UDC 577.150.6
ISOLATION AND CHARACTERIZATION OF FEATHER DEGRADING ENZYMES FROM Bacillus megaterium SN1 ISOLATED FROM GHAZIPUR POULTRY WASTE SITE
© 2012 S. Agrahari, N. Wadhwa
Department of Biotechnology, Jaypee Institute of Information Technology University, Uttar Pradesh, India
e-mail: neeraj.wadhwa@jiit.ac.in Received Junuary 25, 2011
The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30°C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn2+ whereas Ba2+ and Hg2+ inhibited it. Ratio of milk clotting activity to caseinolytic was found to be 520.8 activity for the 30—60% ammonium sulphate fraction in presence of Mn2+ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.
Milk-clotting enzymes, isolated from microbial sources Endothiaparasitica, Bacillus cereus, Mucor pu-sillus lindt and Mucor miehei are used and reported in production of cheese, cottage cheese, sour cream and Emmentaler cheese. The major application of proteases in the dairy industry is in the manufacture of cheese. The milk-coagulating enzymes fall into three main categories, - animal rennets, microbial milk coagulants, and genetically engineered chymosin. In food industry, rennet prepared from the abomasum (fourth stomach from unweaned calves) is used in the production of cheese. Its supply has become less available and expensive. The shortage of calf's rennet has also highly increased due to religious restriction and ethnic regulations against the use of animal secretion in food.
Most commercial proteases (mainly neutral and alkaline) are produced by organisms belonging to the genus Bacillus. Bacterial neutral proteases are active in a narrow pH range (pH 5.0 to 8.0) and have relatively low thermotolerance. Due to their intermediate rate of reaction, neutral proteases generate less bitterness in hydrolyzed food proteins than the animal proteinases and hence they are valuable for use in the food industry. A world shortage of calf rennet due to the increased demand for cheese production has intensified the search for alternative microbial milk coagulants too. The keratinases (EC 3.4.99.11) belong to the group of hydrolases that are important for hydrolyzing feather, hair, wool, collagen and casein. They are large serine or metalloproteases capable of degrading the structure
forming keratinous proteins. Keratin chain is very tightly packed in the a-helix (a-Keratin) and P-sheets (P-keratin) into super-coiled polypeptide chain [1] and produces mechanical stability resistant to common proteolytic enzymes such as pepsin, trypsin and papain. Keratinolytic enzymes are known to have important use in biotechnological processes involving keratin-containing waste from poultry and leather industries, through the development of non-polluting processes [2, 3]. After hydrolysis, the feather can be converted to feed stuffs, fertilizers, glues and films [4].
The aim of the study is to isolate and to characterize extracellular proteases and keratinases by Bacillus megaterium SN1 that can degrade the poultry waste feather and clot milk thus having potential application in bioremediation of feather waste and dairy industry.
MATERIALS AND METHODS
Selection of protease-producing strains on the skim milk agar. Soil isolates showing maximum protease activity were plated on the skim milk agar (10% skim milk powder, 0.1% peptone, 0.5% NaCl and 2% agar). Plates were incubated at 37°C for 24 h and the colonies that showed clear zone were selected and subcul-tured in the LB broth. The bacterial isolate was further incubated in cultivation media checked for protease activity.
Morphological studies of isolated bacterial strains.
Bacterial strain of Bacillus megaterium was identified, maintained and kept as glycerol stock. Bacterial iden-
tification was conducted by morphological, cultural and biochemical tests. Results were compared with Bergey's Manual [5] and Genus Bacillus: Agriculture Handbook [6]. The strain was also identified by chro-mogenic method on the bacillus differential agar M1651 from Himedia (India), recommended for rapid identification of Bacillus species from a mixed culture [7].
Production of enzyme in cultivation media. Seed culture of B. megaterium were prepared in 500 ml Erlenmeyer conical flask containing 100 ml of feather meal medium that composed of (g/l): NH4Cl — 0.5; NaCl - 0.5; K2HPO4 - 0.3; KH2PO4 - 0.4; MgCl2 • 6H2O — 0.1; yeast extract — 0.1 and 10% washed feather, pH 7.5. Cultivation was performed at 30°C at 160 rpm for 72 h and the fresh overnight culture was inoculated in cultivation media. Pigeon feathers (10%), hair (10%) or nail (10%) were also used instead of chicken feathers (10%) to compare the growth of B. megaterium as well as enzyme production after 7 days. Biomass of bacteria was monitored by taking absorbance at 600 nm on spectrophotometer.
Purification of enzyme. Feather meal media with pigeon feather as substrate was selected for keratinase and protease production, the broth was harvested in 72 h of the growth for the enzyme assay. Isolated B. megaterium SN1 was allowed to grow in 500 ml conical flask containing 100 ml of the culture medium at 30°C at 160 rpm for 72 h and fresh culture was inoculated in cultivation media. Cells were harvested by centrifugation (10.000 g, 4°C, 10 min). The 30-60% ammonium sulfate precipitate was obtained from the cell free crude culture broth. The resulting precipitate was collected by centrifugation (10.000 g, 4°C, 30 min) and dissolved in a minimal volume of 10 mM Tris-HCl buffer (pH 8.0) and dialyzed against the same buffer overnight. Then dialysate was loaded on 10 ml Q Sepharose. The 2-4 mM NaCl eluate was collected and protein, protease and keratinase activity were detected in it. All the fractions with high enzyme activity were separately pooled, dialyzed, concentrated by lyo-philization and used for further studies.
Determination of keratinase activity. The kerati-nase activity was assayed by the modified method of Cheng et al. [8] by using keratin as a substrate. The reaction mixture contained 200 ^l of enzyme preparation and 800 ^l of 20 ^g/ml keratin in 10 mM Tris-HCl buffer, pH 8.0. The reaction mixture was incubated at 45°C for 20 min and the reaction was terminated by adding 1 ml of10% chilled trichloroacetic acid. The mixture was centrifuged at 10.000 g for 5 min and the absorbance of the supernatant fluid was determined at 440 nm. All assays were done in triplicate. One unit (U) of enzyme activity was the amount of enzyme that caused a change of 0.01 of absorbance unit at 440 nm in 20 min at 45°C.
Determination of protease activity. Protease activity was assayed in the various fractions by a modified
method of Tsuchida et al. [9] by using casein as substrate. 100 ^l of the enzyme solution was added to 900 ^l of substrate solution (2 mg/ml casein in 10 mM Tris-HCl buffer, pH 8.0).The mixture was incubated at 50°C for 20 min. Reaction was stopped by the addition of an equal volume of 10% chilled trichloroacetic acid and then the reaction mixture was allowed to stand in ice for 15 min to precipitate the insoluble proteins. The supernatant was separated by centrifugation at 10.000 g for 10 min at 4°C; the acid soluble product in the supernatant was neutralized with 5 ml of 0.5 M Na2CO3 solution. The color developed after adding 0.5 ml of 3 fold diluted Folin-Ciocalteau reagent was measured at 660 nm. All assays were done in triplicate. One protease unit was defined as the amount of enzyme that releases 1 ^mol of tyrosine per ml per minute. The specific activity was expressed in the units of enzyme activity per mg of protein.
Determination of milk-clotting activity. It was determined according to the method of Arima [10], which is based on the visual evaluation of the appearance of the first clotting flakes, and expressed in terms of Soxhlet units (SU). One SU is defined as the amount of enzyme which clots 1 ml of a solution containing 0.1 g of the skim milk powder in 40 min at 35°C. In brief, 0.5 ml of tested materials was added to a test-tube containing 5 ml of the reconstituted skim milk solution (10 g of dry skim milk in 100 ml of 10 mM CaCl2 and 10 mM MnSO4) preincubated at 35°C for 5 min. The mixture was mixed well and the clotting time T (s) (the time period starting from the addition of test material to the first appearance of clots of milk solution) was recorded and the clotting activity was calculated using the following formula:
SU = 2400 x 5 x D/T x 0.5; where T - clotting time (s) and D - dilution of the test material.
Protein concentration. Protein concentration of all the crude and dialyzed fractions of 0-30% and 3060% ammonium sulphate was determined by the method of Bradford with bovine serum albumin as a standard [11].
Polyacrylamide gel electrophoresis and zymogra-phy. SDS-PAGE was performed on a slab gel containing 10% (w/v) polyacrylamide by silver staining according to the method ofSwitzer et al. [12]. Casein zy-mography was performed in polyacrylamide slab gels containing SDS and casein (0.12% w/v) as co-polymerized substrate, as described by Choi
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