Purification and Characterization of Alkaline Xylanase Secreted from Paenibacillus macquariensis

Meeta Sharma, Saurabh Mehta, Anil Kumar

School of Biotechnology, Devi Ahilya University, Indore, India.

DOI: 10.4236/aim.2013.31005                

Abstract

An alkaline xylanase secreted by Paenibacillus macquariensis RC 1819 has been purified using ammonium sulfate fractionation, ion exchange chromatography using DEAE-cellulose and gel filtration chromatography over Sephadex G-200 and Sephadex G-100. The purified enzyme had the specific activity, 25.2 units/mg protein with birchwood xylan as a substrate. The purified enzyme showed a single protein band over sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme has been found to be 31,000 ± 2000 as determined by using Sephadex G-200 gel filtration chromatography. The subunit molecular weight has also been found to be ~31,000 as determined using SDS-PAGE indicating monomeric enzyme. The enzyme showed optimum activity at pH 8.6 and temperature, 50°C. The Michaelis constant (Km) of the enzyme for birchwood xylan was 2.2 mg/ml as determined using velocity saturation plot. The metal ions viz. Co⁺² and Mn⁺² stimulated xylanase enzyme activity whereas Hg⁺² inhibited the enzyme activity.

Keywords

Xylanase; Paenibacillus macquariensis; Purification; Michaelis Constant; Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	T. Collins, C. Gerday and G. Feller, “Xylanases, Xylanase Families and Extremophilic Xylanases,” FEMS Microbiology Reviews, Vol. 29, No. 1, 2005, pp. 3-23. doi:10.1016/j.femsre.2004.06.005
[2]	L. Anthony, A. Marcel and R. Eric, “Overproduction and Characterization of Xylanase B from Aspergillus niger,” Canadian Journal of Microbiology, Vol. 51, No. 2, 2005, pp. 177-183. doi:10.1139/w04-116
[3]	S. G. Nair, R. Sindhu and S. Shashidhar, “Fungal Xylanase Production under Solid State and Submerged Fermentation Conditions,” African Journal of Microbiology Research, Vol. 2, No. 4, 2008, pp. 82-86.
[4]	D. Haltrich, B. Nidetzky, K. D. Kulbe, W. Steiner and S. Zupancic, “Production of Fungal Xylanases,” Bioresource Technology, Vol. 58, No. 2, 1996, pp 137-161. doi:10.1016/S0960-8524(96)00094-6
[5]	E. X. F. Filho, “Hemicellulase and Biotechnology,” In: S. G. Pandalai, Ed., Recent Research Development in Microbiology, Research Signpost, Trivandrum, 1998, pp. 165-176.
[6]	C. T. D. Marta, C. A. P. Ana, P. P. Edilberto, N. P. Alexandre and T. F. Telma, “Characterization of Alkaline Xylanases from Bacillus pumilus,” Brazilian Journal of Microbiology, Vol. 31, No. 2, 2000, pp. 90-94.
[7]	M. L. T. M. Polizeli, A. C. S. Rizzatti, R. Monti, H. F. Terenzi, J. A. Jorge and D. S. Amorim, “Xylanases from Fungi: Properties and Industrial Applications,” Applied Microbiology and Biotechnology, Vol. 67, No. 5, 2005, pp. 577-591. doi:10.1007/s00253-005-1904-7
[8]	L. Viikari, M. Ranua, A. Kantelinen, J. Sundquist and M. Linko, “Bleaching with Enzymes,” Proceedings of the 3rd International Conference on Biotechnology, Pulp Paper Industry, Stockholm, 1986, pp. 67-69.
[9]	N. Garg, K. K. Mahatman and A. Kumar, “Xylanase: Applications and Biotechnological Aspects,” LAP Lambert Academic Publishing AG & Co. KG, 2010.
[10]	P. S. Pereira, H. Paveia, M. C. Ferreira and M. R. AiresBarros, “A New Look at Xylanases: An Overview of Purification Strategies,” Molecular Biotechnology, Vol. 24, No. 3, 2003, pp. 257-281. doi:10.1385/MB:24:3:257
[11]	M. Galbe and G. A. Zacchi, “A Review of the Production of Ethanol from Softwood,” Applied Microbiology and Biotechnology, Vol. 59, No. 6, 2005, pp. 618-628.
[12]	Q. A. Beg, M. Kapoor, G. Mahajan and S. Hoondal, “Microbial Xylanases and Their Industrial Applications: A Review,” Applied Microbiology Biotechnology, Vol. 6, No. 3-4, 2001, pp. 326-338. doi:10.1007/s002530100704
[13]	T. N. Nortey, J. F. Patience and R. T. Zijlstra, “Xylanase Supplementation Improves Energy Digestibility of Wheat By-Products in Grower Pigs,” Livestock Science, Vol. 109, No. 1, 2007, pp. 96-99. doi:10.1016/j.livsci.2007.01.092
[14]	H. Uhlig, “Industrial Enzymes and Their Applications,” John Wiley & Sons Ltd., New York, 1998.
[15]	M. Sharma and A. Kumar, “Optimization of Xylanase Secretion from Paenibacillus Macquariensis,” Current Trends in Biotechnology and Pharmacy, Vol. 6, No. 2, 2012, pp. 190-195.
[16]	P. Dheeran, N. Nandhagopal, S. Kumar, Y. K. Jaiswal and D. K. Adhikari, “A Novel Thermostable Xylanase of Paenibacillus Macerans IIPSP3 Isolated from the Termite Gut,” Journal of Industrial Microbiology & Biotechnology, Vol. 39, No. 6, 2012, pp. 851-860. doi:10.1007/s10295-012-1093-1
[17]	P. Shi, J. Tian, T. Yuan, X. Liu, H. Huang, Y. Bai, P. Yang, X. Chen, N. Wu and B. Yao, “Paenibacillus sp. Strain E18 Bifunctional Xylanase-Glucanase with a Single Catalytic Domain,” Applied and Environmental Microbiology, Vol. 76, No. 11, 2010, pp. 2620-2624. doi:10.1128/AEM.00345-10
[18]	G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar,” Analytical Chemistry, Vol. 31, No. 3, 1959, pp. 426-428. doi:10.1021/ac60147a030
[19]	O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, “Protein Measurement with the Folin Phenol Reagent,” Journal of Biological Chemistry, Vol. 193, No. 1, 1951, pp. 265-275.
[20]	U. K. Laemmli, “Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4,” Nature, Vol. 227, No. 5259, 1970, pp. 680-685.
[21]	J. R. Whitaker, “Determination of Molecular Weights of Proteins by Gel Filtration on Sephadex,” Analytical Chemistry, Vol. 35, No. 12, 1963, pp. 1950-1953.
[22]	C. J. O. R. Morris and P. Morris, In: 2nd Edition, Separation Methods in Biochemistry, Pitman Publishing, London, 1976, pp. 93-148.
[23]	K. K. Mahatman, N. Garg, R. Chauhan and A. Kumar, “Production, Purification and Characterization of Xylanase Using Alkalo-Thermophilic Bacillus halodurans KR-1,” Iranica Journal of Energy and Environment, Vol. 1, No. 4, 2010, pp. 265-274.
[24]	F. Vahino and T. A. Nakane, “Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.,” Agricultural and Biological Chemistry, Vol. 45, No. 5, 1980, pp. 1121-1127.
[25]	A. Blanco, T. Vidal, F. J. Colom and J. I. F. Pastor, “Purification and Properties of Xylanase A from Alkali-Tolerent Bacillus sp. Strain BP-23,” Applied and Environmental Microbiology, Vol. 61, No. 12, 1995, pp. 4468-4470.
[26]	S. Nakamura, K. Wakabayashi, R. Nakai, R. Aono and K. Horikoshi, “Purification and Some Properties of an Alkaline Xylanase from an Alkaliphilic Bacillus sp. Strain 41M-1,” Applied and Environmental Microbiology, Vol. 59, No. 7, 1993, pp. 2311-2316.
[27]	A. Gessesse, “Purification and Properties of Two Thermostable Alkaline Xylanase from an Alkaliphilic Bacillus sp.,” Applied and Environmental Microbiology, Vol. 64, No. 9, 1998, pp. 3533-3535.
[28]	B. K. Kubata, T. Suzuki, H. Horitsu, K. Kawai and K. Takamizawa, “Purification and Characterization of Aeromonas carviae ME-1 Xylanase,Which Produces Exclusively Xylobiose from Xylan,” Applied and Envionmental Microbiology, Vol. 60, No. 2, 1994, pp. 531-535.
[29]	T. Araki, N. Enoue and T. Morisita, “Purification and Characterization of β-1,3 Xylanase from a Marine Bacterium, Alcaligenes sp. XY-234,” The Journal of General and Applied Microbiology, Vol. 44, No. 4, 1998, pp. 269-274.
[30]	J. Lin, L. M. Nudlovu, S. Singh and B. Pillay, “Purification and Biochemical Charactrastics of β-D-Xylanase from a Thermophilic Fungus, Thermomyces lanuginosusSSBP,” Biotechnology and Applied Biochemistry, Vol. 30, No. 1, 1999, pp. 73-79.
[31]	J. S. Cho, K. Choi, T. Darden, P. R. Reynolds, J. N. Petitte and S. B. Shears, “Avian Multiple Inositol Polyphosphate Phosphatase in an Active Phytase That Can Be Engineered to Help Ameliorate the Planet’S ‘Phosphate Crisis’,” Journal of Biotechnology, Vol. 126, No. 2, 2006, pp. 248-259.
[32]	T. Collins, M. A. Meuwis, I. Stals, M. Claeyssens, G. Feller and C. Gerday, “A Novel Family 8 Xylanase: Functional and Physiochemical Characterization,” The Journal of Biological Chemistry, Vol. 277, No. 38, 2002, pp. 35133-35139.
[33]	W. Rattiya, P. Pason, K. L. Kyu, K. Sakka, A. Kosugi, Y. Mori and K. Ratanakhanokchai, “Cloning, Sequencing, and Expression of the Gene Encoding a Multidomain endo-1, 4-Xylanase from paenibacillus curdlanolyticus b-6, and Characterization of the Recombinant Enzyme,” Journal of Microbiology and Biotechnology, Vol. 19, No. 3, 2009, pp. 277-285.
[34]	J. Heck, S. Flores, P. Hertzm and M. Ayub, “Optimization of Cellulase-Free Xylanase Activity Produced by Bacillus coagulans BL69 in Solid-State Cultivation,” Process Biochemistry, Vol. 40, No. 1, 2005, pp. 107-112.
[35]	M. B. Wamalwa, G. Zhao, M. Sakka, M. P. Shiundu, T. Kimura and K. Sakka, “High Level Heterologous Expression of Bacillus halodurans Putative Xylanase xyn11A (BHO899) in Kluyveromyces lactis,” Bioscience, Biotechnology and Biochemistry, Vol. 71, No. 3, 2007, pp. 688-693.
[36]	S. Nakamura, Y. Ishiguro, R. Nakai, K. Wakabayashi, R. Aono and K. Horikoshi, “Purification and Characterization of a Thermophilic Alkaline Xylanase from Thermoalkalophilic Bacillus sp. strain TAR-1,” Journal of Molecular Catalysis B: Enzymatics, Vol. 1, No. 1, 1995, pp. 7-15.
[37]	K. B. Bastawade, “Xylan Structure, Microbial Xylanases, and Their Mode of Action,” World Journal of Microbiology and Biotechnology, Vol. 8, No. 4, 1992, pp. 353-368.
[38]	T. Cesar and V. Mrsia, “Purification and Properties of the Xylanase Produced by Thermomyces lanuginosus,” Enzyme and Microbial Technology, Vol. 19, No. 4, 1996, pp. 289-296.
[39]	E. T. Chivero, A. N. Mutukumira and R. Zvauya, “Partial Purification and Characterization of a Xylanase Enzyme Produced by a Micro-Organism Isolated from Selected from Indigenous Fruits of Zimbabwe,” Food Chemistry, Vol. 72, No. 2, 2001, pp. 179-185.
[40]	P. Inkyung and C. Jaiesoon, “Partial Characterization of Extracellular Xylanolytic Activity Derived from Paenibacillus sp. K1J1,” African Journal of Microbiology Research, Vol. 4, No. 12, 2010, pp. 1257-1264.
[41]	C. C. Lee, R. E. Kibblewhite-Accinelli, M. R. Smith, K. Wagschal, W. J. Orts and D. W. S. Wong, “Cloning of Bacillus licheniformis Xylanase Gene and Characterization of Recombinant Enzyme,” Current Microbiology, Vol. 57, No. 4, 2008, pp. 301-305.
[42]	P. Sa-Pereira, M. Costa-Ferreira and M. R. Atres-Barros, “Enzymatic Properties of a Neutral Endo-1,3(4)-b-xylanase Xyl II from Bacillus subtili,” Journal of Biotechnology, Vol. 94, No. 3, 2002, pp. 256-275.
[43]	K. Ratanakhanokchai, K. L. Kyu and M. Tanticharoen, “Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1,” Applied and Environmental Microbiology, Vol. 65, No. 2, 1999, pp. 694-697.
[44]	P. K. A. Muniswaran, P. Selvakumar and N. C. L. Charyulu, “Production of Cellulases from Coconut Coir Pith in Solid State Fermentation,” Journal of Chemical Technology and Biotechnology, Vol. 60, No. 2, 1994, pp. 147151.