NR> Vol.3 No.3, September 2012
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Production, Purification and Characterization of Inulinase from a Newly Isolated Streptomyces sp. CP01

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ABSTRACT

Inulinase is an enzyme catalyzing the hydrolysis of inulin, a plant reserve polysaccharide, into fructoses and fructooligosaccharides which are widely used as food additives. Here we report inulinase from a newly isolated Streptomyces as in the past decade there have been very few reports on inulinases from Streptomyces, especially purification and characterization of these enzymes. Out of 371 Streptomyces isolates, Streptomyces sp. CP01 produced highest inulinase activity of 0.50 U/ml. The enzyme activity was increased to 1.60 U/ml when CP01 was cultivated under the optimal conditions which consisted of using basal medium (Czapek’s Dox) containing 1% (w/v) inulin extract from Jerusalem artichoke’s root tubers and 0.7% (w/v) tryptone at pH8, shaking at 200 rpm and 28℃ for 24 h. The enzyme was purified from culture filtrate to about 67-fold purity by (NH4)2SO4 precipitation followed by four consecutive column chromatography steps. The purified enzyme is a single peptide with approximate molecular mass of 73 kDa as analyzed by gel filtration and 70.8 kDa as assessed by SDS-PAGE. The enzyme is optimally active at 55℃ and pH 6.0, however it still possesses more than 80% of the maximal activity at pH ranging from 5.5 to 9.0. It is stable at temperature up to 50℃ and at broad range of pH from 5.0 to 9.0 for 30 min. Its Km and Vmax values for inulin were 2.34 mM and 440 μmolmin–1mg–1, respectively. This enzyme has potential for industrial application as it is active at moderately high temperature and wide range of pH.

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Cite this paper

N. Laowklom, R. Chantanaphan and P. Pinphanichakarn, "Production, Purification and Characterization of Inulinase from a Newly Isolated Streptomyces sp. CP01," Natural Resources, Vol. 3 No. 3, 2012, pp. 137-144. doi: 10.4236/nr.2012.33018.

References

[1] E. J. Vandamme and D. G. Derycke, “Microbial Inulinases: Fermentation Process, Properties, and Applica-tions,” Advances in Applied Microbiology, Vol. 29, No. 1, 1983, pp.139-176. doi:10.1016/S0065-2164(08)70356-3
[2] I. Trojanova, V. Rada, L. Kokoska and E. Vlkova, “The Bifidogenic Effect of Taraxacum Officinale Root,” Fitoterapia, Vol. 75, No.7-8, 2004, pp. 760-763. doi:10.1016/j.fitote.2004.09.010
[3] A. K. Gupta and N. Kaur, “Fructan Storing Plants a Potential Source of High Fructose Syrups,” Journal of Scientific and Industrial Research, Vol. 56, 1997, pp. 447-452.
[4] S. E. Flemming and J. W. Grootwassink, “Preparation of HighFructose Syrup from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.),” Critical Reviews in Food Science and Nutrition, Vol. 12, No. 1, 1979, pp. 1- 28. doi:10.1080/10408397909527271
[5] K. Szambelan, J. Nowak and Z. Czarnecki, “Use of Zymomonas mobilis and Saccharomyces cerevisiae Mixed with Kluyveromyces fragilis for Improved Ethanol Production from Jerusalem artichoke Tubers,” Biotechnology Letters, Vol. 26, No. 10, 2004, pp. 845-848. doi:10.1023/B:BILE.0000025889.25364.4b
[6] N. Kango, “Production of Inulinase Using Tap Roots of Dandelion (Taraxacum officinale) by Aspergillus niger,” Journal of Food Engineering, Vol. 85, No. 3, 2008, pp. 473-478. doi:10.1016/j.jfoodeng.2007.08.006
[7] R. S. Singh, R. Dhaliwal and M. Puri, “Production of Inulinase from Kluyveromyces marxianus YS-1 Using Root Extract of Asparagus racemosus,” Process Biochemistry, Vol. 41, No. 7, 2006, pp. 1703-1707. doi:10.1016/j.procbio.2006.03.005
[8] W. Gao, Y. Bo, Y. Liu, X., Zhang, J. Wang and L. An, “Characterization of Thermo-Stable Endoinulinase from a New Strain Bacillus smithii T7,” Applied Biochemistry and Biotechnology. Vol. 157, No. 3, 2009, pp. 498-506. doi:10.1007/s12010-008-8313-1
[9] W. Wei, S. Wang, X. Zhu and W. Wan, “Isolation of a Mutant of Kluyveromyces sp. Y-85 Resistant to Catabo- lite Repression,” Journal of Bioscience and Bioengineering, Vol. 87, No. 6, 1999, pp. 816-818. doi:10.1016/S1389-1723(99)80159-5
[10] X. Yu, N. Guo, Z. Chi, F. Gong, J. Sheng and Z. Chi, “Inulinase Overproduction by a Mutant of the Marine Yeast Pichia guilliermondii Using Surface Response Methodology and Inulin Hydrolysis,” Biochemical Engineering Journal, Vol. 43, No. 3, 2009, pp. 266-271. doi:10.1016/j.bej.2008.10.018
[11] P. Selvakumar and A. Pandey, “Solid State Fermentation for the Synthesis of Inulinase from Staphylococcus sp. and Kluyveromyces marxianus,” Process Biochemistry, Vol. 34, No. 8, 1999, pp. 851-855. doi:10.1016/S0032-9592(99)00008-4
[12] A. D. Sharma, S. Kainth and P. K. Gill, “Inulinase Production Using Garlic (Allium sativum) Powder as a Potential Substrate in Streptomyces sp.,” Journal of Food Engineering, Vol. 77, No. 3, 2006, pp. 486-491. doi:10.1016/j.jfoodeng.2005.06.072
[13] P. K. Gill, A. D. Sharma, R. K. Harchand and P. Singh, “Effect of Media Supplements and Culture Conditions on Inulinase Production by an Actinomycete strain,” Bioresource Technology, Vol. 87, No. 3, 2003, pp. 359-362. doi:10.1016/S0960-8524(02)00262-6
[14] M. Hayakawa and H. Nonomura, “Humic Acid Vitamin Agar, a New Medium for the Selective Isolation of Soil Actinomycetes,” Journal of Fermentation Technology, Vol. 65, No. 5, 1987, pp.501-509. doi:10.1016/0385-6380(87)90108-7
[15] T. Kieser, M. J. Bibb, M. J. Buttner, K. F. Chater and D. A. Hopwood, “Practical Streptomyces Genetics,” John Innes Foundation, Norwich, 2000.
[16] A. D. Sharma and P. K. Gill, “Purification and Characterization of Heat Stable Exo-Inulinase from Streptomyces sp.,” Journal of Food Engineering, Vol. 79, No. 4, 2007, pp. 1172-1178. doi:10.1016/j.jfoodeng.2006.04.008
[17] M. Kataoka, K. Ueda, T. Kudo, T. Seki and T. Yoshida, “Application of the Variable Region in 16S rDNA to Create an Index for Rapid Species Identification in the Genus Streptomyces,” FEMS Microbiology Letters, Vol. 151, No. 2, 1997, pp. 249-255. doi:10.1111/j.1574-6968.1997.tb12578.x
[18] W. Lingyun, W. Jianhua, Z. Xiaodong, T. Da, Y. L. Yang, C. G. Cai, F. Tianhua and Z. Fan, “Studies on the Extracting Technical Conditions of Inulin from Jerusalem artichoke Tubers,” Journal of Food Engineering, Vol. 79, No. 3, 2007, pp. 1087-1093. doi:10.1016/j.jfoodeng.2006.03.028
[19] G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars,” Analytical Chemistry, Vol. 31, No. 3, 1959, pp. 426-428. doi:10.1021/ac60147a030
[20] 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. 267-275. doi:10.1038/227680a0
[21] 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.
[22] A. Pandey, C. R. Soccol, P. Selvakumar, V. T. Soccol, N. Krieger and D. Jose. “Recent Developments in Microbial Inulinases, Its Production, Properties and Industrial Applications,” Applied Biochemistry and Biotechnology, Vol. 81, No. 1, 1999, pp. 35-52. doi:10.1385/ABAB:81:1:35
[23] A. Pessoa Jr. and M. Vitolo, “Inulinase from Kluyveromyces marxianus: Culture Medium Composition and Enzyme Extraction,” Brazilian Journal Chemical Engineering, Vol. 16, No. 6, 1999, pp. 237-245.
[24] M. L. Cazetta, P. M. M. Martins, R. Monti and J. Contiero, “Yacon (Polymnia sanchifolia) Extract as a Substrate to Produce Inulinase by Kluyveromyces marxianus var. bulgaricus,” Journal of Food Engineering, Vol. 66, No. 3, 2005, pp. 301-305. doi:10.1016/j.jfoodeng.2004.03.022
[25] J. Sheng, Z. M. Chi, J. Li., L. M. Gao and F. Gong, “Inulinase Production by the Marine Yeast Cryptococcus aureus G7a and Inulin Hydrolysis by the Crude Inulinase,” Process Biochemistry, Vol. 42, No. 5, 2007, pp. 805-811. doi:10.1016/j.procbio.2007.01.016
[26] E. M. Fawzi, “Comparative Study of Two Purified Inulinasees from Thermophile Thielavia terresttris NRRL 8126 and Mesophile Asspergillus foetidus NRRL 337 Grown on Chicorium intybus L.,” Brazilian Journal of Microbiology, Vol. 42, No. 2, 2011, pp. 633-649. doi:10.1590/S1517-83822011000200028
[27] T. Nakamura, Y. Nagatomo, S. Hamada, Y. Nishino and K. Ohta, “Occurrence of Two Forms of Extracellular Endo-Inulinase from Aspergillus niger Mutant 817,” Journal of Fermentation and Bioengineering, Vol. 78, No. 2, 1994, pp. 134-139. doi:10.1016/0922-338X(94)90251-8
[28] H. J. Kwon, S. J. Jeon, D. J. You, K. H. Kim, Y. K. Jeong, Y. H. Kim, Y. M. Kim and B. W. Kim, “Cloning and Characterization of an Exoinulinase from Bacillus polymyxa.,” Biotechnology Letters, Vol. 25, No. 2, 2003, pp. 155-159. doi:10.1023/A:1021987923630
[29] R. T. Kushi, R. Monti and J. Contiero, “Production, Purification and Characterization of an Extracellular Inulinase Came from Kluyveromyces marxianus var. bulgaricus,” Journal of Industrial Microbiology and Biotechnology, Vol. 25, No. 2, 2000, pp. 63-69. doi:10.1038/sj.jim.7000032
[30] N. Kaur, M. Kaur, A. Gupta and R. Singh, “Properties of β-Fructosidases (Invertases and Inulinases) of Fusarium oxysporum Grown on Aqueous Extract of Cichorium intybus Roots,” Journal of Chemical Technology and Biotechnology, Vol. 53, No. 3, 1992, pp. 279-284. doi:10.1002/jctb.280530308
[31] A. E. Cruz-Guerrero, I. Garcia-Pena, E. Barzana, M. Garcia-Garibay and L. Gomez-Ruiz, “Inulinase-Hyperproduction Strains of Kluyveromyces marxianus CDBB-L- 278: A Wild Inulinase Hyperproducing Strain,” Journal of Fermentation and Bioengineering, Vol. 80, No. 2, 1995, pp. 159-163. doi:10.1016/0922-338X(95)93212-3
[32] R. S. Singh, B. S. Sooch and M. Puri, “Optimization of Medium and Process Parameters for the Production of Inulinase from a Newly Isolated Kluyveromyces marxianus YS-1,” Bioresource Technology, Vol. 98, No. 13, 2007, pp. 2518-2525. doi:10.1016/j.biortech.2006.09.011
[33] F. Ertan, F. E. Sanal, A. C. Kaboglu, T. Aktac and E. Baker, “Some Propoties of Inulinase from Rhizoctonia solani,” Journal of Biological Sciences, Vol. 5, No. 3, 2005, pp. 330-335. doi:10.3923/jbs.2005.330.334
[34] P. K. Gill, R. K. Manhas, J. Singh and P. Singh, “Purification and Characterization of Exoinulinase from Aspergillus fumigates,” Applied Microbiology and Biotechnology, Vol. 117, No. 1, 2004, pp. 19-32.
[35] T. Nakamura, A. Shitara, S. Matsuda, T. Matsuo, M. Suiko and K. Ohta, “Production, Purification and Properties of an Endoinulinase of Penicillium sp. TN-88 That Liberates Inulotriose,” Journal of Fermentation and Bioengineering, Vol. 84, No. 4, 1997, pp. 313-318. doi:10.1016/S0922-338X(97)89250-1
[36] J. Sheng, Z. M. Chi, F. Gong and J. Li, “Purification and Characterization of Extracellular Inulinase from a Marine Yeast Cryptococcus aureus G7a and Inulin Hydrolysis by the Purified Inulinase,” Applied Biochemistry and Biotechnology, Vol. 144, No. 2, 2008, pp. 111-121. doi:10.1007/s12010-007-8025-y
[37] F. Gong, Z. M. Chi, J. Sheng, J. Li and X. H. Wang, “Purification and Characterization of Extracellular Inulinase from a Marine Yeast Pichia guilliermondii and Inulin Hydrolysis by the Purified Inulinase,” Biotechnology and Bioprocess Engineering, Vol. 13, No. 5, 2008, pp. 533-539. doi:10.1007/s12257-007-0177-7

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