Production and characterization of multifunctional endoxylanese by Bacillus sp. X13

Topraktan multifonksiyonel endoksilanaz üreten Bacillus sp. X13 suşu izole edilmiştir. Enzim sentezi optimum 37 °C olmak üzere 20 ile 60 °C arasında gerçekleşmiştir. SDS-PAGE analizinde, 66,5 kDa, 80,6 kDa, 95,5 kDa ve 108,4 kDa olarak hesaplanan 4 aktif enzim bandı gözlenmiştir. Enzim optimum aktivitesi 40 °C olup 20-90 °C arasında geniş bir sıcaklık aralığına sahiptir ve maksimum aktivitesi ise pH 6,0 olarak bulunmuştur. İnkübasyon sıcaklığı arttıkça enzim kalan aktivitede giderek artan bir düşüş göstermiştir. Sıcaklık stabilitesi Ca+2 varlığında dahi artmamıştır. Enzim pH 3,6 ile 10,0 arasında 1 saat inkübe edildiğinde kalan aktivite ortalama % 71 olarak elde edilmiştir. Enzim üre ile yüksek oranda inhibe olurken Triton X-100, CaCl2, ZnCl2, KCl, Na2SO3 PMSF ve 2-Merkaptoetanol varlığında çok düşük oranda etkilenmiştir. Bu çalışmada enzimin belirlenen özellikleri bu enzimin endüstriyel kullanımında potansiyel bir tercih sebebi olabilir.

Bacillus sp. X13 suşundan multifonksiyonel endoksilanaz üretimi ve karakterizasyonu

A multifunctional endoxylanase producing Bacillus sp. was isolated from soil. Enzyme synthesis occurred at temperatures between 20 °C and 60 °C with an optimum 37 °C. Analysis of the enzyme by SDS-PAGE revealed 4 active enzyme bands, which were estimated to be 66.5 kDa, 80.6 kDa, 95.5 kDa, and 108.4 kDa. The enzyme has a broad temperature range, between 20 and 90 °C, with an optimum at 40 °C; and maximum activity was at pH 6.0. The enzyme showed a gradual decrease in the remaining activity as the pre-incubation temperature increase. Thermostability was not also increased in the presence of Ca2+. An average of 71% of remaining activity observed when the enzyme incubated between pH 3.6 and 10 for 1 h. The enzyme was highly inhibited by urea, and fairly inhibited by Triton X-100, CaCl2, ZnCl2, KCl, Na2SO3 PMSF, and 2-Mercaptoethanol. The properties of the enzyme presented in this study suggest that this enzyme could be a potential industrial interest.

PDF

___

1. Beg QK, Kapoor M, Mahajan L et al. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56: 326- 338, 2001.
2. Dhillon A, Gupta JK, Kahna S. Enhanced production, purification and characterization of a novel cellulase-poor thermostable, alkalitolerant xylanase from Bacillus circulans AB16. Process Biochem 35: 849- 856, 2000.
3. Asha Poorna C, Prema P. Production and partial characterization of endoxylanase by Bacillus pumilus using agro industrial residues. Biochem Eng J 32: 106- 112, 2006.
4. Heck JX, Hertz PF, Ayup MAZ. Cellulase and Xylanase production by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation. Braz J Microbiol 33: 213- 218, 2004.
5. Subramaniyan S, Prema P. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiol Lett 183:1- 7, 2000.
6. Courtin CM, Delcour AJ. Arabinoxylans and endoxylanase in wheat flour bread making. J Cereal Sci 35: 225- 243, 2002.
7. Breccia JD, Sineriz F, Baigori MD et al. Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens. Enzyme Microb Tech 22: 42- 49, 1998.
8. Gomes I, Gomes J, Steiner W et al. Production of cellulase and xylanase by a wild strain of Tricoderma viride. Appl Microbiol Biotechnol 36: 701-707, 1992.
9. Singh S, Pillay B, Dilsook V et al. Production and properties of hemicellulases by a Thermomyces lanuginosus strain. J Appl Microbiol 88: 975-982, 2000.
10. Subramaniyan S, Prema P. Biotechnology of microbial Xylanases: enzymology, molecular biology and application. Crit Rev Biotechnol 22 (1): 33-46,2002.
11. Gessesse A. Purification and properties of two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Appl Environ Microbiol 64: 3533- 3535, 1998.
12. Voget S, Steele HL, Streit WR. Characterization of metagenomederived halotolerant cellulase. J Biotechnol 126: 26- 36, 2006.
13. Bernhardsdotter ECMJ, Ng JD, Garriott OK et al. Enzymic properties of an alkaline chelator-resistant a-amylase from alkaliphilic Bacillus sp. isolate L1711. Process Biochem 40: 2401- 2408, 2005.
14. Burhan A, Nisa U, Gokhan C et al. Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT- 6. Process Biochem 38: 1397- 1403, 2003.
15. Lo HF, Lin LL, Chen HL et al. Enzymatic properties of a SDSresistant Bacillus sp. TS- 23 a-amylase produced by recombinant Escherichia coli. Process Biochem 36: 743- 750, 2001.
16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680- 685, 1970.
17. Bollag DM, Rozycki MD, Edelstein SJ. Protein methods. Willey- Liss Inc. New York; 1996.
18. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manuel. Cold Spring Harbor Laboratory Pres. New York; 1982.
19. Saul DJ, Williams LC, Grayling RA et al. ceIB, a Gene coding for a bifunctional cellulase from the extreme thermophile “Caldocellum saccharolyticum“. Appl Environ Microbiol 56: 3117- 3124, 1990.
20. Singh J, Batra N, Sobti RC. Purification and characterization of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1. J Ind Microbiol Biotechnol 31: 51- 56, 2004.
21. Lopez C, Blanco A, Pastor FIJ. Xylanase production by a new alkali tolerant Bacillus. Biotechnol Lett 20: 243- 246, 1998.
22. Tseng MJ, Yap MN, Ratanakhanokchai K et al. Purification and characterization of two cellulase free xylanase from an alkaliphilic Bacillus firmus. Enzyme Mibrob Technol 30: 590- 595, 2002.
23. Ryan SE, Nolan K, Thompson R et al. Purification and characterization of a new low molecular weight endoxylanase from Penicillium capsulatum. Enzyme Microb Tehcnol 33: 775- 785, 2003.
24. Gallordo O, Diaz P, Pastor FIJ. Cloning and characterization of xylanase A from the strain Bacillus sp. BP- 7: Comparison with alkaline pI-low molecular weight xylanase of family 11. Curr Microbiol 48: 276- 279, 2004.
25. Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29: 3- 23, 2005.
26. Dey D, Hinge J, Shendye A et al. Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp. Can J Microbiol 38: 436- 442, 1992.
27. Nakamura S, Ishiguro Y, Nakai R et al. Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR- 1. J Mol Cat B Enz 1: 7- 15, 1995.
28. Wejse PL, Ingvorsen K, Mortensen KK. Xylanase production by a novel halophilic bacterium increased 20-fold by response surface methodology. Enzyme Microb Technol 32: 721- 727, 2003.
29. Biely P. Diversity of microbial endo-b-1,4-xylanases. In: Mansfield SD, Saddler JN. Eds. Applications of enzymes to lignocellulosic. American Chemical Society; 2003: pp. 361- 380.
30. Gilkes NR, Claeyssen M, Aebersold R et al. Structural and functional relationships in two families of b-1,4-glycanases. Eur J Biochem 202: 367- 377, 1991.
31. Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316: 695– 696, 1996.
32. White A, Whithers SG, Gilkes NR et al. Crystal structure of the catalytic domain of the b-1,4-glycanase cex from Cellulomonas fimi. Biochem 33: 12546, 1994.