Applied Ecology and Environmental Sciences
ISSN (Print): 2328-3912 ISSN (Online): 2328-3920 Website: Editor-in-chief: Alejandro González Medina
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Applied Ecology and Environmental Sciences. 2021, 9(7), 640-648
DOI: 10.12691/aees-9-7-3
Open AccessArticle

Production, Purification and Characterization of Xylanase Enzyme from Bacillus sp in Solid State Fermentation

Bidare Shivayogiraju Shylesha1, Mahesh Mariswamy2, Shubharani Ramanath2 and Veerapura Narayanappa Yoganandamurthy2,

1Department of Chemistry, Sree Siddaganga College of Arts, Science and Commerce, Tumakuru-572102, (Karnataka), India

2Department of Biotechnology, Azyme Biosciences Private Limited, Bengaluru-560069, (Karnataka), India

Pub. Date: July 12, 2021

Cite this paper:
Bidare Shivayogiraju Shylesha, Mahesh Mariswamy, Shubharani Ramanath and Veerapura Narayanappa Yoganandamurthy. Production, Purification and Characterization of Xylanase Enzyme from Bacillus sp in Solid State Fermentation. Applied Ecology and Environmental Sciences. 2021; 9(7):640-648. doi: 10.12691/aees-9-7-3


Xylanases are extensively applied in paper and pulp industries as well as during preparation of baked products to improve their quality. Additionally, it is also used in coffee, oil and starch industries in order to increase their nutritional values. Soil samples were collected near saw mills in various localities of Bangalore urban to isolate organisms for the production of xylanase using solid state fermentation. Six organisms were isolated using selective media based on their morphological characters. Among them one organism showed maximum production of xylanase enzyme identified as Bacillus sp based on their biochemical test and 16s RNA sequencing. Solid substrate fermentation was carried out using various agro wastes such as sugar cane bagasse, saw dust, paddy husk, wheat straw and orange peel powder. Sugarcane bagasse showed maximum production of enzyme compared to other substrates with different physical parameters such as pH 8, temperature at 35 °C after 72 hrs of incubation. Trace element such as Mg++ enhances the production of enzyme more than 22 % compared with other metal ions like Ca++, Mn++ and Fe++. After production, enzyme purified by using three step methods such ammonium sulphate precipitation, dialysis, ion exchange and gel filtration. Fold purification was increased up to 12 fold, yield 36 % and molecular weight of enzyme was 62 KDa determined using SDS PAGE.

fermentation ion exchange chromatography SDS-PAGE SSF Xylanase

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[1]  Reilly, P.J, “Xylanases: structure and function”, Basic Life Sciences, 18: 111-129. 1981.
[2]  Barlaz, M.A, “Carbon storage during biodegradation of municipal solid waste components in laboratory‐scale landfills”, Global Biogeochemical Cycles, 12(2): 373-380. 1998.
[3]  Singh, A., Yadav, R.D., Kaur, A. and Mahajan, R, “An ecofriendly cost effective enzymatic methodology for deinking of school waste paper”, Bioresource Technology, 120: 322-327. 2012.
[4]  Singh, A., Kaur, A., Dua, A. and Mahajan, R, “An efficient and improved methodology for the screening of industrially valuable xylano-pectino-cellulolytic microbes”, Enzyme Research, Article ID: 725281. 2015.
[5]  Ho, H.L. and Ku, X.N, “Kinetic study of cell growth and production of amylase, cellulase and xylanase by Bacillus subtilis using barley husk as the prime carbon source”, Journal of Advances in Biology & Biotechnology, 14(2): 1-18. 2017.
[6]  Kareem, R.A.W.A., Khushk, I., Bhutto, M.A., Sattar, Q.A., and Ahmed, A, “Xylanase production using fruit waste as cost effective carbon source from thermo-tolerant Bacillus megaterium”, African Journal of Microbiology Research, 8(38): 3463-3470. 2014.
[7]  Ping, L., Wang, M., Yuan, X., Cui, F., Huang, D., Sun, W., Zou, B., Huo, S. and Wang, H, “Production and characterization of a novel acidophilic and thermostable xylanase from Thermoascus aurantiacu”, International Journal of Biological Macromolecules, 109: 1270-1279. 2018.
[8]  Kamble, R.D. and Jadhav, A.R, “Isolation, purification, and characterization of xylanase produced by a new species of Bacillus in solid state fermentation”, International Journal of Microbiology, Article ID: 683193. 2012.
[9]  Mokni-Tlili, S., Ben Abdelmalek, I., Jedidi, N., Belghith, H., Gargouri, A., Abdennaceur, H. and Marzouki, M.N, “Exploitation of biological wastes for the production of value-added hydrolases by Streptomyces sp. MSWC1 isolated from municipal solid waste compost”, Waste Management & Research, 28(9): 828-837. 2010.
[10]  Levasseur, A., Asther, M., and Record, E, “Overproduction and characterization of xylanase B from Aspergillus niger”, Canadian Journal of Microbiology, 51(2): 177-183. 2005.
[11]  Maki, M.L., Broere, M., Leung, K.T. and Qin, W, “Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers”, International Journal of Biochemistry and Molecular Biology, 2: 146-154. 2011.
[12]  Polizeli, M.L.T.M., Rizzatti, A.C.S., Monti, R., Terenzi, H.F., Jorge, J.A. and Amorim, D.S, “Xylanases from fungi: properties and industrial applications”, Applied Microbiology and Biotechnology, 67(5): 577-591. 2005.
[13]  Bindu, B., Jitender, S., Saurabh, S.D. and Ramesh, C.K, “Enhanced production of cellulose-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry”, Enzyme and Microbial Technology, 41(6-7): 733-739. 2007.
[14]  Aysegul, E.Y., Sahin, A., Sahin, F.I. and Mehmet, M, “Isolation of endophytic and xylanolytic Bacillus pumilus strains from Zea mays”, Tarim Bilimleri Dergisi - Journal of Agricultural Sciences, 14(4): 374-380. 2008.
[15]  Rifaat, H.M., Nagieb, Z.A. and Ahmed, Y.M, “Production of xylanases by Streptomyces species and their bleaching effect on rice straw pulp”, Applied Ecology and Environmental Research, 4(1): 151-160. 2005.
[16]  Khandeparker, R.D.S. and Bhosle, N.B, “Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. MTCC 5214 when grown in solid-state fermentation”, Enzyme and Microbial Technology, 39(4): 732-742. 2006.
[17]  Sonia, K.G., Chadha, B.S. and Saini, H.S, “Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation”, Bioresource Technology, 96(14): 1561-1569. 2005.
[18]  Beg, Q.K., Kapoor, M., Mahajan, L. and Hoondal, G.S, “Microbial xylanases and their industrial applications: a review”, Applied Microbiology and Biotechnology, 56(3-4): 326-338. 2001.
[19]  Wakarchuk, W.W., Sung, W.L., Campbell, R.L., Cunningham, A., Watson, D.C. and Yaguchi, M, “Thermo stabilization of the Bacillus circulans xylanase by introduction of disulfide bonds”, Protein Engineering, 7(11): 1379-1386. 1994.
[20]  Panbangred, W., Shinmyo, A., Kinoshita, S. and Okada, H, “Purification and properties of endoxylanase produced by Bacillus pumilus”, Agricultural and Biological Chemistry, 47(5): 957-963. 1983.
[21]  Prade, R.A, “Xylanases: From biology to biotechnology”, Biotechnology and Genetic Engineering Reviews, 13(1): 101-132. 1996.
[22]  Avcioglu, B., Eyupoglu, B. and Bakir, U, “Production and characterization of xylanases of Bacillus strain isolated from soil”, World Journal of Microbiology and Biotechnology, 21: 65-68. 2005.
[23]  Kinegam, S., Tanasupawat, S. and Akaracharanya, A, “Screening and identification of xylanase-producing bacteria from Thai soils”, The Journal of General and Applied Microbiology, 53(1): 57-65. 2007.
[24]  Sanchez, M.M., Fritze, D., Blanco, A., Sproer, C., Tindall, B.J., Schumann, P., Kroppenstedt, R.M., Diaz, P. and Pastor, F.I.J, “Paenibacillus barcinonensis sp. nov., A xylanase-producing bacterium isolated from a rice field in the Ebro river delta”, International Journal of Systematic and Evolutionary Microbiology, 55(2): 935-939. 2005.
[25]  Xu, Z.H., Bai, Y.L., Xu, X., Shi, J.S. and Tao, W.Y, “Production of alkali-tolerant cellulase-free xylanase by Pseudomonas sp. WLUNO24 with wheat bran as the main substrate”, World Journal of Microbiology and Biotechnology, 21: 575-581. 2005.
[26]  Rawashdeh, R., Saadoun, I. and Mahasneh, A, “Effect of cultural conditions on xylanase production by Streptomyces sp. (strain Ib 24D) and its potential to utilize tomato pomace”, African Journal of Biotechnology, 4(3): 251-255. 2005.
[27]  Suto, M., Takebayashi, M., Saito, K., Tanaka, M., Yokota, A. and Tomita, F, “Endophytes as producers of xylanase”, Journal of Bioscience and Bioengineering, 93(1): 88-90. 2002.
[28]  Sneath, P.H.A, “Endospore-forming gram-positive rods and cocci”, In Bergey's Manual of Systematic Bacteriology. Vol 2. Ed., Williams and Wilkins, Baltimore, MD. 9th edition, 1104-1139. 1994.
[29]  Norman, D.L, “Bergey’s Manual of Determinative Bacteriology”, 8th Edition, Buchanan, R.E. and Gibbons, N.E. (eds.). 1974. Williams & Wilkins Co, Baltimore. The Journal of Protozoology, 21(1): 1-7. 1975.
[30]  Bailey, M.J, Biely, P. and Poutanen, K, “Interlaboratory testing of methods for assay of xylanase activity”, Journal of Biotechnology, 23(3): 257-270. 1992.
[31]  Mahesh, M., Somashekhar, R., Bagchi, P. and Puttaiah, E.T, “Optimization for the production of extracellular alkaline phosphatase from Proteus mirabilis”, Journal of Bioprocessing and Biotechniques, 5(3): 1000213. 2015.
[32]  Laemmli, U.K, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature, 227(57): 680-685. 1970.
[33]  Rajashri, D.K. and Anandrao, R.J, “Isolation, purification and characterization of xylanase produced by a new species of Bacillus in solid state fermentation”, International Journal of Microbiology, Article ID 683193. 2012.
[34]  Shivaji, S., Suresh, K., Chaturvedi, P., Dube, S. and Sengupta, S, “Bacillus arsenicus sp. nov., an arsenic-resistant bacterium isolated from a siderite concretion in West Bengal, India”, International Journal of Systematic and Evolutionary Microbiology, 55(3): 1123-1127. 2005.
[35]  Inan, K., Canakci, S. and Beldüz, A.O, “Isolation and characterization of xylanolytic new strains of Anoxybacillus from some hot springs in Turkey”, Turkish Journal of Biology, 35(5): 529-542. 2011.
[36]  Azeri, C., Tamer, A.U. and Oskay, M, “Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp”, African Journal of Biotechnology, 9(1): 63-72. 2010.
[37]  Roy, N. and Rowshanul, H.M, “Isolation and characterization of xylanase producing strain of Bacillus cereus from soil”, Iranian Journal of Microbiology, 1(2): 49-53. 2009.
[38]  Geetha, K. and Gunasekaran, P, “Optimization of nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20”, Biotechnology and Bioprocess Engineering, 15(5): 882-889. 2010.
[39]  Kapilan, R. and Arasaratnam, V, “Paddy husk as support for solid state fermentation to produce xylanase from Bacillus pumilus”, Rice Science, 18(1): 36-45. 2011.
[40]  Beg, Q.K., Bhushan, B., Kapoor, M. and Hoondal, G.S, “Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp”, Enzyme and Microbial Technology, 27(7): 459-466. 2000.
[41]  Chen, W.P., Matsuo, M. and Yasui, T, “Purification and some properties of β-1,3-xylanase from Aspergillus terreus A-07”, Agricultural and Biological Chemistry, 50(5): 1183-1194. 1986.
[42]  Coral, G., Arikan, B., Unaldi, M.N. and Guvenmez, H.K, “Some properties of thermostable xylanase from an Aspergillus niger strain”, Annals of Microbiology, 52(3): 299-306. 2002.
[43]  Malarvizhi, K., Murugesan, K. and Kalaichelvan, P.T, “Xylanase production by Ganoderma lucidum on liquid and solid state fermentation”, Indian Journal of Experimental Biology, 41(6): 620-626. 2003.
[44]  Heck, J.X., Soares, L.H.D.B., Hertz, P.F. and Ayub, M.A.Z, “Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation”, Biochemical Engineering Journal, 32(3): 179-184. 2006.
[45]  Gawande, P.V. and Kamat, M.Y, “Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application”, Journal of Applied Microbiology, 87(4): 511-519. 1999.
[46]  Khandeparker, R. and Bhosle, N.B, “Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp”, Bioresource Technology, 98(4): 897-903. 2007.
[47]  Bisswanger, H, “Enzyme assays”, Perspectives in Science, 1(1-6): 41-55. 2014.
[48]  Honda, H., Kudo, T., Ikura, Y. and Horikoshi, K, “Two types of xylanases of alkalophilic Bacillus sp. No. C-125”, Canadian Journal of Microbiology, 31(6): 538-542. 1985.
[49]  Bernier, R., Desrochers, M.Jr., Jurasek, L. and Paice, M.G, “Isolation and characterization of a xylanase from Bacillus subtilis”, Applied and Environmental Microbiology, 46(2): 511-514. 1983.
[50]  Ohkoshi, A., Kudo, T., Mase, T. and Horikoshi, K, “Purification of three types of xylanases from an alkaliphilic Aeromonas sp”, Agricultural and Biological Chemistry, 49(10): 3037-3038. 1985.
[51]  Kang, M.K., Maeng, P.J. and Rhee, Y.H, “Purification and characterization of two xylanases from alkaliphilic Cephalosporium sp. strain RYM-202”, Applied and Environmental Microbiology, 62(9): 3480-3482. 1996.
[52]  Nagar, S., Mittal, A., Kumar, D., Kumar, L. and Gupta, V.K, “Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation”, New Biotechnology, 28(6): 581-587. 2011.
[53]  Nagar, S., Jain, R.K., Thakur, V.V. and Gupta, V.K, “Biobleaching application of cellulase poor and alkali stable xylanase from Bacillus pumilus SV-85S”, 3 Biotech, 3: 277-285. 2013.
[54]  Sanghi, A., Garg, N., Sharma, J., Kuhar, K., Kuhad, R.C. and Gupta, V.K, “Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation”, World Journal of Microbiology and Biotechnology, 24(5): 633-640. 2008.
[55]  Dhillon, A. and Khanna, S, “Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB16 grown on wheat straw”, World Journal of Microbiology and Biotechnology, 16(4): 325-327. 2000.
[56]  Blanco, A., Vidal, T., Colom, J.F. and Pastor, F.I.J, “Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23”, Applied and Environmental Microbiology, 61(12): 4468-4470. 1995.
[57]  Tseng, M.J., Yap, M.N., Ratanakhanokchai, K., Kyu, K.L. and Chen, S.T, “Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus”, Enzyme and Microbial Technology, 30(5): 590-595. 2002.
[58]  Zhang, G.M., Huang, J., Huang, G.R., Ma, L.X. and Zhang, X.E, “Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina”, Applied Microbiology and Biotechnology, 74(2): 339-346. 2007.