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CenC, a multidomain thermostable GH9 processive endoglucanase from Clostridium thermocellum: cloning, characterization and saccharification studies

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Abstract

The growing demands of bioenergy has led to the emphasis on novel cellulases to improve efficiency of biodegradation process of plant biomass. Therefore, a thermostable cellulolytic gene (CenC) with 3675 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 °C. CenC had a half-life of 24 min at 74 °C, was stable up to 2 h at 60 °C and over a pH range of 5.5–7.5. Enzyme showed high affinity towards various substrates and processively released cellobiose from cellulosic substrates. It efficiently hydrolyzed carboxymethyl cellulose (30 U/mg), β-Glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-β-d-cellobioside (18 U/mg), birchwood 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 K −1m and Kcat K −1m of 7.14 mM, 52.4 µmol 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 °C 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.

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Acknowledgments

This work was supported by a Grant No. 27(54)/2007-DSA (P&D) from the Ministry of Science and Technology, Pakistan.

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    1. Institute of Industrial Biotechnology, GC University, Lahore, 54000, Pakistan

      Ikram ul Haq, Fatima Akram, Mahmood Ali Khan, Zahid Hussain, Ali Nawaz, Kaleem Iqbal & Ali Javed Shah

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    1. Ikram ul Haq
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    2. Fatima Akram
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    3. Mahmood Ali Khan
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    Correspondence to Ikram ul Haq.

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    The authors declare that they have no conflict of interests. We assure the quality and integrity of our research work. This study is completely independent and impartial, all points taken from other authors are well cited in the text. The research work was financially funded by Ministry of Science and Technology, Pakistan.

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    Ikram ul Haq and Fatima Akram have contributed equally to this work.

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    Haq, I.u., Akram, F., Khan, M.A. et al. CenC, a multidomain thermostable GH9 processive endoglucanase from Clostridium thermocellum: cloning, characterization and saccharification studies. World J Microbiol Biotechnol 31, 1699–1710 (2015). https://doi.org/10.1007/s11274-015-1920-4

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