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The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate‐binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N‐terminus, native form (CelO‐BC, 62 kDa); catalytic domain only (CelO‐C, 42 kDa); CBM at the C‐terminus (CelO‐CB, 54 kDa) and CBM attached at both termini (CelO‐BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO‐C, CelO‐CB, CelO‐BC, and CelO‐BCB were 35%, 35%, 30%, and 20%, respectively. The enzyme activities of CelO‐C, CelO‐CB, CelO‐BC, and CelO‐BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per μmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO‐C, CelO‐CB, CelO‐BC, and CelO‐BCB showed KM values of 32, 33, 45, and 43 mg⋅mL–1 and Vmax values of 3571, 3846, 3571, and 4545 U⋅min–1, respectively. CBM3b at the N‐terminus of GH5 linked through a P/T‐rich linker was found to enhance the catalytic activity and thermostability of the enzyme.
Biotechnology and Applied Biochemistry – Wiley
Published: Feb 1, 2023
Keywords: exocellulase O; Clostridium thermocellum; domain engineering; enhanced activity
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