Protein engineering of cellulase for shifting the optimum pH

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Bora B. , Evran S.

9th International Molecular Biology and Biotechnology Congress, Kars, Türkiye, 6 - 10 Aralık 2020, ss.1

  • Basıldığı Şehir: Kars
  • Basıldığı Ülke: Türkiye
  • Sayfa Sayıları: ss.1


Cel12A from Thermotoga maritima is an endoglucanase that catalyzes the hydrolysis of β-1,4 linkages in β-glucans. Biodegredation of structural

biopolymer of plant cell wall is a promising approach in industrial applications. Therefore, there are ongoing efforts to manipulate the structural and

functional properties of cellulases. In this study, we aimed to manipulate the pH optimum of Cel12A. We employed directed evolution approach and

used error-prone PCR to create a random library of mutants. The library was transformed into E. coli by electroporation and the Cel12A-encoding

single colonies were screened on carboxymethyl cellulose agar plates under the manipulated pH conditions. Colonies that were capable of producing

clear zones under acidic conditions were selected. The selected colonies were cultured and the recombinant enzymes were purified by Ni-chelate

affinity chromatography. Catalytic activity of the purified enzymes were assayed by using carboxymethyl cellulose as the substrate. pH optimum, pH

stability, optimum temperature, thermal stability and detergent stability parameters of the mutant enzymes were characterized and compared with the

wild-type enzyme. A triple mutant of Cel12A showed a pH optimum shift from 5.5 to 5.0. Further work is ongoing in order to determine the effect of

single point mutations on the pH shift.