School of Biological Science, University of the Punjab, Lahore, Pakistan.
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
Biotechnol Appl Biochem. 2023 Feb;70(1):290-305. doi: 10.1002/bab.2352. Epub 2022 May 25.
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 K values of 32, 33, 45, and 43 mg⋅mL and V values of 3571, 3846, 3571, and 4545 U⋅min , 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.
过程性纤维素酶（CelO）是热纤维梭菌的一种重要的模块化酶。为了研究碳水化合物结合模块（CBM3b）对 CelO（GH5）催化结构域的影响，通过 CBM3b 的截短和末端融合设计了 CelO 的四个工程衍生酶。这些酶分别为 N 端的 CBM（CelO-BC，62 kDa）、仅含有催化结构域（CelO-C，42 kDa）、C 端的 CBM（CelO-CB，54 kDa）和两端都连接 CBM（CelO-BCB，73 kDa）。所有构建体均被克隆到 pET22b（+）中，并在大肠杆菌 BL21（DE3）star 中表达。CelO-C、CelO-CB、CelO-BC 和 CelO-BCB 的表达水平分别为 35%、35%、30%和 20%。CelO-C、CelO-CB、CelO-BC 和 CelO-BCB 对 1%再生无定形纤维素（RAC）的酶活性分别为 860、758、985 和 1208 个单位/μmole 酶。通过热处理和阴离子交换 FPLC 纯化从大肠杆菌细胞裂解物中部分纯化这些酶。对 RAC 而言，CelO-C、CelO-CB、CelO-BC 和 CelO-BCB 的 K 值分别为 32、33、45 和 43 mg⋅mL，V 值分别为 3571、3846、3571 和 4545 U⋅min。通过富含 P/T 的连接子连接到 GH5 N 端的 CBM3b 被发现可以提高酶的催化活性和热稳定性。
