Abstract
Microbial xylanases and associated enzymes degrade the xylans present in lignocellulose in nature. Xylanase production by Cellulosimicrobium sp. CKMX1, isolated from mushroom compost, produced a cellulase-free extracellular endo-1, 4-β-xylanase (EC 3.2.1.8) at 35 °C and pH 8.0. Apple pomace—an inexpensive and abundant source of carbon—supported maximal xylanase activity of 500.10 U/g dry bacterial pomace (DBP) under solid state fermentation. Culture conditions, e.g., type of medium, particle size of carbon source, incubation period, temperature, initial pH, and inoculum size, were optimized and xylanase activity was increased to 535.6 U/g DBP. CMCase, avicelase, FPase and β-glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with four independent variables (yeast extract, urea, Tween 20 and carboxymethyl cellulose), which resulted in very high levels of xylanase (861.90 U/g DBP). Preliminary identification of the bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16Sr RNA gene sequencing, which identified CKMX1 as Cellulosimicrobium sp. CKMX1. A phylogenetic analysis based on the 16Sr RNA gene sequence placed the isolate within the genus Cellulosimicrobium, being related most closely to Cellulosimicrobium cellulans strain AMP-11 (97% similarity). The ability of this strain to produce cost-effective xylanase from apple pomace on a large scale will help in the waste management of apple pomace.
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References
Altschul SF, Thomas LM, Alejandro AS, Jinghui Z, Zheng Z, Webb M, David JL (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Archana A, Satyanarayana T (1997) Xylanase production by thermophilic Bacillus licheniformis A 99 in solid state fermentation. Enzyme Microb Technol 21:12–17
Bansod SM, Choudhary MD, Srinivasan MC, Rele MV (1993) Xylanase active at high pH from an alkalotolerant Cephalosporium sp. Biotechnol Lett 15:965–970
Battan B, Sharma J, Kuhad RC (2006) High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid state fermentation. World J Microbiol Biotechnol 22:1281–1287
Beg QK, Bhushan B, Kapoor M, Hoondal GS (2000) Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus craft pulp. Enzyme Microb Technol 27:459–466
Beg QK, Kapoor M,Mahajan L,Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–338
Biely PI (1985) Microbial xylanolytic enzymes.Trends Biotechnol 3:286–290
Bocchini DA, Alves-Prado HF, Baida LC, Roberto IC, Gomes E, Da Silva R (2002) Optimization of xylanase production by Bacillus circulans D1 in submerged fermentation using response surface methodology. Process Biochem 38:727–731
Chadha BS, Gulati H, Minhas M, Saini HS, Singh N (2004) Phytase production by the thermophilic fungus Rhizomucarpusilus.World J Microbiol Biotechnol 20:105–109
Corpet F (1988) Multiple sequence alignment with hierarchial clustering. Nucleic Acids Research 16:10881–10890
Ghanem NB, Yusef HH, Mahrouse HK (2000) Production of Aspergillus terreus xylanase in solid state cultures: application of the Plackett-Burman experimental design to evaluate nutritional requirements. Bioresour Technol 73:13–121
Gupta A, Madamwar D (1994) High strength cellulase and β-glucosidase formation from Aspergillus sp. under solid state fermentation. In: Pandey A (ed) Solid state fermentation. Wiley, New Delhi, pp 130–133
Heck J, Flores S, Hertzm P, Ayub M (2005) Optimization of cellulase free xylanase activity by Bacillus coagulans BL69 in solid state cultivation. Process Biochem 30:705–709
Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673–4680
Kim JH, Kim SC, Nam SW (2000) Constitutive overexpression of the endoxylanase gene in Bacillus subtilis. J Microbiol Biotechnol 10:551–553
Maheshwari R, Bhardwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428
Muniswaran PKA, Charyulu NCLN (1994) Solid state fermentation of coconut coir pith for cellulase production. Enzyme Microb Technol 16:436–440
Page RDM (1996) TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358
Pandey A (1990) Improvement in solid state fermentation for glucoamylase production. Biol Wastes 34:11–19
Prakash S, Veeranagouda Y, Kyoung L, Sreeramulu K (2009) Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic Chromohalobacter sp. TPSV 101. World J Microbiol Biotechnol 25:197–204
Rifaat HM, Nagieb ZA, Ahmed YM (2005) Production of xylanases by Streptomyces species and their bleaching effect on rice straw pulp. Appl Ecol Environ Res 4:151–160
Saha BC (2003) Hemicellulose bioconversion. J Ind Microbiol Biotechnol 30:279–291
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Sharma H (1998) Optimization of extracellular xylanase production by Bacillus macerans in solid state fermentation of apple pomace. MSc thesis, Dr Y S Parmar University of Horticulture and Forestry, Nauni-Solan
Sindhu I, Chibber S, Caplash N, Sharma P (2006) Production of cellulase free xylanase from Bacillus megaterium by solid state fermentation for biobleaching of pulp. Curr Microbiol 53: 167–172
Srinivasan MD, Rele MV (1999) Microbial xylanases for paper industry. Curr Sci 77:137–142
Viikari L (1994) Xylanase in bleaching: from an idea to the industry. FEMS Microbiol Rev 13:335–350
Wainø M, Ingvorsen K (2003) Production of β-xylanase and β-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis. Extremophiles 7:87–93
Wang Q, Hou Y, Xu Z, Miao J, Li G (2008) Optimization of cold-active protease production by the psychrophilic bacterium Colwellia spp. NJ341 with response surface methodology. Bioresour Technol 99:1926–1931
Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of 1,4 xylanase in microorganisms functions and applications. Microbiol Rev 52:305–317
Yu X, Hallett SG, Sheppard J, Watson AK (1997) Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletrichumcoccodes spores. Appl Microbiol Biotechnol 47:301–305
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We thank the Department of Science and Technology, under the Ministry of Science and Technology, Government of India, for providing a contingency grant through an Inspire fellowship.
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Walia, A., Mehta, P., Chauhan, A. et al. Optimization of cellulase-free xylanase production by alkalophilic Cellulosimicrobium sp. CKMX1 in solid-state fermentation of apple pomace using central composite design and response surface methodology. Ann Microbiol 63, 187–198 (2013). https://doi.org/10.1007/s13213-012-0460-5
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DOI: https://doi.org/10.1007/s13213-012-0460-5