title: Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis.
abstract: In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 mM dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, PR China.
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.forename: Bangguo
authors.initials: B
authors.lastname: Wu
authors.forename: Jawad
authors.initials: J
authors.lastname: Ullah
authors.forename: Tianxi
authors.initials: T
authors.lastname: Zhang
authors.forename: Jinru
authors.initials: J
authors.lastname: Jia
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Wang
authors.forename: Tianwei
authors.initials: T
authors.lastname: Tan
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D015153:Blotting, Western; D061505:Cell Surface Display Techniques; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D013171:Spores, Bacterial / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D012997:Solvents; C091352:cotG protein, Bacillus subtilis; D000619:Aminohydrolases; C021867:nitrilase