Expression and medium optimization of endoglucanase gene in pET39b (+) vector harboured in Escherichia coli (BL21DE3) /
Endoglucanase is one of the key components of complex multienzyme system collectively known as cellulase, which degrade cellulose to glucose. In this study a Fusarium oxysporum endoglucanase I gene from wtEGI-pET28a plasmid construct was amplified using polymerase chain reaction and subcloned into a...
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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: | Endoglucanase is one of the key components of complex multienzyme system collectively known as cellulase, which degrade cellulose to glucose. In this study a Fusarium oxysporum endoglucanase I gene from wtEGI-pET28a plasmid construct was amplified using polymerase chain reaction and subcloned into a PCRBlunt cloning vector. The endoglucanase I (EG-I) was then subcloned into pET39b(+) expression vector (Novagen,USA) and the new plasmid construct, wtegIpET39b was transformed into E. coli BL21DE3 so as to study the protein expression. Active ~66kDa endoglucanase I was expressed and confirmed by Carboxylmethylcellulose (CMC) assay and SDS-PAGE analyses. Plackett-Burman (PB) design, One-Factor-at-A-Time (OFAT) method and Face Centered Central Composite Design (FCCCD) under response surface methodology (RSM) were consecutively used to optimize the medium components to improve the recombinant endoglucanase production. Based on the PB design, glucose, KH2PO4, cellulose, KCl, NH4Cl and maltose were positively contributing. The optimum levels of the three most contributing factors; glucose, KH2PO4 and cellulose were determined by OFAT and further optimized by FCCCD under response surface methodology (RSM). The maximum endoglucanase I activity of 5.83 U/mL was obtained in an optimum medium containing 1.0% (w/v) glucose, 0.4% (w/v) KH2PO4 and 0.5% (w/v) cellulose which was 5.4 fold higher than the activity (1.08 U/mL) obtained by the PB design and 6.94 fold higher than the commercial media (LB). The validity of the developed model was verified and analysis of variance indicated that the established model was significant (p<0.05). |
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| Item Description: | Abstract 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: | xvi, 106 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 86-98). |