Bacillus licheniformis

Example 4

Inactivation of Rghr2 Regulated Genes and Their Effect on Heterologous Protein Production

The Bli03644, abrB1, yvzC and abh genes were inactivated by insertion of antibiotic marker in a Bra7 strain producing a heterologous α-amylase (i.e., the heterologous P. curdlanolyticus α-amylase disclosed in PCT Publication No. WO2014/164834), wherein the heterologous α-amylase production was determined in the four single knock-out strains (ΔBLi03644, ΔabrB1, ΔyvzC and Δabh) and compared to the parental (control) strain as described in Example 2. For example, as presented in FIG. 7, inactivation of Bli03644, abrB1, yvzC and abh resulted in improved heterologous α-amylase production, while cell growth (OD₆₀₀) was less affected.

Example 1

In this example, the oligopeptide FTLE in chili pepper seeds was extracted as follows:

1) deseeding: fresh chili peppers were taken, and the flesh was separated from the seeds to obtain chili pepper seeds;

2) pulverizing: the chili pepper seeds were pulverized and sieved by an 80 mesh to obtain chili pepper seed powder ;

3) degreasing: the chili pepper seed powder was mixed with n-hexane at a ratio of 1:10 (g/ml); the mixture was stirred and degreased overnight; n-hexane was removed by suction filtration after the degreasing was completed to obtain a chili pepper seed meal;

4) protein extraction: the degreased chili pepper seed meal was dissolved in water at a ratio of 1:10 (w/v, g/mL); the pH value of the solution was adjusted to 9.5 with a NaOH solution to conduct dissolving for 4 h; then the pH value of the solution was adjusted to 4.5 with HCl to conduct precipitating for 2 h; the reaction solution was centrifuged at 8,000 rpm for 20 min, and the precipitate was collected as a crude protein extract;

5) ultra-high pressure assisted enzymolysis: the protein isolated was dissolved in water, and was subjected to an ultra-high pressure treatment at 300 MPa for 30 min; then the product obtained by the ultra-high pressure treatment was subjected to an enzymolysis treatment, in which the enzyme was Bacillus licheniformis, the mass ratio of the enzyme to the substrate was 1:20 (w/w, g/g), the temperature was 40° C., the pH value was adjusted to 8 with 1 mol/L NaOH, and the enzymolysis treatment was performed for 3 h;

6) enzyme inactivation: at the end of the enzymolysis, the enzyme was inactivated at 90° C. for 10 min to obtain a chili pepper seed zymolyte solution;

7) isolation and purification of zymolyte: the chili pepper seed zymolyte solution was passed through a DEAE anion chromatography column, where the mobile phase included deionized water and NaCl; the eluent in a periodfrom 35 min to 45 min was collected; then, isolation and purification were conducted by an ODS-A reverse phase C18 column (hydrophobic column), where the mobile phase included deionized water and 50% methanol, and the eluent in a periodfrom 75 min to 90 min was collected. The peptide fragments in the obtained eluate were subjected to mass spectrometry identification analysis, and information of multiple peptide sequences was obtained.

Example 2

Chemical systhesis was conducted in accordance with the peptide sequences obtained by mass spectrometry identification analysis of Example 1 to obtain synthetic peptides. The effect of each peptide on HepG2 cell proliferation was studied, and the specific steps were as follows:

1) HepG2 cell culture: hepG2 cells were obtained from the ATCC cell bank and were cultured in a DMEM medium containing 10% FBS at 37° C. in a 5% CO₂ cell incubator. Cells were cultured in a 25 cm ² flask, passaged when cells were grown to a density of 70% to 90%, and seeded in a 96-well plate.

2) Peptide fragment treatment: after 24 hours of cell culture in the 96-well plate, the original DMEM medium was aspirated from the wells. DMEM containing peptide fragments at concentrations of 0.1, 0.3, and 0.6 mM were added to each well to continue culturing for 24 hours.

3) Cell proliferation rate measured by MTT method: MTT at a concentration of 5 mg/mL was added to a 96-well plate in 20 μL per well. After incubation for 4 hours, the liquid was aspirated from each well. 150 μL DMSO was added to each well. The absorbance was measured after reacting for 20 min.

The results are shown in the figure. It can be seen that the oligopeptide FTLE has a better HepG2 cell inhibition rate than other oligopeptides, which is helpful for the prevention or treatment of liver cancer.

In the description of this specification, descriptions with reference to the terms “one embodiment”, “some embodiments”, “example”, “specific examples”, or “some examples”, etc. mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this description, schematic representations of the terms above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. The different embodiments or examples and the features of the different embodiments or examples described in this description can be integrated and combined by a person skilled in the art without contradicting each other.

While embodiments of the present disclosure have been shown and described, it will be understood that the above-described embodiments are illustrative and not restrictive and that changes, modifications, substitutions, and variations may be made to the embodiments by those skilled in the art without departing from the scope of the present disclosure.

Example 4

This example illustrates that heat-killed inoculum of Bacillus licheniformis strain ECOBIO_2 does not effectuate lead remediation.

To evaluate the involvement of metabolic process in the bio-precipitation of lead (Pb²⁺), 25 ml of LBP broth with bacterial cells of early lag phase was autoclaved at 121° C. pressure at 20 minutes to kill the cells. Thereafter, the dead cells were harvested by centrifugation at 10,000 rpm for 10 minutes. Harvested cells were inoculated on pre-sterilized LB agar plates to ensure the cellular viability. After 24 hours of incubation by ensuring no growth on LB plates harvested dead cells were used as inoculum. Results shown in FIG. 5F indicated that heat killed bacteria are not able to mediate lead precipitation.