title: Effect of Linker Length and Flexibility on the Clostridium thermocellum Esterase Displayed on Bacillus subtilis Spores.
abstract: In fusion protein design strategies, the flexibility and length of linkers are important parameters affecting the bioactivity of multifunctional proteins. A series of fusion proteins with different linkers were constructed. The effect of temperature, pH, and organic solvents was investigated on the enzymatic activity. Fusion proteins with P1(PTPTPT) and P2((PTPTPT)2) linkers remained highly active with wide temperature range. At pH 9.6, the relative activity of fusion proteins with (PTPTPT)2and S2(EGKSSGSGSESKST) linkers was 70 and 62 % (1.75 and 1.5 times of that of non-linker ones). Fusion proteins with S3((GGGGS)4) linker retained 55 % activity after 5 h of incubation at 80 °C (1.2-fold of that of non-linker fusion proteins and 1.9-fold of GGGGS-linker fusion proteins). Finally, the relative activity of fusion proteins having different linkers was increased with 20 % dimethyl sulfoxide (DMSO) and methanol; relative activity of fusion proteins with EGKSSGSGSESKST linkers was enhanced 1.5- and 2.2-fold, respectively. These results suggest that longer flexible linker can enhance the activity and stability of displayed esterase than shorter flexible linker. Optimizing peptide linkers with length, flexibility, and amino acid composition could improve the thermostability and activity of the displayed enzyme.
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China. hyc@ujs.edu.cn.|State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. hyc@ujs.edu.cn.
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Bangguo
authors.initials: B
authors.lastname: Wu
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Tianxi
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Jinru
authors.initials: J
authors.lastname: Jia
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Lu
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ni
authors.affiliation: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.
authors.forename: Tianwei
authors.initials: T
authors.lastname: Tan
mesh_terms: D020816:Amino Acid Motifs; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000235:genetics* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D057075:Enzyme Assays; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013171:Spores, Bacterial / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004950:Esterases; D000432:Methanol; D004121:Dimethyl Sulfoxide
keywords: Bacillus subtilis; Displayed; Esterase; Flexibility; Linker; Spore