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

• 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.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

Nyckelord

bacillus licheniformis

bacillus megaterium

gene expression

keratinase

feather degradation

biogas production

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