Optimized soluble expression of a novel endoglucanase from Burkholderia pyrrocinia in Escherichia coli
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ORIGINAL ARTICLE
Optimized soluble expression of a novel endoglucanase from Burkholderia pyrrocinia in Escherichia coli Xiaoqing Hu1 · Guangsen Fan2,3,4 · Hui Liao1 · Zhilei Fu2,3 · Chao Ma3 · Hui Ni1 · Xiuting Li2,3,4 Received: 31 March 2020 / Accepted: 29 June 2020 © King Abdulaziz City for Science and Technology 2020
Abstract Burkholderia pyrrocinia B1213, a novel microbe isolated from a Baijiu-producing environment, displayed strong cellulolytic activity on agar plates with glucan as the carbon source and had an activity of 674.5 U/mL after culturing with barley. Genome annotation of B. pyrrocinia identificated a single endoglucanase (EG)-encoding gene, designated as BpEG01790. The endoglucanase BpEG01790 shows 98.28% sequence similarity with an endo-β-1,4-glucanase (EC 3.2.1.4) from Burkholderia stabilis belonging to glycoside hydrolase family 8 (GH8). The gene BpEG01790 has an open reading frame of 1218 bp encoding a 406 amino acid (AA) residue protein (43.0 kDa) with a 40-AA signal peptide. BpEG01790 was successfully cloned into pET28a( +) with and without the signal peptide; however, attempts to overexpress this protein in Escherichia coli BL21(DE3) cells using this expression system failed. BpEG01790 was also cloned into the pCold TF vector. Active BpEG01790 was successfully overexpressed with or without the signal peptide using the pCold TF vector expression system and E. coli BL21 (DE3) cells. Overexpression of recombinant BpEG01790 without the signal peptide was higher compared with the construct that included the signal peptide. Optimization of culture conditions improved the enzyme activity by 12.5-fold. This is the first report describing the heterologous soluble overexpression of an EG belonging to GH8 from B. pyrrocinia using TF as a molecular chaperone. Keywords Burkholderia pyrrocinia · Endoglucanase · GH8 · pCold TF · Soluble expression
Introduction
Xiaoqing Hu and Guangsen Fan contributed equally to this work. * Hui Ni [email protected]; [email protected] * Xiuting Li [email protected]; [email protected] 1
College of Food and Biological Engineering, Jimei University, Yindou Road, Jimei District, Xiamen 361021, Fujian, China
2
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, China
3
School of Food and Health, Beijing Technology and Business University (BTBU), Beijing 100048, China
4
Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU), No 11 Fucheng Street, Haidian District, Beijing 100048, China
Endo-β-1,4-glucanases (EC 3.2.1.4) (EGs) are hydrolytic enzymes that catalyze the endo-hydrolysis of β-1,4-Dglycosidic linkages in cellulose, cereal β-D-glucans and lichenin, which are the main components of the plant cell wall (Ontanon et al. 2019). EGs have attracted noticeable attention because of their potential use in a diverse range of applications, including production of glucan oligosaccharides (Ontanon et al
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