Cloning, Expression, Characterization and Purification of Endoglucanase Gene

Cloning, Expression, Characterization and Purification of Endoglucanase Gene

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Endo-1, 4-B-glucanase is an important enzyme from industrial point of view and its production is considered essential for successful utilization of cellulosic materials (Wu and Lee 1997., Zhang and Lynd 2004). The objective of present research work was cloning, expression, purification and characterization of endoglucanase (bgl C) gene obtained from strain of Bacillus licheniformis ATCC 14580. Isolation of high quality genomic DNA is a prerequisite for molecular techniques (Ansari et al., 2012). The reliability of the method used for the isolation of DNA from biological samples or from reaction mixtures is of significant importance. In the present study a simple DNA isolation protocol developed by Kronstad et al (1983) was used because of its simplicity and cost effectiveness. This method was fast and complete DNA extraction was achieved within 2 h. Many workers have used other methods for the isolation of genomic DNA (Errington, 1984, Ausubel et al., 1987 and Hong et al., 2005). Amplification of endoglucanase gene was performed by polymerase chain reaction using specific primers (forward and reverse) each complementary to one end of target sequence. Primers were designed using software vector NTI. In forward primer, NdeI site a type II restriction endonuclease purified from Neisseria denitrificans was added. NdeI, is a four base cutter with recognition site at 5’-CA↓TATG-3’ and creates overhangs in cut DNA fragments (Chang et al., 2005). It creates “ATG” at the beginning of the gene from where the transcription will start in the cloned gene. Ganiger et al., 2008 also amplified β-1, 6 endoglucanase gene from T. harzianum using PCR. Meera et al., 2011 amplified an intronless gene encoding endoglucanase eng 61 from Aspe...

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...arameters and optimized culture conditions which maximize enzyme yield. As far as the expression level of endoglucanase gene is concerned our results are much better than the recently conducted studies on the expression of endoglucanase of various organisms (Shyamala et al., 2011; Akita et al., 2005; Noronha and Ulhoa 2000). Endoglucanase is an industrially important enzyme involved synergistically in degradation of cellulosic materials into constitutive monomers with other cellulase enzymes. Its broad pH range and higher temperature tolerant ability make it useful for various industrial processes that are proceeded at higher temperature as well as can also help in cutting down cost of various processes. Because of the advantageous properties demonstrated by this enzyme, it can be further exploited for enzymatic hydrolysis of locally produced agriculture biomass.

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