| 1. |
- Alinezhad, S., et al.
(författare)
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Expression of keratinase gene in Bacillus megaterium using an expression vector of pHIS1525.SPlipA and utilization of the resulting recombinant strain for chicken feather degradation prior to biogas production
- 2009
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- An increasing quantity of chickens is being utilized annually in the poultry industry, producing a huge volume of chicken feather waste which presents a high quality supply of keratin. Keratinases possessing high level of keratinolytic activity on insoluble keratin play a crucial role in hydrolyzing chicken feathers. Ever since the discovery of proteolytic ability as well as water solubility of keratinase, many industrial processes regarding keratinase application have been developed. A recently invented application to handle poultry waste is to utilize feathers for biogas production. Obviously, large amount of keratinase is required to break down the keratin prior to further conversion to biogas. Previously, several researches have shown that certain bacteria are able to produce keratinase but it is still a challenge to find out which bacteria is the most reliable source for the production with high efficiency. These challenges gave rise to the molecular biologists to bring the focus on gene cloning to develop recombinant strains resulting in overproduction of keratinase. Over the course of various cloning and expression experiments of similar proteins, it was found that Bacillus megaterium could be a susceptible host cell for keratinase production. In our study, the keratinase gene from the chromosomal DNA of Bacillus licheniformis ATCC®53757 was PCR amplified and subsequently cloned into Bacillus megaterium expression vector, pHIS1525.SPlipA. Bacillus megaterium ATCC®14945 strain was transformed with the recombinant plasmid, pKERHIS1525.SPlipA. The KER gene was expressed under xylose inducible promoter, and the product was then purified using Ni-NTA affinity chromatography. After 18 h of incubation an extracellular keratinase activity of 29U ml-1 was achieved (one unit of activity was determined as the amount of enzyme required to an increase of 0.01 in A420 after 30 min of incubation at 37°C). The recombinant strain was further examined for feather degradation using intact chicken feather waste as carbon source. The chopped chicken feathers were partially degraded by the recombinant strain after three days of incubation and the total macroscopic digestion was ultimately observed after seven days resulting in a yellowish peptide rich fermentation broth. The biogas potential of the hydrolysate will be compared with that of untreated feathers by performing anaerobic batch digestion experiments.
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| 2. |
- Forgács, Gergely, 1983, et al.
(författare)
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Biological treatment of chicken feather waste for improved biogas production
- 2011
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Ingår i: Journal of Environmental Sciences. - : Elsevier BV. - 1001-0742 .- 1878-7320. ; 23:10, s. 1747-1753
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Tidskriftsartikel (refereegranskat)abstract
- A two-stage system was developed which combines the biological degradation of keratin-rich waste with the production of biogas. Chicken feather waste was treated biologically with a recombinant Bacillus megaterium strain showing keratinase activity prior to biogas production. Chopped, autoclaved chicken feathers (4%, W/V) were completely degraded, resulting in a yellowish fermentation broth with a level of 0.51 mg/mL soluble proteins after 8 days of cultivation of the recombinant strain. During the subsequent anaerobic batch digestion experiments, methane production of 0.35 Nm(3)/kg dry feathers (i.e., 0.4 Nm(3)/kg volatile solids of feathers), corresponding to 80% of the theoretical value on proteins, was achieved from the feather hydrolyzates, independently of the pre-hydrolysis time period of 1, 2 or 8 days. Cultivation with a native keratinase producing strain, Bacillus licheniformis resulted in only 0.25 mg/mL soluble proteins in the feather hydrolyzate, which then was digested achieving a maximum accumulated methane production of 0.31 Nm(3)/kg dry feathers. Feather hydrolyzates treated with the wild type B. megaterium produced 0.21 Nm(3) CH(4)/kg dry feathers as maximum yield.
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| 3. |
- Mirabdollah, A., et al.
(författare)
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Optimization of a protoplast transformation method for Bacillus Subtilis, Bacillus megaterium, and Bacillus Cereus by a plasmid pHIS1525.SplipA
- 2009
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- During the past years of gene cloning studies, Escherichia coli has always been a foremost host cell for exogenous genes expressions owing to its high level of protein production and excretion. However, problems relating to low level of extracellular production of some proteins specially the accumulation of cloned proteases within the cells have moved the attentions from E.coli to bacilli bacteria such as B. megaterium, B.subtilis, and B.cereus due to their secretion ability of many different enzymes. Bacillus megaterium is widely used for high-level expression of heterologous proteins with little or no degradation. Bacillus subtilis is a naturally competent host cell for uptake of exogenous DNA, resulting in attractive industrial applications. Bacillus cereus has sporulation capability which makes it suitable for several industrial uses. A conventional approach for transferring DNA into protoplasts or intact cells of bacillus bacteria is chemical transformation, using chemicals through chilling and then shock-heating of the suspension of cells to induce reversible permeabilization of the cell membrane to make it possible for the external DNA to enter into the cells. In most cloning experiments, the transformation with plasmid DNA is performed using Polyethylene glycol (PEG)-induced competence cells. In this study, a PEG-induced protoplast transformation protocol was developed for three different bacillus strains of Bacillus megaterium ATCC®14945, Bacillus Subtilis ATCC®6051, and Bacillus Cereus ATCC®14579. In all cases a plasmid pHIS1525.SPlipA, well working vector in B.megaterium, was applied. Protoplasts were formed in RHAF medium after treating the cells with lysozyme. Two factors, the incubation time and the lysozyme concentration have been found to play the most important role in effective protoplast formation. These two factors were further optimized in this study to elaborate a chemical transformation procedure which can possibly work for other bacillus strains as well. The optical density (A420) and the number of colony-forming units (CFUs) were determined to find the optimal conditions for each strain. The results indicate that PEG-induced protoplast transformation is a sufficient technique when using a plasmid pHIS1525.SPlipA in Bacillus genus.
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