Taha I. Zaghloul

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains.

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.

Enhanced production of lipase by the thermophilic Geobacillus stearothermophilus strain-5 using statistical experimental designs

Statistically based experimental designs were applied to optimize the cultural conditions for the production of a glycerol-inducible lipase from the thermophilic Geobacillus stearothermophilus strain-5. The effect of nineteen culture conditions on enzyme production was evaluated using Plackett-Burman factorial design. Tween 80, K(2)HPO(4), glycerol and glucose were the most significant factors in improving enzyme production. The selected parameters were then further investigated using central composite design to define the optimal process conditions. Maximal enzyme activity (578 U/ml) was reached under the following conditions: glycerol, 2.24% (v/v); Tween 80, 0.76% (v/v); glucose, 0.76% (w/v) and K(2)HPO(4), 0.38% (w/v) which is about five folds the activity in basal medium. A verification experiment was carried out to examine model validation and revealed more than 98% validity.

Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor.

Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully carried out in 14L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were inoculated with activated bacterial cells. The fermentation processes were conducted at 37°C, 700 rpm agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released feathers hydrolysis end products [soluble proteins and NH(2)-free amino groups] and keratinolytic alkaline protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the transfer to larger scale fermentors to carry out feathers biodegradation process.

Isolation, Identification, and Keratinolytic Activity of Several Feather-Degrading Bacterial Isolates

Several feather-degrading bacterial isolates were isolated from Egyptian soil. These isolates were able to degrade chicken feather, when grown on basal medium containing 1% native feather as a source of energy, carbon, and nitrogen. Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is nearly pure keratin, which is not easily degradable by common proteolytic enzymes. The isolates were identified according to the morphological characteristics, biochemical tests, and API 50 CHB Bacillus system. Proteolytic and keratinolytic activities of these isolates were monitored throughout the cultivation of the bacterial isolates on feather. Resulting soluble proteins, which were released as a result of the biodegradation of feather, were demonstrated by SDS-PAGE.

Cloned Bacillus subtilis Alkaline Protease (aprA) Gene Showing High Level of Keratinolytic Activity

The Bacillus subtilis alkaline protease (aprA) gene was previously cloned on a pUB11O-derivative plasmid. High levels of expression and gene stability were demonstrated when B. subtilis cells were grown on the laboratory medium 2XSG. B. subtilis cells harboring the multicopy aprA gene were grown on basal medium, supplemented with 1% chicken feather as a source of energy, carbon, and nitrogen. Proteolytic and keratinolytic activities were monitored throughout the cultivation time. A high level of keratinolytic activity was obtained, and this indicates that alkaline protease is acting as a keratinase. Furthermore, considerable amounts of soluble proteins and free amino acids were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these cells represents an alternative way to improve the nutritional value of feather, since feather waste is currently utilized on a limited basis as a dietary protein supplement for animal feedstuffs. Moreover, the release of free amino acids from feather and the secreted keratinase enzyme would promote industries based on feather waste.

High level of expression and stability of the cloned alkaline protease (aprA) gene in Bacillus subtilis.

Gene expression and plasmid stability of the cloned alkaline protease (aprA) gene in Bacillus subtilis were investigated. B. subtilis cells harboring the multicopy aprA gene were grown on sporulation medium and the activity of the alkaline protease was monitored throughout the cultivation time. Results presented indicate that the expression of the aprA gene occurred late during the stationary phase and the plasmid that carries the aprA gene was segregationally and structurally stable.

Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain

OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150xa0rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH4Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

Recycling of Keratin-containing Materials (Chicken Feather) Through Genetically Engineered Bacteria

Abstract Chicken feather is mainly keratin which is not normally degraded by common proteolytic enzymes. It is generated in large quantities as a by-product of poultry processing industry and little is known about its recycling. We reported the biodegradation of chicken feather waste (CFW) by a recombinant Bacillus subtilis strain harboring a multicopy alkaline protease (aprE) gene which acted as a keratinase. In present work, degradation of CFW was greatly enhanced by optimizing several factors. Pretreatment of CFW with NaOH or two times autoclaving enhanced the process. Cultures supplemented, separately, with 0.1% yeast extract or 0.5% corn oil enhanced the degradation of CFW. Biodegradation was optimized when the cultures contained 2% (w/v) CFW, 5% inoculum size, and incubated at 45°C for 3–4 days. Biodegradation was evaluated by monitoring soluble proteins and NH2-free amino groups that were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these recombinant cells represents an alternative way to improve the nutritional value of feather. Moreover, the release of soluble proteins and amino acids from feather as well as the secreted keratinase enzyme would promote several industries based on feather waste.

Water Splitting for High‐Yield Hydrogen Production Energized by Biomass Xylooligosaccharides Catalyzed by an Enzyme Cocktail

Green hydrogen production through water splitting at low temperatures is highly desired for hydrogen economy. Herein, we demonstrate an inu2005vitro non‐natural enzymatic pathway to utilize the chemical energy stored in xylooligosaccharides from biomass to split water to produce a nearly theoretical yield of H2 (i.e., ≈9.5 H2 per xylose plus water). This pathway was constructed on the basis of the novel activities of phosphopentomutase catalyzing the conversion of d‐xylose 1‐phosphate into d‐xylose 5‐phosphate and of ribose 5‐phosphate isomerase catalyzing the conversions of d‐xylose 5‐phosphate and d‐xylulose 5‐phosphate. This study suggests that the discovery of novel promiscuous enzyme activities is important to implement complicated biotransformations catalyzed by synthetic enzymatic pathways.

Enhanced stability of the cloned Bacillus subtilis alkaline protease gene in alginate-immobilized B. subtilis cells

Expression and stability of the cloned Bacillus subtilis alkaline protease (aprE)gene was monitored throughout the growth of free and alginate-immobilized B. subtilis cells. The time as well as the level of expression of the aprE gene in alginate-immobilized cells was found to be close to that of free cells. The multicopy plasmid that carries the aprE gene was stably maintained in alginate-immobilized cells. Plasmid stability was greatly enhanced, it reached 83% and 8% after ten growth cycles for alginate-immobilized and free cells in the absence of stress, respectively. Data presented demonstrate that immobilization of B. subtilis recombinant cells would partially solve the problem of plasmid instability in B. subtilis.