Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans

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1 ISSN No: Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans V.PRIYANGA Department bio technology, Periyar University Salem *Corresponding author s Received: 23/12/2015, Revised: 27/02/2016 and Accepted: 29/04/2016 Abstract Feathers are rich in aminoacids and can be employed as a dietary protein supplement for animal feed. Microbial degradation of for improving their nutritional value. The feather sample were collected from hatchery near Namakkal, Bacillus cereus was isolated from poultry plant wastewater, showed high keratinolytic activity when cultured on feather meal medium. Optimum keratinolytic activity was observed at 40 o C and ph 8.0. The enzyme also showed to be stable between 40 and 50 o C. Degradation assay was done and it shows maximum degradation. SDS- PAGE was performed and the molecular weight of the protein is 108 kda. The Bacillus cereus isolated might be useful in leather, keratin waste treatment, animal feeding industry, and also cosmetic industry. Key words: *Reviewed by Organizing committee of 4 th National Conference On Emerging Trends and New Challenges in Biotechnology 1. INTRODUCTION 101

2 Keratins are the largest and most complex family of cytoskeletal intermediate filament proteins of animal cells, particularly epithelia [1]. Keratins are grouped into hard keratins (feather, hair, hoof and nail) and soft keratins (skin and callus) according to the sulfur content [2]. Hard keratins are insoluble and resistant to degradation by common proteolytic enzymes, such as trypsin, pepsin and papain because of their high degree of cross-linking by disulfide bonds, hydrogen bonding and hydrophobic interactions [3]. World-wide poultry processing plants produce millions of tons of feathers as a waste product annually, which consists of approximately 90% keratin. Feathers represent 5-7% of the total weight of mature chickens. These feathers constitute a sizable waste disposal problem. Several different approaches have been used for disposing of feather waste, including land filling, burning, natural gas production and treatment for animal feed[4,5,6]. The biotechnological processing of feathers for the production of feather meal, in contraposition to chemical processing, present advantages such the preservation of essential amino acids (methionine, lysine, histidine), found in sub-excellent levels[6,7]. Feathers consist primarily of keratin and are generated in large quantities as a waste byproduct in commercial poultry processing industries. The mechanical stability of keratin and its resistance to microbial degradation depend on the tight packing of the protein chain in α helix (α -keratin) and β sheet (β keratin) structures and their linkage by cystine bridges due to a high degree of cross-linkage by disulfide bonds, hydrogen bonding and hydrophobic interactions[8,9]. Many keratinases from species of Bacillus [10], Pseudomonas [11] fungi[12] and Actinomycetes[13] has been reported and some of them were purified and characterized. The aim of this study was to select keratin-degrading bacteria from JSC Biocentras collection and poultry processing plant wastewater, and to study their possibility to degrade chicken feathers. Keratin utilization has been reported in a wide variety of organisms including non-filamentous and filamentous bacteria [14, 15] helminthes, water moulds [16] and filamentous fungi. 102

3 The present study reports the isolation of a new Bacillus cereus with the ability to degrade raw feathers. The microorganism demonstrated a potential processes due to the high enzyme production and feather degradation. 2.Materials and Methods A. Sample Collection The feather samples were collected from Hatcheries Near Namakkal. They were stored in sample bags at low temperature. B. Isolation of Keratinolytic the Microorganism The samples were flooded in PBS saline solution 0.85%, after serially diluted 10ˉ6 dilution factor was used for isolation of keratinolytic bacteria. The samples were plated on feather meal agar and incubated at 37 C for 24 hours. After incubation the different colonies were isolated by pure culture methods. The dominant culture colonies were identified by biochemical techniques [17]. The morphological and physiological characteristics of the isolated bacterium were compared with Bergey's Manual of Systematic Bacteriology [18]. C. Growth Determination of Isolated Bacteria: The optimal growth conditions of the isolate were determined using feather meal agar, incubated an orbital shaker at 160 rev/min, in flasks containing 100 ml of the medium [19]. The following temperatures are used 30, 40, 50 C and ph; 6.0, 7.0, 8.0, 9.0. Bacterial growth was monitored by measuring the colony forming units (cfu). Samples of 4 ml were withdrawn at each 24 hours and diluted to 10-6 in saline solution (0.85) and loaded triplicate onto nutrient agar plates. Plates were incubated at 30 o C for 24 hrs and counts done among 30 to 100 colonies. D. Enzyme Assay Keratynolitic activity was determined with keratin azure as a substrate. The reaction mixture contained 0.2 ml of culture supernatant and 0.8 ml of 0.4 % (w/v) keratin azure in 10mM Tris -Hcl buffer (ph, 6, 7, 8 and 9). The mixture was incubated at 50 o C for 1 to 6 hours and the centrifuge rev/min -1 for 15 minutes and supernatant were measured at 650 nm [20]. E. Protein Estimation 103

4 Protein estimation was done as described by using Bovine serum Albumin at the rate of 1mg/ml as the standard. Different concentrations of the standard ranging from 0.1 to 1mg/ml were taken and made up to 1 mg/ml. Then 5ml of alkaline copper reagent was added, mixed well and allowed to stand for 10 minutes at room temperature. Then 0.5ml of diluted Folin's phenol reagent was added and mixed well. The mixture was incubated for 30 minutes at room temperature. The absorbance at 650nm was read spectrophotom etrically.the protein concentrations of enzyme were estimated [21]. F. Degradation assay The extent of keratin degradation by the microbial cultures in broth was determined by spectrophotometer at the maximum absorbance of the respective keratin[4 G. Sodium-Dodecylsulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) One dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) was carried out following the modified method of Laemmli et al., (1970). SDS-PAGE was run on vertical slab gel system. Proteins were electrophoresed on 10% separating gel (0.75 mm thickness) overlaid with 4% stacking gel [22]. E. Effect of the ph and Temperature on Enzyme Activity The effect of the ph was studied by assaying the enzyme using PBS buffers ph 6.0, 7.0, 8.0 and 9.0; The effect of the temperature was measured by incubating the enzyme at temperatures at 30 o C,40 C and 50 o C[10]. 3.RESULT AND DISCUSSION A.Isolation of Keratinolytic Microorganisms After being incubated for 48 hours, a plate containing feather meal agar showed the growth of a single colony, which was re-isolated using the same pure culture medium and it was shown in plate I. They were then subjected to biochemical analysis and the results were given in table 1[23]. The isolate dominant bacterial culture was identified as Bacillus subtilis, based on the morphological and biochemical analysis compared with Bergey's Manual [24]. 104

5 Table. 1 Biochemical results of the isolate. S. No TESTS RESULT 1 Gram staining + 2 Motility Motile 3 Catalase + 4 Citrate Utilization - 5 Indole - 6 Methyl Red - 7 Voges Proskauer (VP) + 4 Citrate Utilization - 5 Indole - 6 Methyl Red - 7 Voges Proskauer (VP) + B. Growth Determination of Isolated Bacteria Maximum growth was observed at ph 7.0 and 40ºC. These results were tabulated in table 2 and Figure 1, showed the effect of ph and temperature on the growth of the isolate. These results are very close to the ones obtained by using Streptomyces pactum [25] Streptomyces fradiae[26] Streptomyces S.K1-02 [20] Streptomyces sp. 594 [27,28] and Streptomyces thermoviolaceus strain SD8 [29]. Table. 2 Effect of ph and Temperature on the Growth of the Isolate Temperature ph6 ph7 ph8 ph

6 C. Enzyme assay: The enzyme assay carried out using Keratin Azure. The enzyme activities was tabulated in table 3 and the results was given in Figure 2. Similar results were obtained by Bacillus licheniformes PWD1[27], and also by using Clostridium bifermentans[28]. the enzyme obtained from Bacillus cereus are less thermostable, but according to the results obtained in this work a good stability (40 to 60 O C) was achieved using this Streptomyces, which is similar to the previously isolated Streptomyces S.K1-02[29]. Table. 3 Enzyme Activities Time(hrs) ph6 ph7 ph8 ph D. Protein Estimation The protein content in enzyme was found to be 2.86 mg /ml E. Degradation assay The isolated bacterial samples were checked for the extent of keratin degradation in liquid media gave maximum degradation. Other authors have considered that keratinolytic bacteria generally have optimum growth and feather degradation activity at high temperatures [25,32]. F. Molecular Weight Analysis by SDS-PAGE The enzyme proteins were run on SDS-PAGE along with the molecular weight 106

7 marker. While comparing the lane 1 with lane 3, the intracellular protein profile showed the presence of stress protein, but a better expression of the 108 k Da. 4. Conclusion The feather sample were collected from hatchery near Namakkal, Bacillus cereus was isolated from poultry plant wastewater, showed high keratinolytic activity when cultured on feather meal medium. Optimum keratinolytic activity was observed at 40 o C and ph 8.0. The enzyme also showed to be stable between 40 and 50 o C. Degradation assay was done and it shows maximum degradation. SDS-PAGE was performed and the molecular weight of the protein is 108 kda. The Bacillus cereus isolated might be useful in leather, keratin waste treatment, animal feeding industry, and also cosmetic industry. REFERENCES 1. J.A.Scott, W.A.Untereiner Determination of keratin degradation by fungi using keratin azure. Med Mycol 42(2004) 239-/ R.Gupta, P.Ramnani Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70(2006) A.M.Farag, M.A.Hassan Purification characterization and immobilization of keratinase from Aspergillus oryzae. Enzyme Microbial Technol 34(2004) E.H.Brutt, J.M.Ichida Bacteria useful for degrading Keratin. United States Patent No. 6(2001) D.M.T. Tapia, J.Contiero Production and partial characterization of kerotinase produced by a microorganism isolated from poultry processing plant waste water. Afr J Biotechnol 7(2008) A.A.Onifade, A.L.Sane, L.Musalla,N.S. Zarban Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresource Technol 66(1998) A.Anbu, A.Gopinath, A.Hilda, T.Lakshmi, G.Annadurai Purification of keratinase from poultry farm isolate - Scopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme Microbiol Technol 36(2005) G.D.Haki, S.K.Rakshit Developments in industrially important thermostable enzymes. Bioresource Technol 89(2003) A.Amara, A.Ehab Serour Wool Quality Improvement Using Thermophilic Crude Proteolytic Microbial Enzymes American-Eurasian. J Agric Environ Sci 3(2008)

8 10. H.Korkma, H.Hür, S.Dinçer Characterization of alkaline keratinase of Bacillus licheniformis strain HK-1 from poultry waste. Ann Microbiol 54( 2004) S.Tork, M.M.Aly, L.Nawar Biochemical and Molecular Characterization of a New Local Keratinase Producing Pseudomonas sp., Asian J Biotechnol 2(2010) I.H.Soomoro, Y.F.Kazi, M.Zardari, A.H.Shar Isolation of keratinophilic fungi from soil in Khairpur city, Sindh, Pakistan. Bangladesh J Microbiol 24( 2007) H.Gradisar, J.Friedrich, I.Krizaj, R.Jerala Similarities and Specifities of Fungal Keartinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to some Known Proteases. Appl Environ Microbiol 71(2005) V.Matikevicienė, D.Masiliuniens, S.Grigiskis Environmental Technology Resources Proceedings of the 7th International Scientific and Practical Conference.1(2009) 15. K.Morihara, T.Oka, H.Tsuzuki Multiple proteolytic enzymes of Streptomyces fradiae production, isolation, and preliminary characterization. Biochim Biophys Acta 139(1967) R.L.Seymour, T.W.Johnson Saprolegniaceae: A keratinophilic Aphanomyces from soil. Mycologia 65(1973) M.Cortezi, J.Contiero, J.B.Cristian, B.Roberta, R.Monti Characterization of a Feather Degrading by Bacillus amyloliquefaciens Protease: A New Strain, World J Agric Sci 4(2008) J.D.Holts Bergey's Manual of Systematic Bacteriology. 9th edn, Willians and Wilkins, Baltimore M.T.Daniel, G.T. M. Lucia Production and partial characterization of keratinase produced by a microorganism isolated from poultry processing plant waste water. Afr J Biotechnol 7(2008) F.Letournou, P.Soussotte, P.Bressollier, P.Branland Keratinokytic activity of Streptomyces sp. S.K1-02: a new isolated strain. Lett Appl Microbiol 26(1998) O.H.Lowry, N.J.Rosebrough, A.L.Farr, R.J.Randall Protein measurement with the folin phenol reagent. J Biol Chem193(1951) U.K.Laemmli Cleavage of structural proteins during the assembly of bacteriophage T 4, Nature (Lond) 227(1790) B.Lou, X.Zheng Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. J Zhejiang University Sci 9(2008) S.J.Stewart Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington B.Bockle, B.Galunsky, R.Muller Characterization of a keratinolytic serine protienase from Streptomyces pactum DS Appl Environ Microbiol 61(1995) R.A.Young,R.E. Smith Degradation of feather keratin by culture filtrates of Streptomyces fradiae Can J Microbiol 21(1975)

9 27. L.A.IAzeredo, M.B.Lima,R.R.R. Coleho, D.M.G.Freire Thermophilic protease production by Streptomyces sp., in submerged and solid state fermentation using feather meal. J Appl Microbiol 100(2006) X.Fuhong, C.Yapeng, X.Yang, Y.Jing, X.Zhiquan, L.Yuanming, Q.Shijun Purification and characterization of four keratinases produced by Streptomyces sp. strain 16 in native human foot skin medium. Bioresource Technol 101(2010) R.R.Chitte, V.K.Nalawade, S.Dey Keratinolytic activity from the broth of a featherdegrading thermophilic Streptomyces thermoviolaceus strain SD8. Lett Appl Microbiol 28(1999) S.W.Cheng, H.M.Hu, S.W.Shen, H.Takagi, Y.Tsai Production and characterization of keratinase of a feather degrading Bacillus licheniformes PWD-1. Biosci Biotechnol Biochem 59(1995) G.T.Macfarlane, S.Macfarlane Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC Appl Environ Microbiol 58(1992) C.M.Williams, C.S.Richter, J.M.Mackenzie J.C.H.Shih Isolation, identification and characterization of a feather-degrading bacterium. Appl Environ Microbiol 56(1990)

DEGRADATION OF KERATIN CONTAINING WASTES BY BACTERIA WITH KERATINOLYTIC ACTIVITY KERATĪNU SATUROŠO ATKRITUMU DEGRADĒŠANA AR BAKTĒRIJU

DEGRADATION OF KERATIN CONTAINING WASTES BY BACTERIA WITH KERATINOLYTIC ACTIVITY KERATĪNU SATUROŠO ATKRITUMU DEGRADĒŠANA AR BAKTĒRIJU ISSN 1691-5402 ISBN 978-9984-44-027-9 Environment. Technology. Resources Proceedings of the 7 th International Scientific and Practical Conference. Volume 1 Rēzeknes Augstskola, Rēzekne, RA Izdevniecība,