Hu Xiaoqing, Fan Guangsen, Liao Hui, Fu Zhilei, Ma Chao, Ni Hui, Li Xiuting
College of Food and Biological Engineering, Jimei University, Yindou Road, Jimei District, Xiamen, 361021 Fujian China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing, 100048 China.
3 Biotech. 2020 Sep;10(9):387. doi: 10.1007/s13205-020-02327-w. Epub 2020 Aug 11.
B1213, a novel microbe isolated from a -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 identificated a single endoglucanase (EG)-encoding gene, designated as . The endoglucanase BpEG01790 shows 98.28% sequence similarity with an endo-β-1,4-glucanase (EC 3.2.1.4) from belonging to glycoside hydrolase family 8 (GH8). The gene 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. was successfully cloned into pET28a( +) with and without the signal peptide; however, attempts to overexpress this protein in BL21(DE3) cells using this expression system failed. 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 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 using TF as a molecular chaperone.
B1213是从产环境中分离出的一种新型微生物，在以葡聚糖为碳源的琼脂平板上表现出很强的纤维素分解活性，用大麦培养后活性为674.5 U/mL。对其进行基因组注释鉴定出一个编码内切葡聚糖酶（EG）的基因，命名为。内切葡聚糖酶BpEG01790与来自属于糖苷水解酶家族8（GH8）的一种内切-β-1,4-葡聚糖酶（EC 3.2.1.4）的序列相似性为98.28%。基因有一个1218 bp的开放阅读框，编码一个406个氨基酸（AA）残基的蛋白质（43.0 kDa），带有一个40个AA的信号肽。已成功将其克隆到带有和不带有信号肽的pET28a(+)中；然而，使用该表达系统在BL21(DE3)细胞中过表达该蛋白质的尝试失败了。也被克隆到pCold TF载体中。使用pCold TF载体表达系统和BL21(DE3)细胞，成功地在有或没有信号肽的情况下过表达了活性BpEG01790。与包含信号肽的构建体相比，没有信号肽的重组BpEG01790的过表达更高。培养条件的优化使酶活性提高了12.5倍。这是第一篇描述使用TF作为分子伴侣从异源可溶性过表达属于GH8的EG的报告。
