Elsevier

Process Biochemistry

Volume 39, Issue 1, 30 September 2003, Pages 5-11
Process Biochemistry

Production and separation of exo- and endoinulinase from Aspergillus ficuum

https://doi.org/10.1016/S0032-9592(02)00264-9Get rights and content

Abstract

The production of both exo- and endoinulinase by Aspergillus ficuum JNSP5-06 was investigated. Optimum fermentation conditions were found to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4·7H2O, 0.05%; ZnSO4·7H2O, 0.01%; initial pH 6.5. A new and convenient method was developed to separate the inulinases by native-polyacrylamide gel electrophoresis (PAGE). Eight protein bands were obtained. Three bands were identified as exoinulinase and two bands were endoinulinase using TLC and HPLC.

Introduction

Inulin, a fructose polymer found as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory and dahlia, represents a potential source of fructose and inulooligosaccharides that can be used as sweeteners and functional food additives. This fructan consists of a linear chain of fructose (β-2,1-link) with a terminal glucose unit [1]. Inulin is depolymerized by two types of inulinase: exoinulinase (β-d-fructan fructohydrolase, EC 3.2.1.80), endoinulinase (2,1-β-d-fructan fructanohydrolase, ECβ-3.2.1.7). Exoinulinase can successively release fructose from the non-reducing β-2,1 end of inulin. The complete hydrolysis of inulin by this enzyme yields 95% fructose syrup under optimized condition [2]. Endoinulinase acts on the internal linkage of inulin randomly to release inulo-triose, -tetraose and -pentaose as the major products [3].

Most microbial inulinases are exo-acting enzymes. Nakamura et al. [4] first reported that a strain of Aspergillus niger excreted two distinct inulin-hydrolysing enzymes, endo- and exoinulinase. Thereafter, Penicillium purpurogenum and Chrysosporium pannorum were reported to produce both endo- and exoinulinase [5], [6]. A. ficuum, one of the industrially important fungi, also excreted endoinulinase as well as exoinulinase [7].

Because of the synergistic action of the two enzymes [8], fructose is easily obtained; however, it is difficult to determine whether the enzymes coexist. Similarly, it is difficult to separate the two enzymes completely by conventional methods as they are very similar in properties [9]. This paper describes the production of inulinase by an inulinase-producing strain, A. ficuum, and the development of a new and convenient method to separate and identify exo- and endoinulinases.

Section snippets

Micro-organisms and culture conditions

The strain isolated from a soil sample was identified as A. ficuum JNSP5-06. The strain was maintained on solid medium at 4 °C. The basal medium contained the following: 2% inulin, 2% peptone, 1.2% (NH4)H2PO4, 0.5% NaCl, 0.05% MgSO4·7H2O, initial pH 5.5.

Chemicals

Inulin purchased from Orafiti Company. Other chemicals were all of analytical grade.

Assay of enzyme activities

Enzymes were assayed by measuring the concentration of reducing sugars released from inulin and sucrose. The reaction mixture containing 1 ml of diluted crude

Effect of different carbon sources on the production of inulinase

On growing A. ficuum in media containing different carbon sources, the maximum inulinase production was observed with medium containing inulin (Table 1). Fructose, which is believed to be the primary inducer of inulinase [13], produced an inulinase yield that was about 30% of that observed with inulin. This is an indication that inulin is a potential inducer of inulinase.

Effect of different nitrogen sources on the production of inulinase

Inulinase activity increased following the replacement of peptone by yeast extract (Table 2). Penicillium sp. TN-88 was also

Discussions

The medium composition used for inulinase production depends on the type of micro-organism involved. A. ficuum, a thermostable industrial strain, produced inulinase in response to the presence of inulin. Yeast extract and (NH4)H2PO4 in the medium were suitable organic and inorganic nitrogen sources, respectively, for the synthesis of inulinase while Mg2+ and Zn2+ further increased the inulinase level. After 5 days fermentation under optimal conditions inulinase activity attained 25 U/ml, which

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