scholarly journals Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier

2010 ◽  
Vol 76 (17) ◽  
pp. 5926-5933 ◽  
Author(s):  
Sébastien Potot ◽  
Cláudia R. Serra ◽  
Adriano O. Henriques ◽  
Ghislain Schyns
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Recombinant Proteins ◽  
Animal Feed ◽  
Active Form ◽  
Bioactive Molecules ◽  
Full Potential ◽  
Spore Surface ◽  
Outer Coat ◽  
Feed Enzymes

ABSTRACT The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric β-glucuronidase enzyme from Escherichia coli.

scholarly journals Characterization of the Bacillus subtilis Spore Morphogenetic Coat Protein CotO

2005 ◽  
Vol 187 (24) ◽  
pp. 8278-8290 ◽  
Author(s):  
D. C. McPherson ◽  
H. Kim ◽  
M. Hahn ◽  
R. Wang ◽  
P. Grabowski ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Late Phase ◽  
Coat Proteins ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Fluorescence And Electron Microscopy ◽  
Complex Protein ◽  
Bacillus Spores ◽  
Critical Model

ABSTRACT Bacillus spores are protected by a structurally and biochemically complex protein shell composed of over 50 polypeptide species, called the coat. Coat assembly in Bacillus subtilis serves as a relatively tractable model for the study of the formation of more complex macromolecular structures and organelles. It is also a critical model for the discovery of strategies to decontaminate B. anthracis spores. In B. subtilis, a subset of coat proteins is known to have important roles in assembly. Here we show that the recently identified B. subtilis coat protein CotO (YjbX) has an especially important morphogenetic role. We used electron and atomic force microscopy to show that CotO controls assembly of the coat layers and coat surface topography as well as biochemical and cell-biological analyses to identify coat proteins whose assembly is CotO dependent. cotO spores are defective in germination and partially sensitive to lysozyme. As a whole, these phenotypes resemble those resulting from a mutation in the coat protein gene cotH. Nonetheless, the roles of CotH and CotO and the proteins whose assembly they direct are not identical. Based on fluorescence and electron microscopy, we suggest that CotO resides in the outer coat (although not on the coat surface). We propose that CotO and CotH participate in a late phase of coat assembly. We further speculate that an important role of these proteins is ensuring that polymerization of the outer coat layers occurs in such a manner that contiguous shells, and not unproductive aggregates, are formed.

Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis

2017 ◽  
Vol 27 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Huayou Chen ◽  
Zhi Chen ◽  
Bangguo Wu ◽  
Jawad Ullah ◽  
Tianxi Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Proteins ◽  
Rational Design ◽  
Thermotoga Maritima ◽  
Sodium Dodecyl ◽  
Ph Stability ◽  
Activity Measurement ◽  
Spore Surface ◽  
Outer Coat ◽  
The Stability

In the present study, fusion genes composed of <i>Thermotoga maritima</i> MSB8 nitrilase and <i>Bacillus subtilis</i> 168 outer coat protein CotG were constructed with various peptide linkers and displayed on <i>B. subtilis</i> DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of <i>B. subtilis</i> 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)<sub>3</sub> demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)<sub>2</sub>. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)<sub>2</sub> and (EAAAK)<sub>3</sub>. Fusions with linker (EAAAK)<sub>2</sub> demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)<sub>3</sub>. In the presence of 1 m<smlcap>M</smlcap> dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)<sub>3</sub>, 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 <i>B. subtilis</i>.

scholarly journals Functional Regions of the Bacillus subtilis Spore Coat Morphogenetic Protein CotE

1999 ◽  
Vol 181 (22) ◽  
pp. 7043-7051 ◽  
Author(s):  
Tamara Bauer ◽  
Shawn Little ◽  
Axel G. Stöver ◽  
Adam Driks
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Coat Protein ◽  
Protein Assembly ◽  
Spore Coat ◽  
Coat Proteins ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Coat Protein Assembly

ABSTRACT The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat’s outer layer. We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.

scholarly journals Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

1978 ◽  
Vol 253 (19) ◽  
pp. 6694-6701
Author(s):  
L. Munoz ◽  
Y. Sadaie ◽  
R.H. Doi
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Spore Coat ◽  
Precursor Synthesis ◽  
Spore Coat Protein

scholarly journals Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

2006 ◽  
Vol 188 (21) ◽  
pp. 7609-7616 ◽  
Author(s):  
Alicia Monroe ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Cross Linking ◽  
Spore Coat ◽  
Bacillus Subtilis Spore ◽  
Binding Partners ◽  
Lysine Residues ◽  
Cross Links ◽  
Hydrolysis Of ◽  
Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

Effect of temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37

2021 ◽  
Vol 66 (1) ◽  
pp. 72-79
Author(s):  
Thuoc Doan Van ◽  
Hung Nguyen Phuc
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Animal Feed ◽  
Culture Conditions ◽  
Effect Of Temperature ◽  
Physical Parameters ◽  
Original Activity ◽  
Optimum Ph ◽  
Production Activity ◽  
Ph Range

The effect of physical parameters such as temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37 was investigated. The results indicated that the optimum culture conditions for enzyme activity were pH 7.0 and 35 oC. The optimum pH and temperature for enzyme activity were 6.0 and 70 oC. The crude enzyme was found to be stable in the pH range of 5.0 to 7.0. The enzyme was stable for 1 h at a temperature from 30 to 80 oC; nearly 100% of enzyme activity remained at temperatures of 30 - 40 oC, and about 34% of original activity remained at a temperature of 80 oC. These features demonstrated that α-amylase from B. subtilis V37 can be applied in many areas such as the food, fermentation, and animal feed industries.

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