Aspergillus wentii Kaba İnulinaz Aktivitesinin Ön Karakterizasyonu

İnülinazlar fonksiyonel besin maddeleri olarak kullanılan inülin ve fruktooligosakkaritlerin enzimatik hidrolizi ile fruktoz şurubu üretiminde kullanılabilen önemli bir enzim grubudur. Bu çalışmada inulinaz eldesi için yeni bir kaynak olan ve üretim ortamında tek karbon kaynağı olarak yer elması kullanılarak üretilen Aspergillus wentii inulinazının kaba enzimatik ekstraktlarının kısmi karakterizasyonunun gerçekleştirilmesi hedeflendi. Derycke ve Wandamme (1984)’ın kullandığı, karbon kaynağı olarak sadece inülin içeren tarama medyumu, inülin yerine yer elması tozu kullanılarak yeniden düzenlendi ve A. wentii’nin inülinaz üretimi için kullanıldı. A. wentii inulinazı’nın aktivite ve stabilite açısından kısmi karakterizasyonu gerçekleştirildi. Kaba enzimatik ekstrakt için optimum pH, sıcaklık ve inkübasyon süresi sırası ile pH 6,0, 35˚C ve 10 dk olarak belirlendi. A. wentii inülinazı için Km değeri yaklaşık 1×10-4 M, Vmax değeri ise 6,134 mmol/ml/dk olarak hesaplandı. Enzimin pH 3,0-6,0 aralığında aktivitesinin kararlı olduğu (%96,8) ve 50˚C ye kadar aktivitesini koruduğu ve 80˚C’de %62,3 bağıl aktiviteye sahip olduğu görüldü. Elde edilen sonuçlar A. wentii inulinazının subtratı ile olan ilişkisi, iyi bir pH ve termal kararlılığa sahip olması göz önüne alındığında endüstriyel olarak kullanılabilir bir enzim olduğunu doğrulamaktadır. A. wenti inulinazı inulin içeren tarımsal ürünlerden saf fruktoz şurubu hazırlanması için yeni ve uygun bir kaynak olabilir.

Anahtar Kelimeler:

Aspergillus wentii, İnulinaz, yerelması, kısmi karakterizasyonu

PRELIMINARY CHARACTERIZATION OF CRUDE INULINASE ACTIVITY OF ASPERGILLUS WENTII

This study aims to perform a partial characterization of the crude enzymatic extracts of Aspergillus wentii inulinase produced by using the Jerusalem artichoke as the only carbon source in a production medium, which is a new source for obtaining inulinase. The screening medium that contained inulin as the only carbon source used by Derycke and Wandamme (1984) was redesigned to include Jerusalem artichoke powder instead of inulin and was used for the inulinase production of A. wentii. The crude inulinase produced by A.wentii was preliminarily characterized for its activity and stability. Optimum pH, temperature and incubation times for the crude enzymatic extract were determined as pH 6.0, 35°C and 10 min, respectively. The Km and the Vmax values were calculated as approximately 1×10-4 M, and 6.134 mmol/ml/min for A. wentii inulinase, respectively. The enzyme activity was observed to be stable at the intervals of pH 3.0-6.0 (96.8%), to maintain its activity until 50°C, and to have 62.3% relative activity at 80°C. The results confirm that A. wentii inulinase can be an enzyme with industrial treatments due to its relationship with its substrate and its good pH stability and thermal stability. A. wentii inulinase can be a new and usable source for preparation of pure fructose from inulin-containing agricultural products.

Keywords:

Aspergillus wentii, Inulinase, Jerusalem artichoke, partial characterization.,

PDF

___

Arand, M., Golubev, A.M., Neto, J.R.B, Polikarpov, I.W.R.& Korneeva, O.S. 2002. Purification, characterization, Gene Cloning and Preliminary X-ray Data of the Exo-Inulinase from Aspergillus awamori. Biochemical Journal, 362: 131-113.
Artık, N., Mert, İ.& Bayındırlı L. 2011. Karbonhidratlar, Mısır Şekeri ve Gıda Endüstrisinde Kullanımı., TGDF Yayınları, Ankara.
Cazetta, M.L., Martins P.M.M., Monti, R. & Contiero, J.M.R. 2005. Yacon extract as substrate to produce inulinase by Kluyveromyces marxianus var. Bulgaricus. Journal of Food Enginering, 66: 301-305.
Chen, H.Q., Chen, X.M., Li, Y., Wang, J., Jin, ZY., Xu, X.M., Zhao, J.W., Chen, T.X. & Xie, Z.J. 2009.Purification and characterization of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06. Food Chemistry, 115:1206-1212.
Chi, Z., Zhang, T., Liu, G., Yue, L. 2009. Inulinase-expressing microorganisms and applications of inulinases. Microbiology Biotechnology, 82: 211-220.
Derycke, D,G. & Vandamme, E.J. 1984. Production and properties of A. niger inulinase. Journal of Chemical Technology and Biotechnology, 34: 45-51.
Ertan, F., Sanal (E), F., Kaboğlu (Ç), A., Aktaç, T.& Bakar, E. 2005. Some properties of inulinase from Rhizoctonia solani. J.Biol.Science, 5(3) : 330-334.
Fullbrook, PD. 1996. Practical Applied Kinetics. In: Godfrey T, West S (eds) Industrial enzymology. 2nd edition. Stockholm Press, New York, pp 483–540.
Ge, XY., Zhang, W.G. 2005. Effects of octadecanoylsucrose derivatives on the production of inulinase by Apergillus niger SL-09. World Journal of Microbiology and Biotechnology, 21:1633-1638.
Gill, PK., Sharma, A.D., Harchand, RK. & Singh, P. 2003. Effect of media supplements and culture conditions on
inulinase production by an actinomycete strain. Bioresource Technology, 87: 359-362.
Gupta, AK., Gill, A. & Kaur, N. 1998. A HgCI2 insensitive and thermally stable Inulinase from Aspergillus oryzae. Phytochemistry, 49:55-58.
Kang, S., Chang, Y.C., Oh, S.J. & Kim, S. 1998. Purification and properties of an endo-inulinase from an Arthrobacter sp. Biotechnology. Letter,. 20: 983-986.
Kango, N.& Jain, S.C. 2011. Production and properties of Microbial Inulinases: Recent Advances. Food Biotechology, 25: 165-212.
Kıran, E.Ö., Çömlekçioğlu, U. & Dostbil, N. 2006. Mikrobiyal Enzimler ve Endüstride Kullanım Alanları. Kahramanmaraş Sütçüimam Üniversitesi Fen ve Mühendislik Dergisi, 9:12.
Kim, K.K., Nascimento, A.S., Golubev, A.M., Polikarpov, I., Kim, CS., Kang, S. & Kim S. 2008. Catalytic mechanism of inulinase from Arthrobacter sp. S37. Biochemical and Biophysical Research Communications, 371: 600-605.
Kim, M.K., Kim, Y.H., Kim, H.R., Kim, B.I., Byun, S.M. & Uhm T. 1994.Thermal stability of an acidic inulinase from Scytalidium acidophilum, Biotechnology Letters., 16: 965-966.
Kumar, V.V., Premkumar, M.P., Sathyaselvabala, V.K., N.J, Dineshkirupha, S.& Sivanesan, S. 2011. Aspergillus niger exo-inulinase purification by three phase partitionmig. Engineering in Life Science, 11:607-614.
Lima, DM., Oliveira, R.Q., Uetanabaro, A.P., Goes-Neto, A., Rosa, C.A.& Assis, S.A. 2009. Thermostable inulinases secreted by yeast and yeast-like strains from the Brazilian semi-arid region. International Journal of Food Sciences and Nutrition, 60: Suppl 7, 63-71.
Mahmoud, D.A.R., Mahdy El Sayed, M.E., Shousha, W.G.H., Refaat Hala W.,& Abdel Fattah, A.F. 2011. Raw garlic as a new substrate for inulinase production in comparison to dry garlic. Australian Journal of Basic and Applied Sciences , 5: 453-462.
Mutanda, T.B., Wilhelmi, B.& Whiteley, C.G. 2009. Controlled production of fructose by an exoinulinase from Aspergillus ficuum. Applied Biochemistry and Biotechnology, 159: 65-77.
Nakamura, T., Shitara A., Matsuda, S., Matsuo, T., Suiko M.& Ohta, K. 1997. Production, purification and properties of an endoinulinase of Penicillium sp. TN-88 that liberaties inulotriose. Journal of Fermentation and Bioengineering, 84: 313-318.
Ortega, N., Diego, S., Mateos, M.P.& Busto, M.D. 2004. Kinetic properties and thermal behaviour of polygalacturonase used in fruit juice clarification. Food Chemistry, 88: 209-217.
Ricca, E., Calabro, V., Curcia, S., Basso A.& Gardassi, L. 2010. Fructose Production by Inulinase Covalently Immobilized on Sepabeads in Batch and Fluidized Bed Bioreactor. International Journal of Molecular Sciences, 11: 1180-1189.
Sanal E, F., Ertan, F.& Aktac, T. 2005. Production of Exo-inulinase from Alternaria alternata growth on Jerusalem Artichoke and some biochemical properties. Journal of Biological. Science, 5:(4), 497-505.
Selvakumar, P.& Pandey, A. 1999. Isolation and Characterization of inulinase producing strains from Rhizosphere soil. Journal of Scientific and Industrial Research, 57: 621-624.
Sguarezi, C., Longo, C., Ceni, G., Boni, G., Silva, M. Luccio, M., Mazutti, M.A., Maugeri, F., Rodrigues, M. & Treichel, H. 2009. Inulinase Production by Agro-Industrial Residues:Optimization of Pretreatment of Substrates and
Production Medium. Food Bioprocess Technology, 2: 409-414.
Sharma, A.D. & Gill, P.K. 2007. Purification and characterization of heat stable exo-inulinase from Streptomyces sp. Journal of Food Enginering, 79:1172-1178.
Sheng, J., Chi Z., Gong, F. & Li J. 2008. Purification and characterization of extracellular inulinase from a marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase. Applied Biochemistry and Biotechnology, 144: 111-121.
Sing, P. & Gill, P.K. 2006. Production of inulinases: Recent Advances. Food Technology and Biotechnology, 44: 152-162.
Treichel, H., Mazutti, M.A., Maugeri, F. & Rodrigues, M.I. 2009. Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor. Journal of Industrial Microbiology and Biotechnology, 36: 895-900.
Tsujimoto, Y., Watanabe, A., Nakano, K., Watanabe, K., Matsui H., Tsuji, K., Suzuki,Y. & Tsukihara, T. 2003. Gene cloning, expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus KP1289. Applied. Microbiology. Biotechnology, 62: 180-185.
Zhang, T., Gong, F., Peng, Y. & Chi Z. 2009. Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase. Process Biochemistry, 44:1335-1339.
Zittan, L. 1981. Enzymatic Hydrolysis of Inulin-An Alternative Way to Fructose Production. Starch, 33; 373-377.