Recombinant Bacillus Subtilis Research Articles

Potential of Marine Bacterial Metalloprotease A69 in the Preparation of Peanut Peptides with Angiotensin-Converting Enzyme (ACE)-Inhibitory and Antioxidant Properties.

Marine bacterial proteases have rarely been used to produce bioactive peptides, although many have been reported. This study aims to evaluate the potential of the marine bacterial metalloprotease A69 from recombinant Bacillus subtilis in the preparation of peanut peptides (PPs) with antioxidant activity and angiotensin-converting enzyme (ACE)-inhibitory activity. Based on the optimization of the hydrolysis parameters of protease A69, a process for PPs preparation was set up in which the peanut protein was hydrolyzed by A69 at 3000 U g-1 and 60 °C, pH 7.0 for 4 h. The prepared PPs exhibited a high content of peptides with molecular weights lower than 1000 Da (>80%) and 3000 Da (>95%) and contained 17 kinds of amino acids. Moreover, the PPs displayed elevated scavenging of hydroxyl radical and 1,1-diphenyl-2-picryl-hydrazyl radical, with IC50 values of 1.50 mg mL-1 and 1.66 mg mL-1, respectively, indicating the good antioxidant activity of the PPs. The PPs also showed remarkable ACE-inhibitory activity, with an IC50 value of 0.71 mg mL-1. By liquid chromatography mass spectrometry analysis, the sequences of 19 ACE inhibitory peptides and 15 antioxidant peptides were identified from the PPs. These results indicate that the prepared PPs have a good nutritional value, as well as good antioxidant and antihypertensive effects, and that the marine bacterial metalloprotease A69 has promising potential in relation to the preparation of bioactive peptides from peanut protein.

Whole-cell biocatalysis for ε-poly-l-lysine production by a food-grade recombinant Bacillus subtilis

ε-Poly-l-lysine (ε-PL), a natural food preservative with various advantages, is primarily produced by Streptomyces. It has attracted considerable attentions for the outstanding antibacterial activity, safety, heat stability, water solubility and other remarkable properties. In this study, a food-grade recombinant Bacillus subtilis was constructed for the biocatalysis of ε-PL. Firstly, the d-alanine racemase gene (alrA) was deleted from the genome of Bacillus subtilis 168 to construct an auxotrophic B. subtilis 168 (alrA-). Based on the shuttle plasmid pMA5, a food-grade plasmid pMA5a was constructed by replacing the genes of kanamycin resistance (Kanr) and ampicillin resistance (Ampr) with alrA and the gene encoding α-peptide of β-galactosidase (lacZα), respectively. Subsequently, codon-optimized ε-PL synthase gene (pls) and P-pls were ligated into pMA5a and transformed in E. coli DH5α and expressed in B. subtilis 168 (alrA-). Finally, the whole-cell biocatalysis conditions for ε-PL production by B. subtilis 168 (alrA-)/pMA5a-pls were optimized, and the optimal conditions were 30°C, pH 4, l-lysine concentration of 0.6 g/L, bacterial concentration of 15 % (w/v) and a catalytic time of 7 h. The ε-PL production reached a maximum of 0.33 ± 0.03 g/L. The product was verified to be ε-PL by HPLC and tricine-SDS-PAGE. The information obtained in this study shows critical reference for the food-grade heterologous expression of ε-PL.

Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.

Design-build-test of recombinant Bacillus subtilis chassis cell by lifespan engineering for robust bioprocesses

Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. Bacillus subtilis is widely used in industry for its food safety properties, but challenges remain in the limitations of microbial fermentation. This study proposes a novel strategy based on lifespan engineering to design robust B. subtilis chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis, tolerate toxic substrates, and get a higher mass transfer efficiency. The modified chassis cells could produce high levels of l-glutaminase, and tolerate hydroquinone to produce α-arbutin efficiently. In a 5 L bioreactor, the l-glutaminase enzyme activity of the final strain CRE15TG was increased to 2817.4 ± 21.7 U mL−1, about 1.98-fold compared with that of the wild type. The α-arbutin yield of strain CRE15A was increased to 134.7 g L−1, about 1.34-fold compared with that of the WT. To our knowledge, both of the products in this study performed the highest yields reported so far. The chassis modification strategy described in this study can Improve the utilization efficiency of chassis cells, mitigate the possible adverse effects caused by excessive metabolic modification of engineered strains, and provide a new idea for the future design of microbial cell factories.

Recombinant Bacillus subtilis expressing functional peptide and its effect on the growth, antioxidant capacity and intestine in Megalobrama amblycephala

P4 is a functional small peptide screened after enzymatic hydrolysis of cotton meal. Bacillus subtilis CM66 (OB) underwent recombination to yield Bacillus subtilis CM66-P4’ (RB), capable of secreting P4. This study aimed to assess the effects of RB on the growth, antioxidant capacity and intestine in blunt snout bream (Megalobrama amblycephala) through in vitro and in vivo experiments. Seven groups were formed: a control group (CG) with no additional bacteria, three groups with different concentrations of Bacillus subtilis CM66 (2 × 107, 2 × 108, 2 × 109 CFU/kg - referred to as LOG, MOG, HOG), and three groups with corresponding concentrations of Bacillus subtilis CM66-P4’ (2× 107, 2 × 108, 2 × 109 CFU/kg - referred to as LRG, MRG, HRG). After 8 weeks of feeding, the results showed that OB and RB had no significant impact on growth performance. Compared with CG and MOG, the MRG group exhibited a significant increase in protease and total anti-oxidative capacity (T-AOC) in the intestine (P < 0.05). The mRNA expression of mucin-2 in MOG and MRG were significantly increased (P < 0.05). Compared with CG, muscle layer thickness (MLT) of MOG and MRG were significantly increased (P < 0.05), villus height (VH) and goblet cells numbers of MRG were increased (P < 0.05). Species richness and diversity of intestinal microbes were significantly increased in MOG and MRG (P < 0.05). P4 demonstrated a significant alleviation of the increase in sod expression in intestinal epithelial cells (IECs) treated with H2O2 and mitigated the decrease in cells activity (P < 0.05). In conclusion, P4 effectively mitigated the impact of H2O2 on IECs in blunt snout bream. Dietary supplementation of 2 × 108 CFU/kg RB (secreting P4) improved the intestinal microbes and intestinal barrier, resulting in enhanced antioxidant capacity.

Valorization of chicken feather waste using recombinant bacillus subtilis cells by solid-state fermentation for soluble proteins and serine alkaline protease production

Feather waste, a by-product of the poultry industry, is rich in proteins, peptides, and amino acids. Improper disposal of feathers can cause environmental pollution. Solid-state fermentation (SSF) is a viable alternative to submerged fermentation due to its simplicity, productivity, and lower cost. The study goal is a biorefinery of chicken feather waste supplemented with wheat bran using a recombinant Bacillus subtilis strain to produce soluble proteins and a serine alkaline protease. Plackett-Burman Design and Central Composite Design were utilized in a statistical-mathematical model to optimize the process. Multi-factorial design optimization resulted in 80 % substrate degradation efficiency, an alkaline protease with dual activities (1423 proteolytic units and 190 keratinolytic units), 214 mg soluble proteins/g substrate, and 87 % model validation. Scaling up the SSF process to 50 g of substrate significantly enhanced the end products of feather biodegradation to 1616 proteolytic units, 2844 keratinolytic units, and 127 mg soluble proteins/g substrate. Aim and scope of the manuscriptThe aim of the present study is to utilize chicken feather waste (alone or supplemented with other materials) through recombinant Bacillus subtilis cells using solid state fermentation (SSF) at a laboratory scale. The plan study provides a promising waste management in the environmental field concerning biodegradation of such recalcitrant keratinous wastes supplemented with agricultural residues via recombinant microorganism. On semi-pilot scale, high production and quality of soluble protein, protease, and keratinase activity were produced according to the statistically optimised first stage fermentation in the laboratory scale. The bioconversion process took place as a major goal to obtain valuable products, with low utilities and energy requirements. Therefore, this will consider as an economically feasible and environmentally friendly alternative. Moreover, this study is considered as first step fermentation for feather waste to pave the road for directing it to a second step fermentation for biogas production and bioenergy generation through bio-electrochemical systems (Manuscript under publication).

Both recombinant Bacillus subtilis Expressing PCV2d Cap protein and PCV2d-VLPs can stimulate strong protective immune responses in mice

Porcine circovirus type 2 (PCV2) is one of the most serious pathogens in pig herds worldwide. The Capsid protein (Cap), a structural protein of PCV2, is involved in the host's immune response; it induces neutralizing-antibody production and has good immunogenicity. The main PCV2 subtype currently prevalent in the Chinese pig herd is PCV2d. In this study, We constructed a recombinant Bacillus subtilis (B. subtilis) capable of secreting Cap protein, named pHT43-Cap/B. subtilis; we concentrated the supernatant of the recombinant bacteria and observed virus-like particles (VLPs) of PCV2d formed by Cap protein under transmission electron microscopy, named PCV2d-VLPs. The immunocompetence of the pHT43-Cap/B. subtilis and PCV2d-VLPs were then assessed by oral administration and by intramuscular injection into mice, respectively. The results showed that the levels of PCV2d-Cap protein-specific IgG in the serum and of PCV2d-Cap protein-specific sIgA in the small intestinal fluid of pHT43-Cap/B. subtilis immunized mice were elevated compared to the control group, both of them highly significant (p < 0.01), and the corresponding serum-specific IgG antibodies were effective in neutralizing PCV2d virulence. The virus load in the liver of the immunized mice was significantly lower than that in the control group (p < 0.01), as was the virus load in the spleen and lungs of the immunized mice (p < 0.05). In addition, the serum levels of PCV2d-Cap-specific IgG in mice immunized with PCV2d-VLPs by intramuscular injection were significantly elevated compared to the control group (p < 0.05), and the viral load in all tissues was significantly lower in immunized mice (p < 0.05). In conclusion, the recombinant bacterium pHT43-Cap/B. subtilis can induce effective mucosal and humoral immunity in mice, PCV2d-VLPs can induce humoral immunity in mice, and both vaccines have good immunogenicity; these results provide a theoretical and material basis for the development of a new vaccine against PCV2d.

Environmentally Responsible Bioengineering for Spore Surface Expression of Helicobacter pylori Antigen

The development of genetic technologies and bioengineering are creating an increasing number of genetically engineered microorganisms with new traits for diverse industrial applications such as vaccines, drugs and pollutant degraders. However, the destiny of genetically engineered bacterial spores released into the environment as long-life organisms has remained a big environmental challenge. In this study, an environmentally responsible and sustainable gene technology solution based on the concept of thymine starvation is successfully applied for cloning and expression of a Helicobacter pylori antigen on Bacillus subtilis spore surface. As an example, a recombinant Bacillus subtilis strain A1.13 has been created from a gene fusion of the corresponding N-terminal fragment of spore coat protein CotB in B. subtilis and the entire urease subunit A (UreA) in H. pylori and the fusion showed a high stability of spore surface expression. The outcomes can open the door for developing highly safe spore vectored vaccines against this kind of pathogen and contributing to reduced potential risks of genetically engineered microorganisms released in the environment.

Creating a Vaccine-like Supplement against Respiratory Infection Using Recombinant Bacillus subtilis Spores Expressing SARS-CoV-2 Spike Protein with Natural Products.

Vaccination is the most effective method of combating COVID-19 infection, but people with a psychological fear of needles and side effects are hesitant to receive the current vaccination, and alternative delivery methods may help. Bacillus subtilis, a harmless intestinal commensal, has recently earned a strong reputation as a vaccine production host and delivery vector, with advantages such as low cost, safety for human consumption, and straightforward oral administration. In this study, we have succeeded generating "S spores" by engineering B. subtilis with spore coat proteins resembling the spike (S) protein of the ancestral SARS-CoV-2 coronavirus. With the addition of two immunostimulating natural products as adjuvants, namely Astragalus membranaceus (Fisch.) Bge (AM) and Coriolus versicolor (CV), oral administration of S spores could elicit mild immune responses against COVID-19 infection without toxicity. Mucosal IgA against the S protein was enhanced by co-feeding with AM and CV in an S spores-inoculated mouse model. Faster and stronger IgG responses against the S protein were observed when the mice were fed with S spores prior to vaccination with the commercial COVID-19 vaccine CoronaVac. In vitro studies demonstrated that AM, CV, and B. subtilis spores could dose-dependently activate both macrophages and dendritic cells by secreting innate immunity-related IL-1β, IL-6, and TNF-α, and some other proinflammatory chemokines and cytokines. In conclusion, the combination of S spores with AM and CV may be helpful in developing a vaccine-like supplement against respiratory infection.

Construction of recombinant SAG22 Bacillus subtilis and its effect on immune protection of coccidia

Avian coccidiosis causes huge economic losses to the global poultry industry. Vaccine is an important means to prevent and control coccidiosis. In this study, Bacillus subtilis was selected as the expression host strain to express anti Eimeria tenella surface protein SAG22. The synthesized surface protein SAG22 gene fragment of E. tenella was ligated with Escherichia coli-bacillus shuttle vector GJ148 to construct the recombinant vector SAG22-GJ148. And then the recombinant Bacillus strain SAG22-DH61 was obtained by electrotransfer. The results of SDS-PAGE and Western Blot showed that the recombinant protein SAG22 was successfully expressed intracellularly. The immunoprotective effect of recombinant Bacillus strain SAG22-DH61 on broiler chickens was evaluated in 3 identically designed animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. Each batch of experiments was divided into 4 groups: blank control group (NC), blank control group+infected E. tenella (CON), addition of recombinant SAG22-DH61 strain+infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain+infected with E. tenella (GJ148-DH61). The animal experiments results showed that the average weight gain of the SAG22-DH61 group was higher than that of the infected control group, and the difference was significant in the d 14 and 28 attack tests (P < 0.05); the oocyst reduction rate of the SAG22-DH61 group was much higher than that of the GJ148-DH61 group (P < 0.05); the intestinal lesion count score of the SAG22-DH61 group was much lower than that of the GJ148-DH61 group (P < 0.05). In addition, the SAG22-DH61 group achieved highly effective coccidia resistance in the d 14 attack test and moderately effective coccidia resistance in both the d 21 and 28 attack tests. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

Preparation and characterization of silica nanoparticles as an efficient carrier for two bio‐detergents based enzymes

AbstractBio‐detergents are new bio‐friendly formulas that contain biobased ingredients, including enzymes. In the present study, alkaline protease and α‐amylase were immobilized via physisorption onto silica nanoparticles (SNPs). The derivatized SNPs served as major components of a prepared bio‐detergent. Alkaline protease was produced by the recombinant Bacillus subtilis cells that carry the protease genes on a multiple‐copy plasmid, while α‐amylase was commercially purchased. SNPs were prepared by the sol–gel method and well‐characterized through the Brunauer–Emmett–Teller (BET) method, scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta potential, X‐ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The adsorption capacity of the SNPs was determined via colorimetry through the adsorption of methylene blue dye (MB), with approximately 97% adsorption achieved under the conditions employed. The Langmuir isotherm well‐described the adsorption of MB on SNPs. High immobilization yield for the enzymes was obtained, and the storage stability of SNP‐alkaline protease and SNP‐α‐amylase was good, reaching 65% and 85% of their initial activities after 6 weeks of storage at 4°C, respectively. The immobilized enzymes could be reused for 7 cycles. Additionally, the immobilized enzymes retained residual activity to a greater extent than free enzymes in simulated basic detergent solutions. SNPs containing adsorbed alkaline protease and α‐amylase were mixed with a basic detergent solution, and the washing efficiency of some proteinous and starchy stains was examined through Hunter Lab spectrophotometry. The latter experiments demonstrated that the immobilized enzymes performed well during the washing process.

Bacillus subtilis vector based oral rabies vaccines induced potent immune response and protective efficacy in mice.

Rabies is a worldwide epidemic that poses a serious threat to global public health. At present, rabies in domestic dogs, cats, and some pets can be effectively prevented and controlled by intramuscular injection of rabies vaccine. But for some inaccessible animals, especially stray dogs, and wild animals, it is difficult to prevent with intramuscular injection. Therefore, it is necessary to develop a safe and effective oral rabies vaccine. We constructed recombinant Bacillus subtilis (B. subtilis) expressing two different strains of rabies virus G protein, named CotG-E-G and CotG-C-G, immunogenicity was studied in mice. The results showed that CotG-E-G and CotG-C-G could significantly increase the specific SIgA titers in feces, serum IgG titers, and neutralizing antibodies. ELISpot experiments showed that CotG-E-G and CotG-C-G could also induce Th1 and Th2 to mediate the secretion of immune-related IFN-γ and IL-4. Collectively, our results suggested that recombinant B. subtilis CotG-E-G and CotG-C-G have excellent immunogenicity and are expected to be novel oral vaccine candidates for the prevention and control of wild animal rabies.

Dietary addition of recombinant Bacillus subtilis expressing a fungal phytase increases phosphorus fixation in muscle of Pacific white shrimp Litopenaeus vannamei

Dietary addition of recombinant Bacillus subtilis expressing a fungal phytase increases phosphorus fixation in muscle of Pacific white shrimp Litopenaeus vannamei

Oral administration of recombinant Bacillus subtilis expressing a multi-epitope protein induces strong immune responses against Salmonella Enteritidis

The S. Enteritidis causes serious economic losses to the poultry industry every year. Vaccines that induce a mucosal immune response may be successful against an S. Enteritidis infection because mucosa plays an important role in preventing S. Enteritidis from entering the body. In order to develop novel and potent oral vaccines based on Bacillus subtilis (B. subtilis) to control the spread of S. Enteritidis in the poultry industry, we constructed a B. subtilis that can secrete a multi-epitope protein (OmpC-FliC-SopF-SseB-IL-18). Oral immunization of chickens was performed, and serum antibodies, mucosal antibodies, specific cellular immunity and serum cytokines were detected. Immunizing chicks with S. Enteritidis was evaluated. The results showed high levels of specific IgG in addition to high levels of specific secretory immunoglobulin A (sIgA) in chickens who received oral administrations of recombinant B. subtilis. Additionally, recombinant B. subtilis may significantly increase the levels of IL-2 and T cell-mediated immunity. Recombinant B. subtilis effectively protected chickens against S. Enteritidis and reduced pathological damage to the spleen and jejunum. Our study’s outcomes indicate that the expression of the multi-epitope protein OmpC-FliC-SopF-SseB-IL-18 by B. subtilis could generate a mucosal vaccine candidate for animals to defend against S. Enteritidis in the future.

Second-generation bioethanol production from phytomass after phytoremediation using recombinant bacteria-yeast co-culture

• P. vittata phytomass can be a promising feedstock for bioethanol production. • Shaking with 1% HNO 3 pretreatment removed greater than 80% As from P. vittata phytomass. • Recombinant bacteria-yeast co-culture system resulted in 48.5% bioethanol yield. • Developed microbial system has a great potential for bioethanol production via CBP. Sustainable management of harvested hyperaccumulating plants used in phytoremediation of toxic metals is considered quite challenging. The biomass of hyperaccumulators can be an attractive feedstock for bioethanol production, while removal of toxic metals and efficient ethanol conversion systems are necessary to ensure safe and optimum bioethanol production. This study aimed to develop a compatible and efficient consolidated bioprocessing system (CBP) to produce bioethanol from phytomass of arsenic (As)-hyperaccumulator Pteris vittata . A recombinant kefir yeast Kluyveromyces marxianus (KR7 strain) contained six cellulase genes and two recombinant Bacillus subtilis (Type 1 and Type 2 strains) carrying cellulosomal genes in different arrangements were used, and the saccharification and fermentation of P. vittata phytomass were investigated through one-step (KR7 strain only) and co-culture ( B. subtilis + KR7 strain) methods. The results of reducing sugar showed that KR7 strain possesses higher saccharification ability than both B. subtilis strains. While the co-culture of B. subtilis Type 2 strain with KR7 strain gave the highest ethanol yield, which achieved 48.5% ethanol yield from P. vittata phytomass after 3 d cultivation. In addition, simple pretreatment by shaking P. vittata with 1% of HNO 3 efficiently removed more than 80% of As from the phytomass, suggesting its applicability and feasibility for efficient As extraction to ensure efficient and non-toxic ethanol production. Our results confirm that the developed recombinant bacteria-yeast co-culture possesses a great potential for optimum bioethanol production from As-hyperaccumulators-derived phytomass via CBP.

Fermentation optimization for production of lactoferrin N-lobe by recombinant Bacillus subtilis

To achieve an efficient preparation of lactoferrin N-lobe, we optimized the fermentation process for a recombinant Bacillus subtilis pMA0911-D60Y/Y92D producing lactoferrin N-lobe. The IOD of the lactoferrin N-lobe reached 68.03% under the optimized cultural conditions, that is using glucose and tryptone as the best carbon and nitrogen source, respectively, and conduct the fermentation under pH 7.0, 28 ℃, for 25.5 h. An optimized fermentation process was obtained through fermentation optimization on a 10 L fermenter. That is, culturing the recombinant strain at 30 ℃, pH 7.5 within 0-7 h, and switching to induction at 28 ℃, pH 7.5 within 7-25 h for production of lactoferrin N-lobe, using an agitation speed of 300 r/min throughout the fermentation. After the fermentation, the cells were collected and disrupted, followed by purification of the lactoferrin N-lobe to homogeneity by using HisTrap HP-affinity and a SuperdexTM 200 (10/300 GL)-affinity chromatography. The purified lactoferrin N-lobe proteins with over 94% purity were obtained. One liter culture of recombinant B. subtilis pMA0911-D60Y/Y92D produced 23.5 mg of pure protein. This study may facilitate the fermentative production of the recombinant lactoferrin N-lobe.

Hyaluronic acid production and characterization by novel Bacillus subtilis harboring truncated Hyaluronan Synthase

Hyaluronic Acid (HA) is a natural biopolymer that has important physiological and industrial applications due to its viscoelastic and hydrophilic characteristics. The responsible enzyme for HA production is Hyaluronan synthase (HAS). Although in vitro structure–function of intact HAS enzyme has been partly identified, there is no data on in vivo function of truncated HAS forms. In the current study, novel recombinant Bacillus subtilis strains harboring full length (RBSFA) and truncated forms of SeHAS (RBSTr4 and RBSTr3) were developed and HA production was studied in terms of titer, production rate and molecular weight (Mw). The maximum HA titer for RBSFA, RBSTr4 and RBSTr3 was 602 ± 16.6, 503 ± 19.4 and 728 ± 22.9 mg/L, respectively. Also, the HA production rate was 20.02, 15.90 and 24.42 mg/L.h−1, respectively. The findings revealed that RBSTr3 produced 121% and 137% more HA rather than RBSFA and RBSTr4, respectively. More interestingly, the HA Mw was about 60 kDa for all strains which is much smaller than those obtained in prior studies.

Surface Display of Peptides Corresponding to the Heptad Repeat 2 Domain of the Feline Enteric Coronavirus Spike Protein on Bacillus subtilis Spores Elicits Protective Immune Responses Against Homologous Infection in a Feline Aminopeptidase-N-Transduced Mouse Model.

Although feline coronavirus (FCoV) infection is extremely common in cats, there are currently few effective treatments. A peptide derived from the heptad repeat 2 (HR2) domain of the coronavirus (CoV) spike protein has shown effective for inhibition of various human and animal CoVs in vitro, but further use of FCoV-HR2 in vivo has been limited by lack of practical delivery vectors and small animal infection model. To overcome these technical challenges, we first constructed a recombinant Bacillus subtilis (rBSCotB-HR2P) expressing spore coat protein B (CotB) fused to an HR2-derived peptide (HR2P) from a serotype II feline enteric CoV (FECV). Immunogenic capacity was evaluated in mice after intragastric or intranasal administration, showing that recombinant spores could trigger strong specific cellular and humoral immune responses. Furthermore, we developed a novel mouse model for FECV infection by transduction with its primary receptor (feline aminopeptidase N) using an E1/E3-deleted adenovirus type 5 vector. This model can be used to study the antiviral immune response and evaluate vaccines or drugs, and is an applicable choice to replace cats for the study of FECV. Oral administration of rBSCotB-HR2P in this mouse model effectively protected against FECV challenge and significantly reduced pathology in the digestive tract. Owing to its safety, low cost, and probiotic features, rBSCotB-HR2P is a promising oral vaccine candidate for use against FECV/FCoV infection in cats.

Safety and Immunogenicity of Inactivated Bacillus subtilis Spores as a Heterologous Antibody Booster for COVID-19 Vaccines.

The coronavirus diseases 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have threatened the world for more than 2 years. Multiple vaccine candidates have been developed and approved for emergency use by specific markets, but multiple doses are required to maintain the antibody level. Preliminary safety and immunogenicity data about an oral dose vaccine candidate using recombinant Bacillus subtilis in healthy adults were reported previously from an investigator-initiated trial in Hong Kong. Additional data are required in order to demonstrate the safety and efficacy of the candidate as a heterologous booster in vaccinated recipients. In an ongoing, placebo-controlled, observer-blinded, fixed dose, investigator-initiated trial conducted in the Macau, we randomly assigned healthy adults, 21 to 62 years of age to receive either placebo or a Bacillus subtilis oral dose vaccine candidate, which expressed the spike protein receptor binding domain of SARS-CoV-2 on the spore surface. The primary outcome was safety (e.g., local and systemic reactions and adverse events); immunogenicity was a secondary outcome. For both the active vaccine and placebo, participants received three courses in three consecutive days. A total of 16 participants underwent randomization: 9 participants received vaccine and 7 received placebo. No observable local or systemic side-effect was reported. In both younger and older adults receiving placebo, the neutralizing antibody levels were gradually declining, whereas the participants receiving the antibody booster showed an increase in neutralizing antibody level.

Investigating the Bioconversion of Tyrosol to Hydroxyltyrosol by Recombinant Bacillus Subtilis Spores and the Anticancer Effects of Hydroxytyrosol on MCF-7

چکیده: مقدمه: تیروزول (2و 4- هیدروکسی فنیل اتانول ) از ترکیبات فنلی طبیعی موجود در زیتون است که اثرات آنتی اکسیدانی و ضد سرطانی آن شناخته شده است. بخش قابل توجهی از این ترکیبات فنلی در فرایند تولید روغن از زیتون خارج شده و وارد آب استفاده شده در فرایند و نهایتا وارد پساب کارخانه می‌شوند. در حالی که می‌توان این ترکیبات را به مواد فراسودمند و مکمل‌های غذایی تبدیل کرد. در این مطالعه تیروزول در اثر تبدیل زیستی با استفاده از آنزیم تیروزیناز تثبیت شده در سطح اسپور به هیدروکسی تیروزول تبدیل و اثر ضد سرطانی ترکیب حاصل بر روی سلول‌های سرطان سینه رده MCF-7 بررسی شد. مواد و روش‌ها: اسپورهای سویه نوترکیب Bacillus subtilis DB104 (pSDJH-cotE-tyr) که در پژوهش قبلی با استفاده از روش‌های مهندسی ‌ژنتیک و تکنیک نمایش پروتئین در سطح ایجاد شده بود، در محیط اسپورزایی تهیه شد. با استفاده از اسپورها واکنش تبدیل زیستی تیروزول به هیدروکسی تیروزول انجام و روند واکنش با استفاده از کروماتوگرافی مایع با فشار بالا مورد ارزیابی قرار گرفت. اثر ضد سرطانی هیدروکسی تیروزول بر روی رده سلولی MCF-7 با استفاده از فلوسیتومتری مطالعه شد. نتایج: نتایج حاصل از کروماتوگرافی مایع با فشار بالا نشان داد که، 1 میلی‌مولار از تیروزول پس از 60 دقیقه توسط تیروزیناز بیان شده در سطح اسپور به 1 میلی مولار هیدروکسی تیروزول تبدیل شد. همچنین اثرات ضدسرطانی هیدروکسی تیروزول حاصل بیش از 80 درصد در سلول‌های سرطانیMCF-7 پس از 48 ساعت مشاهده گردید. بحث و نتیجه گیری: براساس نتایج حاصل، ازتیروزیناز تثبیت شده درسطح اسپور می‌توان به عنوان آنزیمی فعال جهت تولید ترکیبات فرا سودمند با خاصیت ضد سرطانی از مشقات روغن زیتون استفاده کرد. اثرات ضد سرطانی هیدروکسی تیروزول تولید شده در اثر تبدیل زیستی تیرزول بر سلول‌های سرطانی MCF-7 ، بر اساس مطالعات انجام شده بر سایر سلول‌های سرطانی تائید شده است.