Cloning, Expression and Characterization of Highly Active Recombinant Thermostable Cellulase from Thermotoga naphthophila
Cloning, Expression and Characterization of Highly Active Recombinant Thermostable Cellulase from Thermotoga naphthophila
Aisha Khalid1, Muhammad Tayyab1,*, Abdual Rauf Shakoori2, Abu Saeed Hashmi1, Tahir Yaqub3, Ali Raza Awan1, Muhammad Wasim1, Sehrish Firyal1, Zaheer Hussain4 and Munir Ahmad5
ABSTRACT
Current study deals with the production, purification and characterization of recombinant thermostable cellulase from Thermotoga naphthophila. PCR using the genomic DNA of T. naphthophila as template resulted in amplification of 1 kb cellulase gene. The amplified cellulase gene was cloned in pTZ57R/T and sub-cloned in pET28a. The expression of recombinant cellulase was analyzed using BL21 CodonPlus (DE3) cells as expression host. The expression studies resulted in the production of recombinant enzyme as soluble protein. The recombinant protein was purified by affinity column chromatography. The characterization studies of purified protein demonstrated the optimal enzyme activity at 90°C and pH 4.8. The presence of cobalt enhanced the cellulase activity and 2.5 mM cobalt was recorded the optimal concentration for the maximal cellulase activity. SDS-PAGE analysis confirmed the molecular weight of recombinant protein as 39 kDa. The protein was found thermostable which retained more than 70% residual activity with an incubation of 1.67h at 90 °C in the presence of cobalt. Presence of ionic and non-ionic detergents showed an inhibitory effect on the enzyme activity. Kinetic studies of recombinant protein demonstrated the km and Vmax values of 0.22 mg/mL and 2500 µmoles/min respectively with the specific activity of 12464 Umg-1, using carboxymethyl cellulose as substrate. The deinking potential of recombinant cellulase to remove ink from the paper makes this enzyme a suitable candidate for its use in paper Industry. We are reporting a new member of M42 Family of aminopeptidases. The stability of this recombinant cellulase at wide range of temperature, pH, its high level activity and its paper deinking potential makes it a suitable candidate for its use in paper industry.
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