Journal
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
Volume 31, Issue 11, Pages 1699-1710Publisher
SPRINGER
DOI: 10.1007/s11274-015-1920-4
Keywords
Thermostable; Processive endoglucanase; Module protein; Saccharification; Clostridium thermocellum
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Funding
- Ministry of Science and Technology, Pakistan [27(54)/2007-DSA (PD)]
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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 A degrees C. CenC had a half-life of 24 min at 74 A degrees C, was stable up to 2 h at 60 A degrees 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), beta-Glucan Barley (94 U/mg); also showed activity towards p-nitrophenyl-beta-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 K-m, V-max, K-cat, V-max K (m) (-1) and K-cat K (m) (-1) of 7.14 mM, 52.4 A mu mol 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 A degrees 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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