Journal of Chromatography & Separation Techniques
Open Access
ISSN: 2157-7064
+44 1300 500008
ISSN: 2157-7064
+44 1300 500008
Ikram ul Haq, Fatima Akram and Ali Nawaz
GC University, Pakistan
Posters & Accepted Abstracts: J Chromatogr Sep Tech
The growing demands of bioenergy have led to the emphasis on novel cellulases to improve efficiency of biodegradation process of plant biomass. Therefore, a thermostable cellulolytic gene (CenC) with 3,675 bp was cloned from Clostridium thermocellum and over-expressed in Escherichia coli strain BL21 CodonPlus. It was attested that CenC belongs to glycoside hydrolase family 9 (GH9) with four binding domains, a processive endoglucanase. CenC was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 137.11 kDa, by purification steps of heat treatment combined with ion-exchange chromatography. Purified enzyme displayed optimal activity at pH 6.0 and 70�?ºC. CenC had a half-life of 24 min at 74�?ºC, was stable up to 2 hours at 60�?ºC and over a pH range of 5.5-7.5. Enzyme showed high affinity towards various substrates and processively released cellobiose from cellulosic substrates confirmed by using HPLC technique. It efficiently hydrolyzed carboxymethyl cellulose (30 U/mg), �?²-glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-�?²-D-cellobioside (18 U/mg), birch wood xylan (19 U/mg), beechwood xylan (17.5 U/mg), avicel (9 U/mg), Whatman filter paper (11 U/mg) and laminarin (3.3 U/mg). CenC exhibited Km, Vmax, Kcat, Vmax Km-1 and Kcat Km-1 of 7.14 mM, 52.4 �?¼mol mg-1 min-1, 632.85 s-1, 7.34 min-1 and 88.63, respectively used CMC as substrate. Recombinant CenC saccharified pretreated wheat straw and bagasse to 5.12% and 7.31%, respectively at pH 7.0 and 45�?ºC after 2 h incubation. Its thermostability, high catalytic efficiency and independence of inhibitors make CenC enzyme an appropriate candidate for industrial applications and cost-effective saccharification process.
Email: ikmhaq@yahoo.com