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Background RESULTS Conclusion Lignin peroxidase is closely related to agriculture and food as it improves the quality of feedstuffs, facilitates the degradation of lignin in agricultural wastes, and degrades azo dyes that have similar complex structures to lignin. However, the current status of homologous or heterologous expression of lignin peroxidase is unsatisfactory and needs to be modified with the help of immobilization and directed evolution to maximize its potential. Directed evolution technology is an effective strategy for designing and improving enzyme characteristics, and Bacillus subtilis spore surface display technology is an efficient method for preparing immobilized enzymes.A colorimetric dye decolorization assay using Congo red as a substrate was developed and optimized for high‐throughput screening of spore surface display in a 96‐well plate. After two rounds of screening, a superior mutant strain was selected from 2700 mutants. Its highest catalytic activity was 196.36%. The amino acid substitution sites were identified as N120D and I242T.The mechanism for the enhanced catalytic activity was explained using protein modeling and functional analysis software. This study provides insights into the rational design of lignin peroxidase and its application in food and agriculture. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a widespread Gram-positive facultative aerobic bacterium that is recognized as generally safe. It has shown significant application value and great development potential in the animal farming industry. As a probiotic, it is frequently used as a feed growth supplement to effectively replace antibiotics due to its favourable effects on regulating the intestinal flora, improving intestinal immunity, inhibiting harmful microorganisms, and secreting bioactive substances. Consequently, the gut health and disease resistance of farmed animals can be improved. Both vegetative and spore forms of B. subtilis have also been utilized as vaccine carriers for delivering the antigens of infectious pathogens for over a decade. Notably, its spore form is regarded as one of the most prospective for displaying heterologous antigens with high activity and stability. Previously published reviews have predominantly focused on the development and applications of B. subtilis spore surface display techniques. However, this review aims to summarize recent studies highlighting the important role of B. subtilis as a probiotic and vaccine carrier in maintaining animal health. Specifically, we focus on the beneficial effects and underlying mechanisms of B. subtilis in enhancing disease resistance among farmed animals as well as its potential application as mucosal vaccine carriers. It is anticipated that B. subtilis will assume an even more prominent role in promoting animal health with in-depth research on its characteristics and genetic manipulation tools. [ABSTRACT FROM AUTHOR]

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range. [ABSTRACT FROM AUTHOR]

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin–neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease. [ABSTRACT FROM AUTHOR]

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range.

The development of efficient mucosal vaccines is strongly dependent on the use of appropriate vectors. Various biological systems or synthetic nanoparticles have been proposed to display and deliver antigens to mucosal surfaces. The Bacillus spore, a metabolically quiescent and extremely resistant cell, has also been proposed as a mucosal vaccine delivery system and shown able to conjugate the advantages of live and synthetic systems. Several antigens have been displayed on the spore by either recombinant or non-recombinant approaches, and antigen-specific immune responses have been observed in animals immunized by the oral or nasal route. Here we review the use of the bacterial spore as a mucosal vaccine vehicle focusing on the advantages and drawbacks of using the spore and of the recombinant vs. non-recombinant approach to display antigens on the spore surface. An overview of the immune responses induced by antigen-displaying spores so far tested in animals is presented and discussed. [ABSTRACT FROM AUTHOR]

Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces.

Abstract Backgrounds The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. Results p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. Conclusions This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin–neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

Backgrounds: The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. Results: p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. Conclusions: This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40. [ABSTRACT FROM AUTHOR]

Lacticaseibacillus rhamnosus GG (LGG) can promote intestinal health by modulating the immune responses of the gastrointestinal tract. However, knowledge about the immunomodulatory action of LGG-derived soluble factors is limited. In our previous study, we have displayed LGG-derived p75 protein on the spore surface of Bacillus subtilis. The objective of this study was to determine the effect of spore-displayed p75 (CotG-p75) on immune system by investigating transcriptional response of Caco-2 cells stimulated by CotG-p75 through RNA-sequencing (RNA-seq). RNA-seq results showed that CotG-p75 mainly stimulated genes involved in biological processes, such as response to stimulus, immune regulation, and chemotaxis. KEGG pathway analysis suggested that many genes activated by CotG-p75 were involved in NF-ĸB signaling and chemokine signaling pathways. CotG-p75 increased cytokines and chemokines such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, CCL20, CCL22, and IL1B essential for the immune system. In particular, CotG-p75 increased the expression levels of NF-ĸB-related genes such as NFKBIA, TNFAIP3, BIRC3, NFKB2, and RELB involved in immune and inflammatory responses. This study provides genes and pathways involved in immune responses influenced by CotG-p75. These comprehensive transcriptome profiling could be used to elucidate the immunomodulatory action of CotG-p75. [ABSTRACT FROM AUTHOR]

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1b, IL-6, IL-10, TNF-a and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines. [ABSTRACT FROM AUTHOR]

A Lacticaseibacillus rhamnosus GG-derived protein, p75, is one of the key molecules exhibiting probiotic activity. However, the molecular mechanism and transcriptional response of p75 in human intestinal epithelial cells are not completely understood. To gain a deeper understanding of its potential probiotic action, this study investigated genome-wide responses of HT-29 cells to stimulation by spore-displayed p75 (CotG-p75) through a transcriptome analysis based on RNA sequencing. Analysis of RNA-seq data showed significant changes of gene expression in HT-29 cells stimulated by CotG-p75 compared to the control. A total of 189 up-regulated and 314 down-regulated genes was found as differentially expressed genes. Gene ontology enrichment analysis revealed that a large number of activated genes was involved in biological processes, such as epithelial cell differentiation, development, and regulation of cell proliferation. A gene–gene interaction network analysis showed that several DEGs, including AREG, EREG, HBEGF, EPGN, FASLG, GLI2, CDKN1A, FOSL1, MYC, SERPINE1, TNFSF10, BCL6, FLG, IVL, SPRR1A, SPRR1B, SPRR3, and MUC5AC, might play a critical role in these biological processes. RNA-seq results for selected genes were verified by reverse transcription-quantitative polymerase chain reaction. Overall, these results provide extensive knowledge about the transcriptional responses of HT-29 cells to stimulation by CotG-p75. This study showed that CotG-p75 can contribute to cell survival and epithelial development in human intestinal epithelial cells. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed Pycnoporus sanguineus CDH (ps CDH) on the surface of Bacillus subtilis spores for the first time. Enzymatic characterization revealed that spore surface display enhanced the tolerance of ps CDH to high temperature (80 °C) and low pH levels (3.5) compared to free ps CDH. Furthermore, we found that glycerol, lactic acid, and malic acid promoted the activity of immobilized spore-displayed ps CDH; glycerol has a more significant stimulating effect, increasing the activity from 16.86 ± 1.27 U/mL to 46.26 ± 3.25 U/mL. After four reuse cycles, the ps CDH immobilized with spores retained 48% of its initial activity, demonstrating a substantial recovery rate. In conclusion, the spore display system, relying on cotG, enables the expression and immobilization of CDH while enhancing its resistance to adverse conditions. This system demonstrates efficient enzyme recovery and reuse. This approach provides a novel method and strategy for the immobilization and stability enhancement of CDH. • Integration of CDH fixation and stability enhancement through spore surface display. • Glycerol, lactic acid, and malic acid promote the activity of spore-displayed CDH. • CDH immobilized with spores demonstrating a substantial recovery rate. [ABSTRACT FROM AUTHOR]

Abstract Lactobacillus rhamnosus p75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted from B. subtilis was confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3–7 and maintained at 70% at 50 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics.

Abstract Background Bacillus subtilis spores have been commonly used for the surface display of various food-related or human antigens or enzymes. For successful display, the target protein needs to be fused with an anchor protein. The preferred anchored proteins are the outer-coat proteins of spores; outer-coat proteins G (CotG) and C (CotC) are commonly used. In this study, mutant trehalose synthase (V407M/K490L/R680E TreS) was displayed on the surface of B. subtilis WB800n spores using CotG and CotC individually or in combination as an anchoring protein. Results Western blotting, immunofluorescence, dot blot, and enzymatic-activity assays detected TreS on the spore surface. The TreS activity with CotC and CotG together as the anchor protein was greater than the sum of the enzymatic activities with CotC or CotG alone. The TreS displayed on the spore surface with CotC and CotG together as the anchoring protein showed elevated and stable specific activity. To ensure spore stability and prevent spore germination in the trehalose preparation system, two germination-specific lytic genes, sleB and cwlJ, were deleted from the B. subtilis WB800n genome. It was demonstrated that this deletion did not affect the growth and spore formation of B. subtilis WB800n but strongly inhibited germination of the spores during transformation. The conversion rate of trehalose from 300 g/L maltose by B. subtilis strain WB800n(ΔsleB, ΔcwlJ)/cotC-treS–cotG-treS was 74.1% at 12 h (350 U/[g maltose]), and its enzymatic activity was largely retained, with a conversion rate of 73% after four cycles. Conclusions The spore surface display system based on food-grade B. subtilis with CotC and CotG as a combined carrier appears to be a powerful technology for TreS expression, which may be used for the biotransformation of d-maltose into d-trehalose.

A Lacticaseibacillus rhamnosus GG-derived protein, p75, is one of the key molecules exhibiting probiotic activity. However, the molecular mechanism and transcriptional response of p75 in human intestinal epithelial cells are not completely understood. To gain a deeper understanding of its potential probiotic action, this study investigated genome-wide responses of HT-29 cells to stimulation by spore-displayed p75 (CotG-p75) through a transcriptome analysis based on RNA sequencing. Analysis of RNA-seq data showed significant changes of gene expression in HT-29 cells stimulated by CotG-p75 compared to the control. A total of 189 up-regulated and 314 down-regulated genes was found as differentially expressed genes. Gene ontology enrichment analysis revealed that a large number of activated genes was involved in biological processes, such as epithelial cell differentiation, development, and regulation of cell proliferation. A gene–gene interaction network analysis showed that several DEGs, including AREG, EREG, HBEGF, EPGN, FASLG, GLI2, CDKN1A, FOSL1, MYC, SERPINE1, TNFSF10, BCL6, FLG, IVL, SPRR1A, SPRR1B, SPRR3, and MUC5AC, might play a critical role in these biological processes. RNA-seq results for selected genes were verified by reverse transcription-quantitative polymerase chain reaction. Overall, these results provide extensive knowledge about the transcriptional responses of HT-29 cells to stimulation by CotG-p75. This study showed that CotG-p75 can contribute to cell survival and epithelial development in human intestinal epithelial cells.

Lactobacillus rhamnosus p75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted from B. subtilis was confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3–7 and maintained at 70% at 50 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics. [ABSTRACT FROM AUTHOR]

Introduction: Bacillus subtilis spore surface display technique has long been used to display antigens and enzymes for medical and industrial proposes. In this technique, the enzyme is genetically immobilized on the spore surface and one of the capabilities of spore displayed enzyme is the reusability. In this study, spore displayed tyrosinase was used for the bioconversion of soybean extracted daidzein to more hydroxylated, anticancer compound, 3ʹ-ODI. Materials and methods: Bacillus subtilis DB104 (pSDJH-cotE-tyr) which was constructed in our previous study was used as an enzyme source. The reaction was done in 37°C for 1 hour. To detect the product of the reaction, high-performance liquid chromatography was used. Results: The results revealed that 1mM of daidzein was converted to about 1mM 3ʹ-ODI during 60 min by 4*108 spores. The retained activity of the spore displayed tyrosinase was also detected about 58% after three times usage. Discussion and conclusion: Spore surface displayed enzymes have created a new way to improve enzyme stability and reusability. Our results showed that active tyrosinase on the surface of the spores has the potential to be used in industrial conditions to produce more hydroxylated isoflavones. [ABSTRACT FROM AUTHOR]

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background. [ABSTRACT FROM AUTHOR]

Summary: Bacillus species are widely recognized as good industrial platform strains, which can produce a variety of valuable products including enzymes and functional proteins. Bacillus expression systems gain various competitive edges over other expression systems, with respect to nontoxicity, convenience for gene modification and high yield of target proteins. Recently, a number of Bacillus expression systems have been developed, and various strategies were conducted to improve the yields of target proteins. In this review, we focused on the strategies of host strain optimization for heterologous protein production, including secretion pathway optimization, RNA and protein stability enhancement, cell growth facilitation, genome streamlining and transcriptional regulator engineering. In addition, Bacillus spore surface display and food‐grade expression systems were developed to expand the application of Bacillus expression system in recent years. Finally, the challenges and prospects of Bacillus expression system were discussed regarding the recent progresses, challenges and trends in this field. [ABSTRACT FROM AUTHOR]

In this paper, we report removing heavy metal using Bacillus subtilis spore surface display system. We used CotE protein as an anchoring motif because of its high abundance and location in the outer coat layer. And we inserted His12 (double histidine 6 tag) at the C-terminal end of anchoring motif. The proper expression of CotE-His12 fusion protein (22.8 kDa) was confirmed by western blot. We confirmed the surface expression of the CotE-His12 fusion protein using flow cytometry. We tried Ni2+ and Cd2+ adsorption with recombinant spore DB104 (pCotE-His12) and DB104 spore. The amount of adsorbed Ni2+ was 18.2 nmol/mg for DB104 spore and 82.4 nmol/mg for DB104 (pCotE-His12) spore. In the case of Cd2+, the adsorbed amount was 32.6 nmol/mg for DB104 spore and 79.1 nmol/mg for DB104 (pCotE-His12) spore. This means that our spore displayed His12 system can be generally applied for the removal of various kind of heavy metals in the field. [ABSTRACT FROM AUTHOR]

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis. [ABSTRACT FROM AUTHOR]

Abstract: We report a spore‐based biocatalysis platform capable of producing and self‐assembling active multimeric enzymes on a spore surface with a high loading density. This was achieved by co‐expressing both a spore surface‐anchoring scaffoldin protein containing multiple cohesin domains and a dockerin‐tagged enzyme of interest in the mother cell compartment during Bacillus subtilis sporulation. Using this method, tetrameric β‐galactosidase was successfully displayed on the spore surface with a loading density of 1.4 × 104 active enzymes per spore particle. The resulting spore biocatalysts exhibited high conversion rates of transgalactosylation in water/organic emulsions. With easy manufacture, enhanced thermostability, excellent reusability, and long‐term storage stability at ambient temperature, this approach holds a great potential in a wide range of biocatalysis applications especially involving organic phases. [ABSTRACT FROM AUTHOR]

Porcine rotavirus is a major cause of acute viral gastroenteritis in suckling piglets, and vaccination is considered to be an effective measure to control these infections. The development of a live mucosal vaccine using Bacillus subtilis spores as an antigen delivery vehicle is a convenient and attractive vaccination strategy against porcine rotavirus. In this study, a shuttle vector was constructed for the spore surface display of the spike protein VP8* from porcine rotavirus (the genotype was G5P[7]). A successful display of the CotB-VP8* fusion protein on the spore surface was confirmed by Western blot and immunofluorescence microscopy analysis. The capacity for immune response generated after immunization with the recombinant strain was evaluated in a mouse model. The intestinal fecal IgA and serum IgG were detected by enzyme-linked-immunosorbent serologic assay (ELISA). Importantly, recombinant strain spores could elicit strong specific mucosal and humoral immune responses. These encouraging results suggest that recombinant B. subtilis BV could provide a strategy for a potential novel application approach to the development of a new and safe mucosal subunit vaccine against porcine rotavirus.

Taking the advantages of inert and stable nature of endospores, we developed a biocatalysis platform for multiple enzyme immobilization on Bacillus subtilis spore surface. Among B. subtilis outer coat proteins, CotG mediated a high expression level of Clostridium thermocellum cohesin (CtCoh) with a functional display capability of ∼104 molecules per spore of xylose reductase-C. thermocellum dockerin fusion protein (XR-CtDoc). By co-immobilization of phosphite dehydrogenase (PTDH) on spore surface via Ruminococcus flavefaciens cohesin-dockerin modules, regeneration of NADPH was achieved. Both xylose reductase (XR) and PTDH exhibited enhanced stability upon spore surface display. More importantly, by altering the copy numbers of CtCoh and RfCoh fused with CotG, the molar ratio between immobilized enzymes was adjusted in a controllable manner. Optimization of spore-displayed XR/PTDH stoichiometry resulted in increased yields of xylitol. In conclusion, endospore surface display presents a novel approach for enzyme cascade immobilization with improved stability and tunable stoichiometry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:383-389, 2017 [ABSTRACT FROM AUTHOR]

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. [ABSTRACT FROM AUTHOR]

In the present study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. An Escherichia coli– Bacillus subtilis shuttle vector (pHS-CotG-nit) was constructed for the spore surface display of the nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The successful display of CotG-nit fusion protein on the spore surface of B. subtilis was verified by western blot analysis and activity measurement. The optimal temperature and pH of the spore surface-dispalyed nitrilase were observed to be 50 °C and pH 8.0, respectively, which were higher than the free nitrilase (45 °C and pH 7.5). The analysis of thermal and pH stability indicated that the spore surface-displayed nitrilase retained 79% and 97% at 75 °C and pH 8.0 after 1 h of incubation, whereas it were 32% and 52%, respectively, for free nitrilase. Furthermore, the reusability experiments indicated that the activity of the spore surface-displayed nitrilase was not significantly decreased throughout the reusability process, which still retained 83% of the initial activity at the fifth cycle. Above all, these results suggested that surface display of enzymes on the spore of B. subtilis might be an effective method for enzyme immobilization and help to meet the ever-increasing industrial demand for preparation and stabilization of biocatalysts. [ABSTRACT FROM AUTHOR]

Background: LinB, as a Haloalkane dehalogenase, has good catalytic activity for many highly toxic and recalcitrant compounds, and can realize the elimination of chemical weapons HD in a green non-toxic mode., Objectives: In order to display Haloalkane dehalogenase LinB on the surface of Bacillus subtilis spore., Methods: We have constituted the B. subtilis spore surface display system of halogenated alkanes dehalogenase LinB by gene recombination., Results: Data revealed that LinB can display on spore surface successfully. The hydrolyzing HD analogue 2-chloroethyl ethylsulfide (2-CEES) activity of displayed LinB spores was 4.30±0.09 U/mL, and its specific activity was 0.78±0.03U/mg. Meanwhile, LinB spores showed a stronger stress resistance activity on 2-CEES than free LinB. This study obtained B. subtilis spores of LinB (phingobium japonicum UT26) with enzyme activity that was not reported before., Conclusion: Spore surface display technology uses resistance spore as the carrier to guarantee LinB activity, enhances its stability, and reduces the production cost, thus expanding the range of its application., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

Two model proteins of an enhanced green fluorescent protein (EGFP) and a tetrameric β-Galactosidase (β-Gal) from Bacillus stearothermophilus IAM11001 were fused to the C-terminus of the crust proteins Y (CotY) and Z (CotZ) from B. subtilis 168. Surface-expression of the resulting fusion proteins CotY-EGFP, CotZ-EGFP, CotY-β-Gal, and CotZ-β-Gal were then evaluated. Fluorescence intensity analysis of the transformed strains indicated that the CotY-EGFP and CotZ-EGFP fusion proteins were successfully displayed on spores. β-Gal was anchored on the surface of B. subtilis spores, confirmed based on western blotting and immunofluorescence microscopy. Results of a β-Gal assay showed that when fused to CotY and CotZ, the obtained enzyme activities of spore-displayed β-Gals were 0.12 and 0.25 U/mg of spores (dry weight), respectively. CotY and CotZ were, thus, shown to be suitable candidate carriers for display of multimeric enzymes on the spore surface. [ABSTRACT FROM AUTHOR]

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 ( PCV2 ) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein ( Cap ) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Li, Dai, Liu, Khalique, Wang, Zeng, Zhang, Ni, Zeng, Jing and Pan.)

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101( ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture. [ABSTRACT FROM AUTHOR]

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An additional application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic production of chemicals, and the delivery of biomolecules of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanalytical and biotechnological applications, and the use of spores as vehicles for vaccines and therapeutic agents. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2CTD, in vivo both free and spore-adsorbed BclA2CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2CTD could act as a putative mucosal antigen targeting C. difficile spores. [ABSTRACT FROM AUTHOR]

Porcine rotavirus is a major cause of acute viral gastroenteritis in suckling piglets, and vaccination is considered to be an effective measure to control these infections. The development of a live mucosal vaccine using Bacillus subtilis spores as an antigen delivery vehicle is a convenient and attractive vaccination strategy against porcine rotavirus. In this study, a shuttle vector was constructed for the spore surface display of the spike protein VP8* from porcine rotavirus (the genotype was G5P[7]). A successful display of the CotB-VP8* fusion protein on the spore surface was confirmed by Western blot and immunofluorescence microscopy analysis. The capacity for immune response generated after immunization with the recombinant strain was evaluated in a mouse model. The intestinal fecal IgA and serum IgG were detected by enzyme-linked-immunosorbent serologic assay (ELISA). Importantly, recombinant strain spores could elicit strong specific mucosal and humoral immune responses. These encouraging results suggest that recombinant B. subtilis BV could provide a strategy for a potential novel application approach to the development of a new and safe mucosal subunit vaccine against porcine rotavirus. [ABSTRACT FROM AUTHOR]

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE , cotG , and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His 6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN 3 , the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

Nitrilase is a valuable type of hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from Acidovorax facilis 72W was inserted into an Escherichia coli-Bacillus subtilis shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and 50°C, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the 10 th cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by E. coli (pET-28a-nit). Generally, the display of A. facilis 72W nitrilase on the spore surface of Bacillus subtilis may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.

The production of d-allulose is usually conducted via isolated-enzyme or whole-cell biocatalysis reactions. In the present study, an innovative biocatalyst, d-psicose 3-epimerase (DPEase) from Clostridium scindens ATCC 35704, presented on the surface of Bacillus subtilis spores, was applied for d-allulose production. DPEase was fused at the C-terminus of the anchoring protein, CotZ, via a peptide linker, and trophic genes were used as selection markers during the chromosomal integration. The optimal temperature and pH of the fusion protein CotZ-DPEase were 55 °C and pH 7.5-8.0, respectively, and the anchored DPEase exhibited high thermostability. Under optimal conditions, 30 g/L of recombinant spores can produce 85 g/L d-allulose from 500 g/L d-fructose after 12 h, and 60% of the yield was maintained after five cycles of utilization. Therefore, this biocatalyst system, capable of expressing and immobilizing DPEase on the spore surface of B. subtilis, was an appropriate alternative for d-allulose production.