Production, Purification and Characterization of Inulinase from a Newly Isolated Streptomyces sp. CP01

Nirobol Laowklom, Rungtrakarn Chantanaphan, Pairoh Pinphanichakarn

Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.

DOI: 10.4236/nr.2012.33018                

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 K_m and V_max 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.

Keywords

Characterization; Inulinase; Production; Purification; Streptomyces

Conflicts of Interest

The authors declare no conflicts of interest.

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