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Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier.

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 beta-glucuronidase enzyme from Escherichia coli.

SUBMITTER: Potot S

PROVIDER: S-EPMC2935070 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier.

Potot Sébastien S Serra Cláudia R CR Henriques Adriano O AO Schyns Ghislain G

Applied and environmental microbiology 20100702 17

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 resista ...[more]

PMID: 20601499

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Project description:Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Corroborating the proteomic analyses, electron microscopy analyses show a significantly thinner outer coat layer of the spores cultured on the solid agar medium.

Dataset Information

Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier.

Publications

Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier.

Similar Datasets

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