thus the most abundant biopolymer on earth. Endoglucanase is one of the key constituent of a complex multienzyme system collectively known as cellulase which will degrade cellulose into glucose. Cellulase has been used in various industries such...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Engineering, International Islamic University Malaysia,
2012
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| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
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| Summary: | Cellulose is the main component in plant cell wall and thus the most abundant biopolymer on earth. Endoglucanase is one of the key constituent of a complex multienzyme system collectively known as cellulase which will degrade cellulose into glucose. Cellulase has been used in various industries such as textile, detergent, food and feed, pulp and paper, and recently in bio-fuel industries. In the present work, the --1.2 kb gene encoding for the endoglucanase I from Fusarium oxysporum has been amplified from pET28a-wtEG-I plasmid construct by polymerase chain reaction (PCR) and cloned into pKLAC2 expression vector. The newly constructed plasmid pKLAC2-EG-I was then transformed into Kluyveromyces /actis GG799 cells expression system for protein over-expression. The -44. 7 kDa active EG-1 was expressed and confirmed by SOS-PAGE and enzyme assay and then purified via ionexchange chromatography using Fast Protein Liquid Chromatography (FPLC) system. Plackett-Burman d~ign, One-Factor-at-A-Time (OFAT) method and central composite design (CCD) under response surface methodology (RSM) were used to optimize the medium composition to improve recombinant EG-1 production from K lactis. Seven variables showed positive effect on increasing endoglucanase I production by Plackett-Burman including four types of nitrogen source (yeast extract, peptone, KN03, and (NH4)iS04), two types of carbon source (glucose and soluble starch) and one phosphate source (KH2P04). Three factors that have the highest positive effects (KN03, (N"4)2S04 and glucose) were chosen and analyzed using the OFAT method to determine the optimum levels to be used in the media for high endoglucanase I production. Based on the OFAT method, the optimum levels of the variables were, ammonium sulfate 3 g/L, potassium sulfate 4 g/L and glucose 1.5 %. The three factors chosen were used for optimization by central composite design, whereas the other factors were maintained at their optimum level. Statistical analysis showed that the optimum medium containing 2 g/L of potassium nitrate, I g/L of ammonium sulfate and 0.5 % of glucose gave the highest endoglucanase I production of 53.98 U/mL. Several analyses such as ANOV A, t-test and p-values were used to evaluate the model as well as the optimization process. The EG-1 production increased from 24.02 U/mL using basic commercial media to 29.75 U/mL by OFAT and then to 53.98 U/mL using CCD under RSM, which gave an improvement in the production by 1.24 fold and 1.82 fold, respectively. |
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| Item Description: | Abstracts in English and Arabic. "A dissertation submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)." --On t.p. |
| Physical Description: | xviii, 106 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 85-97). |