Production and separation of exo- and endoinulinase from Aspergillus ficuum
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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2018, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :This hydrolysis may be conducted by an enzyme called exoinulinase, a kind of a β-d-fructan fructohydrolase (EC3.2.1.80), which is an extracellular glycoprotein that widely spread in plants and microorganisms, such as fungi, yeast, and bacteria [18,19]. It could split the terminal fructose units with successively releasing β-d-fructose [20,21]. Under anaerobic condition, the similar stepwise hydrolysis process was also observed but with reduced hydrolysis rate when DP5 was incubated in human gut microbiota (data not shown).
Fructose production from Jerusalem artichoke using mixed inulinases
2018, Agriculture and Natural ResourcesCitation Excerpt :Both fructose and inulo-oligosaccharides can be produced from inulin by the enzymatic action of an exo-inulinase (β-D-fructan fructohydrolase, EC 3.2.1.80) acting either alone or in synergy with an endo-inulinase (2,1-β-D-fructan fructanohydrolase, EC 3.2.1.7) (Zhengyu et al., 2005; Ricca et al., 2007; Sirisansaneeyakul et al., 2007a; Chen et al., 2009; Lima et al., 2011; Mutanda et al., 2014). Chemical hydrolysis of inulin to fructose is possible, but produces unwanted by-products and coloration, so the use of inexpensively produced microbial inulinases is preferred for producing fructose and inulo-oligosaccharides from inulin as enzymatic hydrolysis of inulin may yield up to 95% pure fructose (Jing et al., 2003; Ricca et al., 2007). Inulinases are divided into exo- and endo-inulinases depending on their modes of action on inulin, where exo-inulinases act by successively splitting off terminal fructose units in sucrose, raffinose, and inulin to liberate fructose (Sirisansaneeyakul et al., 2007a).