Trichophyton sp. Suşundan Keratinaz Üretimi, Saflaştırılması ve Kısmi Karakterizasyonu
Yıl 2020,
, 1135 - 1143, 31.10.2020
Dönay Parlak
,
Hüseyin Tanış
,
Ashabil Aygan
Öz
Keratinaz aktivitesi gösteren Trichophyton sp. Tr-9 suşu keratin tozu- feather meal içeren minimal besiyerinde büyütürek enzim üretimi gerçekleştirilmiştir. Süpernatanttan Amonyum sülfat çöktürme sonrası, Sephadeks G-100 ve DEAE Sepharose kolon kromatografi uygulamaları ile enzim saflaştırılması yapılmıştır. Maksimum keratinolitik aktivite pH 7.5 ve 37°C’de gözlenmiştir. Enzim pH 5.5-8.0 ve 20°C-40°C sıcaklık aralıklarında stabilite gösterirken, enziminin moleküler ağırlığı SDS page ve zimogram analizlerinde yaklaşık 34 kDa olarak hesaplandı. CaCI2 (5mM) keratinaz aktivitesini (%148) stimüle edici etki gösterdi. Diğer taraftan EDTA (5mM) ve SDS (%1) sırasıyla %49, % 49 etki göstererek kısmen inhibe etti, PMSF (1mM ve 5mM) ile tam inaktivasyon elde edildi. Sonuç olarak, enzimin fiziko-kimyasal özellikleri birçok endüstriyel ve biyoteknolojik uygulamalarda kullanışlı olabileceğini göstermiştir.
Destekleyen Kurum
K.S.Ü. BAP
Teşekkür
Bu çalışma KSU Bilimsel araştırmalar birimi tarafından 2009/4-6M Nolu proje ile desteklenmiştir.
Kaynakça
- Anbu P, Hilda A, Sur HW, Hur BK, Jayanthi S 2008. Extracellular keratinase from Trichophyton sp. HA-2 isolated from feather dumping soil. Int Biodeterior Biodegrad 62:287-292.
- Apodaca G, McKerrow, JH 1985. Purification and Characterization of a 27,000- Mr Extracellular Proteinase from Trichophyton rubrum. Infect. Immun,57(10): 3072–3080.
- Arikan B, Unaldi N, Coral G, Colak O, Aygan A, Gulnaz O 2003. Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochemistry, 38:1397-1403.
- Asahi M, Lindquist R, Fukuyama K, Apodaca G, Epstein W.L, McKerrow JH 1985. Purification and Charaterization of Major Extracellular Proteinases from Trichophyton rubrum. Biochem. J., 232:139–144.
- Balint B, Bagi Z, Toth A, Rakhelye G, Perei K, Kovacs KL 2005. Utilization of Keratin-Containing Biowaste to Produce Biohydrogen. Applied Microbiology and Biotechnology, 69:404-410.
- Bernal C, Vidal L, Valdivieso E, Coello N 2003. Keratinolytic Activity of Kocuria rosea. World J. Microbiol. Biotechnol., 19:255–261.
- Bernal C, Cairo J, Coello N 2006. Purification and characterization of a novel exocellular keratinase from Kocuria rosea. Enzyme Microbial Technol 38:49–54.
- Bhat MK 2000. Cellulases and Related Enzymes Inbiotechnology. Biotechnology Advances, 18:355-383.
- Cai, CG, Lou, BG, Zheng XD 2008. Keratinase Production and Keratin Degradation by a Mutant Strain of Bacillus subtilis. Zhejiang Univ. Sci. B, 9:60–67.
- Day WC, Toncic P, Stratman SL., Leuman U, Harmon SR 1968. Biochim. Biophys. Acta, 167: 596-606.
- Dubey R, Adhikary S, Kumar J, Sinha N 2010. Isolation, Production, Purification, Assay and Characterization of Alkaline Protease Enzyme from Aspergillus niger and Its Compatibility with Commercial Detergents, Developmental Microbiology and Molecular Biology, 1(1):75-94.
- Esawy MA 2007. Isolation and Partial Characterization of Extracelluler Keratinase from a Novel Mesophilic Streptomyces albus AZA. Research Journal of Agriculture and Biological Sciences, 3(6):808-817.
- Fakhfakh-Zouari N, Hmidet N, Haddar A, Kanoun S, Nasri M 2010. A Novel Serine Metallokeratinase from a Newly Isolated Bacillus pumilus A1 Grown on Chicken Feather Meal: Biochemical and Molecular Characterization. Appl Biochem Biotechnol, 162:329–344.
- Farag AM, Hassan MA 2004 Purification, Characterization and İmmobilization of a Keratinase from Aspergillus orizae. Enzyme Microb. Technol., 34:85–93.
- Ferrero MA, Castro GR, Abate CM, Baigori MD, Sineriz F1996. Thermostable Alkaline Proteases of Bacillus licheniformis MIR 29: Isolation, Production and Characterization. Appl MicrobiolBiotechnol., 45:327-332.
- Friedrich J, Gradisar H, Vrecl M, Pogacnik A 2005. In vitro Degradation of Porcine Skin Epidermis by a Fungal Keratinase of Doratomyces microsporus. Enzyme Microb. Technol., 36:455–460.
- Gradisar H, Friedrich J, Krizaji I, Jerala R 2005. Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of and Doratomyces microsporus to Some Known Proteases. Appl. Environ. Microbiol., 71:3420–3426.
- Gupta R, Gupta K, Saxena RK, Khan S 1999. Bleach-stable, alkaline protease from Bacillus sp. Biotechnol Letts 21:135–138.
- Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B 2003. Microbial α amylases: a Biotechnological Perspective. Process Biochem., 1-18.
- Gousterova A,Braikova D,Goshev I,Christov P,Tishinov K,Vasileva-Tonkova E, Haertlé T, Nedkov P2005,Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis Lett Appl Microbiol.40(5):335-40.
- Habbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa M, Badis A,Ladjama A 2014. Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost. J. Biosci. Bioeng. 117:413-421.
- Horikoshi K 1999. Alkaliphiles: Some Applications of their Products for Biotechnology. Microbiol. Mol. Bio., 63:735-750.
- Korkmaz H, Unaldı MN, Aslan B, Coral G, Arikan B, Dinçer S, Colak O 2003. Keratinolytic activity of Streptomyces strain BA7, a new isolate from Turkey. Ann Microbiol., 53: 85-93.
- Kreplak L, Doucet J, Dumas P, Briki F 2004. New Aspects of the α- helix to β-Sheet Transition in Stretched Hard α -Keratin Fibers. Biophys. J., 87:640–647.
- Langeveld JPM, Wang JJ, Van de Wiel DFM, Shih GC, Garsen J, Bossers A, Shih JCH 2003. Enzymatic Degradation of Prion Protein in Brain Stem from Infected Cattle and Sheep.JID., 188:1782–1789.
- Marcondes NR, Taira CL, Vandresen DC, Svidzinski TIE, Kadowaki MK, Peralta RM 2008. New Feather-Degrading Filamentous Fungi. Microb. Ecol., 56:13–17.
- Mckittrick J, Chen PY, Bodde SG, YangW,Novitskaya EE, Meyers MA 2012. The Structure, Functions, and Mechanical Properties of Keratin. JOM, Vol. 64, No. 4.
- Moallaei H, Zaini F, Larcher G, Beucher B, Bouchara JP 2006. Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii. Mycopathologia, 161:369–375.
- Mohamedin AH 1999. Isolation, Identification and some Cultural Conditions of a Protease Producing Thermophilic Streptomyces strain Grown on Chicken Feathers as a substrate. Int. Biodeterior. Biodegrad., 43:13–21.
- Mohorcic M, Torkar A, Friedrich J, Kristl J, Murdan S 2007.An Investigation into Keratinolytic Enzymes to Enhance Ungual Drug Delivery. Int. J. Pharm., 332:196–201.
- Moreira-Gasparin FG, Souza CGM, Costa AM, Alexandrino AM, Bracht CK, Boer CG, Peralta RM 2009. Purification and Characterization of an Efficient poultry Feather Degrading-Protease from Myrothecium verrucaria. Biodegradation, 20:727–736.
- Muhsin TM, Hadi RB 2002. Degradation of Keratin Substrades by Fungi Isolated from Sewage Sludge. Mycopathologia, 154: 185-189.
- Nam GW, Lee DW, Lee HS, Lee NJ, Kim B, ChoeEA, Hwang JK, Suhartono, MT, Pyun YR 2002. Native-Feather Degradation by Fervidobacterium islandicum AW-1, a Newly Isolated Keratinase-Producing Thermophilic Anaerobe. Arch. Microbiol., 178:538–547.
- Okafor JI, Ada N 2000. Keratinolytic Activity of five Human Isolates of the Dermatophytes. Journal of Common Diseases, 32,300–305.
- Onifade AA, Al-Sane NA, Al-Musallam, AA, Al-Zarban, S 1998. Potentials for Biotechnological Applications of Keratin-Degrading Microorganisms and their Enzymes for
Nutritional Improvement of Feathers and Other Keratins as Livestock Feed Resources. Bioresour. Technol., 66:1–11.
- Pandian S, Sundaram J, Panchatcharam P 2012. Isolation, Identification and Characterization of Feather Degrading Bacteria. European Journal of Experimental Biology, 2(1):274-282.
- Qin LM, Dekio S, Jidoi J 1992. Some Biochemical Characteristics of a Partially Purified Extracellular Keratinase from Trichophyton schoenleinii. Zentralbl Bakteriol., 277:236–244.
- Rabilloud T, Blisrick T, Heller M, Luche S, Aebersold R, Lunardi J, Braun‐Breton C 1999.Analysis of membrane proteins by two‐dimensional electrophoresis: Comparison of the proteins extracted from normal or Plasmodium falciparum ‐ infected erythrocyte ghostsElectrophoresis,20, 3603-3610.
- Raju K C, Neogi U, Saumya R, Goud N R 2007. Studies on Extracellular Enzyme Keratinase Dermatophyte Microsporum gypseum. International Journal of Biological Chemistry, 1(3): 174-178.
- Rao MB, Tanksale AM, Ghatge MS, Deshpande VV 1998. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiol. Mol. Biol. Rev., 62(3):597-635.
- Riffel A, Ortolan S, Brandelli A 2003. De-hairing Activity of Extracellular Proteases Produced by Keratinolytic Bacteria. Journal of Chemical Technology & Biotechnology, 78(8): 855-859.
- Riffel A, Brandelli A, Bellato S, de M, Gustavo HMF, Eberlin MN, Tavares FCA 2007. Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J Biotechnol 128:693–703.
- RojanavanichV,YoshiikeT, Tsuboi R, Takamori K, Ogawa, H 1990. Purification and Characterization of an Extracellular Proteinase from Hendersonula toruloidea. Infect. Immun., 58(9): 2856–2861.
- Suntornsuk W, Suntornsuk L 2003. Feather Degradation by Bacillus sp. FK 46 in Submerged Cultivation. Bioresour. Technol., 86:239–243.
- Takami H, Nogi Y, Horikoshi K 1999. Reidentification of Keratinase Producing Facultatively Alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles, 3:293-296.
- Tanış H, Cihangir N 2009. Klinik İzolatlardan Elde Edilen Trichophyton rubrum ve Trichophyton mentagrophytes’in Proteaz Aktivitelerinin Araştırılması. F.Ü. Sağ.Bil. Tıp Derg., 23(3):137-144.
- Tsuboi R, Ko IJ, Takamori K, Ogawa H 1989. Isolation of a Keratinolytic Proteinase from Trichophyton mentagrophytes with Enzymatic Activity at Acidic pH. Infect. Immun.,57:3479–3483.
- Vidyasagar M, Prakash S, Jayalakshmi SK, Sreeramulu K. 2006. Optimization of cultural conditions for the production of halo thermophilic protea sefrom halophilic bacterium Chromohalobacter sp. TVSP101.World. J Microbiol Biotechnol 23:655–662
- Voet D, Voet JG, Pratt CW2008. Fundamentals of Biochemistry: Life at the Molecular Level.3rd. ed. New York: Wiley.
Isolation and Partial Characterizatıon of Keratinase from Trichophyton sp.
Yıl 2020,
, 1135 - 1143, 31.10.2020
Dönay Parlak
,
Hüseyin Tanış
,
Ashabil Aygan
Öz
Keratinase enzyme production was accomplished from Trichophyton sp. Tr-9 in minmal medium containing feather meal. Enzyme was purified with Ammonium sulfate, Sephadeks G-100 and DEAE Sepharose column. Maxiumum keratinolitik activity was obtained at pH 7.5 and 37°C. Enzyme was higly stabil between pH 5.5-7.5 and 20-40oC. With the SDS-Page analysis of the enzyme, molecular weight of the enzyme was calculated as 34 kDa. CaCI2 (5mM) had a stimultory effect (148%) on enzyme. On the other hand, EDTA (5mM) and SDS (%1) inhibited enzyme acitivity up to 49% and 49%, respectively. PMSF (1- 5mM) has strongly inhibited enzyme. As a result, physico-chemical properties of the enzymeshowed that Trichophyton sp. Tr-9 could be useful in various industrial and biotechnological applications.
Kaynakça
- Anbu P, Hilda A, Sur HW, Hur BK, Jayanthi S 2008. Extracellular keratinase from Trichophyton sp. HA-2 isolated from feather dumping soil. Int Biodeterior Biodegrad 62:287-292.
- Apodaca G, McKerrow, JH 1985. Purification and Characterization of a 27,000- Mr Extracellular Proteinase from Trichophyton rubrum. Infect. Immun,57(10): 3072–3080.
- Arikan B, Unaldi N, Coral G, Colak O, Aygan A, Gulnaz O 2003. Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochemistry, 38:1397-1403.
- Asahi M, Lindquist R, Fukuyama K, Apodaca G, Epstein W.L, McKerrow JH 1985. Purification and Charaterization of Major Extracellular Proteinases from Trichophyton rubrum. Biochem. J., 232:139–144.
- Balint B, Bagi Z, Toth A, Rakhelye G, Perei K, Kovacs KL 2005. Utilization of Keratin-Containing Biowaste to Produce Biohydrogen. Applied Microbiology and Biotechnology, 69:404-410.
- Bernal C, Vidal L, Valdivieso E, Coello N 2003. Keratinolytic Activity of Kocuria rosea. World J. Microbiol. Biotechnol., 19:255–261.
- Bernal C, Cairo J, Coello N 2006. Purification and characterization of a novel exocellular keratinase from Kocuria rosea. Enzyme Microbial Technol 38:49–54.
- Bhat MK 2000. Cellulases and Related Enzymes Inbiotechnology. Biotechnology Advances, 18:355-383.
- Cai, CG, Lou, BG, Zheng XD 2008. Keratinase Production and Keratin Degradation by a Mutant Strain of Bacillus subtilis. Zhejiang Univ. Sci. B, 9:60–67.
- Day WC, Toncic P, Stratman SL., Leuman U, Harmon SR 1968. Biochim. Biophys. Acta, 167: 596-606.
- Dubey R, Adhikary S, Kumar J, Sinha N 2010. Isolation, Production, Purification, Assay and Characterization of Alkaline Protease Enzyme from Aspergillus niger and Its Compatibility with Commercial Detergents, Developmental Microbiology and Molecular Biology, 1(1):75-94.
- Esawy MA 2007. Isolation and Partial Characterization of Extracelluler Keratinase from a Novel Mesophilic Streptomyces albus AZA. Research Journal of Agriculture and Biological Sciences, 3(6):808-817.
- Fakhfakh-Zouari N, Hmidet N, Haddar A, Kanoun S, Nasri M 2010. A Novel Serine Metallokeratinase from a Newly Isolated Bacillus pumilus A1 Grown on Chicken Feather Meal: Biochemical and Molecular Characterization. Appl Biochem Biotechnol, 162:329–344.
- Farag AM, Hassan MA 2004 Purification, Characterization and İmmobilization of a Keratinase from Aspergillus orizae. Enzyme Microb. Technol., 34:85–93.
- Ferrero MA, Castro GR, Abate CM, Baigori MD, Sineriz F1996. Thermostable Alkaline Proteases of Bacillus licheniformis MIR 29: Isolation, Production and Characterization. Appl MicrobiolBiotechnol., 45:327-332.
- Friedrich J, Gradisar H, Vrecl M, Pogacnik A 2005. In vitro Degradation of Porcine Skin Epidermis by a Fungal Keratinase of Doratomyces microsporus. Enzyme Microb. Technol., 36:455–460.
- Gradisar H, Friedrich J, Krizaji I, Jerala R 2005. Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of and Doratomyces microsporus to Some Known Proteases. Appl. Environ. Microbiol., 71:3420–3426.
- Gupta R, Gupta K, Saxena RK, Khan S 1999. Bleach-stable, alkaline protease from Bacillus sp. Biotechnol Letts 21:135–138.
- Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B 2003. Microbial α amylases: a Biotechnological Perspective. Process Biochem., 1-18.
- Gousterova A,Braikova D,Goshev I,Christov P,Tishinov K,Vasileva-Tonkova E, Haertlé T, Nedkov P2005,Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis Lett Appl Microbiol.40(5):335-40.
- Habbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa M, Badis A,Ladjama A 2014. Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost. J. Biosci. Bioeng. 117:413-421.
- Horikoshi K 1999. Alkaliphiles: Some Applications of their Products for Biotechnology. Microbiol. Mol. Bio., 63:735-750.
- Korkmaz H, Unaldı MN, Aslan B, Coral G, Arikan B, Dinçer S, Colak O 2003. Keratinolytic activity of Streptomyces strain BA7, a new isolate from Turkey. Ann Microbiol., 53: 85-93.
- Kreplak L, Doucet J, Dumas P, Briki F 2004. New Aspects of the α- helix to β-Sheet Transition in Stretched Hard α -Keratin Fibers. Biophys. J., 87:640–647.
- Langeveld JPM, Wang JJ, Van de Wiel DFM, Shih GC, Garsen J, Bossers A, Shih JCH 2003. Enzymatic Degradation of Prion Protein in Brain Stem from Infected Cattle and Sheep.JID., 188:1782–1789.
- Marcondes NR, Taira CL, Vandresen DC, Svidzinski TIE, Kadowaki MK, Peralta RM 2008. New Feather-Degrading Filamentous Fungi. Microb. Ecol., 56:13–17.
- Mckittrick J, Chen PY, Bodde SG, YangW,Novitskaya EE, Meyers MA 2012. The Structure, Functions, and Mechanical Properties of Keratin. JOM, Vol. 64, No. 4.
- Moallaei H, Zaini F, Larcher G, Beucher B, Bouchara JP 2006. Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii. Mycopathologia, 161:369–375.
- Mohamedin AH 1999. Isolation, Identification and some Cultural Conditions of a Protease Producing Thermophilic Streptomyces strain Grown on Chicken Feathers as a substrate. Int. Biodeterior. Biodegrad., 43:13–21.
- Mohorcic M, Torkar A, Friedrich J, Kristl J, Murdan S 2007.An Investigation into Keratinolytic Enzymes to Enhance Ungual Drug Delivery. Int. J. Pharm., 332:196–201.
- Moreira-Gasparin FG, Souza CGM, Costa AM, Alexandrino AM, Bracht CK, Boer CG, Peralta RM 2009. Purification and Characterization of an Efficient poultry Feather Degrading-Protease from Myrothecium verrucaria. Biodegradation, 20:727–736.
- Muhsin TM, Hadi RB 2002. Degradation of Keratin Substrades by Fungi Isolated from Sewage Sludge. Mycopathologia, 154: 185-189.
- Nam GW, Lee DW, Lee HS, Lee NJ, Kim B, ChoeEA, Hwang JK, Suhartono, MT, Pyun YR 2002. Native-Feather Degradation by Fervidobacterium islandicum AW-1, a Newly Isolated Keratinase-Producing Thermophilic Anaerobe. Arch. Microbiol., 178:538–547.
- Okafor JI, Ada N 2000. Keratinolytic Activity of five Human Isolates of the Dermatophytes. Journal of Common Diseases, 32,300–305.
- Onifade AA, Al-Sane NA, Al-Musallam, AA, Al-Zarban, S 1998. Potentials for Biotechnological Applications of Keratin-Degrading Microorganisms and their Enzymes for
Nutritional Improvement of Feathers and Other Keratins as Livestock Feed Resources. Bioresour. Technol., 66:1–11.
- Pandian S, Sundaram J, Panchatcharam P 2012. Isolation, Identification and Characterization of Feather Degrading Bacteria. European Journal of Experimental Biology, 2(1):274-282.
- Qin LM, Dekio S, Jidoi J 1992. Some Biochemical Characteristics of a Partially Purified Extracellular Keratinase from Trichophyton schoenleinii. Zentralbl Bakteriol., 277:236–244.
- Rabilloud T, Blisrick T, Heller M, Luche S, Aebersold R, Lunardi J, Braun‐Breton C 1999.Analysis of membrane proteins by two‐dimensional electrophoresis: Comparison of the proteins extracted from normal or Plasmodium falciparum ‐ infected erythrocyte ghostsElectrophoresis,20, 3603-3610.
- Raju K C, Neogi U, Saumya R, Goud N R 2007. Studies on Extracellular Enzyme Keratinase Dermatophyte Microsporum gypseum. International Journal of Biological Chemistry, 1(3): 174-178.
- Rao MB, Tanksale AM, Ghatge MS, Deshpande VV 1998. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiol. Mol. Biol. Rev., 62(3):597-635.
- Riffel A, Ortolan S, Brandelli A 2003. De-hairing Activity of Extracellular Proteases Produced by Keratinolytic Bacteria. Journal of Chemical Technology & Biotechnology, 78(8): 855-859.
- Riffel A, Brandelli A, Bellato S, de M, Gustavo HMF, Eberlin MN, Tavares FCA 2007. Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J Biotechnol 128:693–703.
- RojanavanichV,YoshiikeT, Tsuboi R, Takamori K, Ogawa, H 1990. Purification and Characterization of an Extracellular Proteinase from Hendersonula toruloidea. Infect. Immun., 58(9): 2856–2861.
- Suntornsuk W, Suntornsuk L 2003. Feather Degradation by Bacillus sp. FK 46 in Submerged Cultivation. Bioresour. Technol., 86:239–243.
- Takami H, Nogi Y, Horikoshi K 1999. Reidentification of Keratinase Producing Facultatively Alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles, 3:293-296.
- Tanış H, Cihangir N 2009. Klinik İzolatlardan Elde Edilen Trichophyton rubrum ve Trichophyton mentagrophytes’in Proteaz Aktivitelerinin Araştırılması. F.Ü. Sağ.Bil. Tıp Derg., 23(3):137-144.
- Tsuboi R, Ko IJ, Takamori K, Ogawa H 1989. Isolation of a Keratinolytic Proteinase from Trichophyton mentagrophytes with Enzymatic Activity at Acidic pH. Infect. Immun.,57:3479–3483.
- Vidyasagar M, Prakash S, Jayalakshmi SK, Sreeramulu K. 2006. Optimization of cultural conditions for the production of halo thermophilic protea sefrom halophilic bacterium Chromohalobacter sp. TVSP101.World. J Microbiol Biotechnol 23:655–662
- Voet D, Voet JG, Pratt CW2008. Fundamentals of Biochemistry: Life at the Molecular Level.3rd. ed. New York: Wiley.