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The State of the Art in the Production of Fructose From Inulin Enzymatic Hydrolysis

October 2, 2007

By Ricca, Emanuele Calabro, Vincenza; Curcio, Stefano; Iorio, Gabriele

ABSTRACT The present work reviews the main advancements achieved in the last decades in the study of the fructose production process by inulin enzymatic hydrolysis. With the aim of collecting and clarifying the majority of the knowledge in this area, the research on this subject has been divided in three main parts: a) the characteristics of inulin (the process reactant); b) the properties of the enzyme inulinase and its hydrolytic action; c) the advances in the study of the applications of inulinases in bioreactors for fructose production. Many vegetable sources of inulin are reported, including information about their yields in terms of inulin. The properties of inulin that appear relevant for the process are also summarized, with reference to their vegetable origin.

The characteristics of the inulinase enzyme that catalyzes inulin hydrolysis, together with the most relevant information for a correct process design and implementation, are described in the paper. An extended collection of data on microorganisms capable of producing inulinase is reported. The following characteristics and properties of inulinase are highlighted: molecular weight, mode of action, activity and stability with respect to changes in temperature and pH, kinetic behavior and effect of inhibitors. The paper describes in detail the main aspects of the enzyme hydrolysis reaction; in particular, how enzyme and reactant properties can affect process performance. The properties of inulinase immobilized on various supports are shown and compared to those of the enzyme in its native state.

Finally, a number of applications of free and immobilized inulinases and whole cells in bioreactors are reported, showing the different operating procedures and reactor types adopted for fructose production from inulin on a laboratory scale.

KEYWORDS inulin, fructose, enzyme reactions, inulinase

INTRODUCTION

Fructose is a monosaccharide present in a large number of fruits, particularly in apples and tomatoes, in honey, and has been also detected in some mammal’s semen (Percival, 1976; Collins and Munasinghe, 1987). It is the sweetest natural sugar. The chemical formula of fructose is C^sub 6^H^sub 12^O^sub 6^. It can be observed in two enantiomers: D-fructose and L-fructose; the latter cannot be found in nature. Each of them has a linear configuration, but they can rearrange into different interchangeable structures with closed ring configurations. The double bond to oxygen stands on beta position; for this reason D-fructose and L-fructose are often called beta-D-fructose and beta-L-fructose. At normal conditions D- fructose is crystalline and uncolored (Percival, 1976).

Due to its sweetness, fructose is emerging in the world of sweeteners facing the competition with sucrose, which causes problems related to corpulence, cariogenicity, arteriosclerosis and diabetes (Vandamme and Derycke, 1983). The worldwide demand for fructose syrups has increased in recent years due to the inclusion of high fructose content (55%) corn syrup in popular soft drinks. So far its high cost limits its commercial diffusion. A low-cost process for fructose production would be an important result, not only in a public health scenario, but also from an economic point of view, since fructose is approximately 1.5-2 times sweeter than sucrose on an equal weight basis (Fleming and GrootWassink, 1979); this means that any ongoing industrial operation in which sucrose is adopted as a sweetener can be run identically by using a quantity of fructose reduced by a factor 1.5-2.

At present, fructose is being obtained by sucrose inversion thanks to the enzyme invertase or by glucose isomerization carried out by glucose isomerase (Gupta et al., 1992; Curcio et al., 2005). The latter is the main process used in industry today, but is characterized by a strong thermodynamic limitation: the values of the equilibrium concentrations of fructose and glucose are around 50% (Zittan, 1981). As a consequence, the final sugar syrup obtained from this reaction contains about 42% fructose, 50% glucose and 8% oligosaccharides. The subsequent concentration of fructose syrups is carried out by column chromatography techniques, which are very costly (Kochhar et al., 1999); alternatively some additives such as borate may be used to move the thermodynamic equilibrium to 80% fructose concentration, but problems arise with this procedure due to possible toxicity from additives (Fleming and GrootWassink, 1979). Another possibility is the production of fructose by acidic hydrolysis of inulin; in fact, inulin can be hydrolyzed at pH values of 1-2, and 80-100[degrees]C for 1-2 h. In any case, this process did not achieve great development due to the undesirable coloring and flavoring of inulin hydrolysis products and the increase in ash content whose removal, by means of ion exchange resins, is very expensive (Kochhar et al, 1999).

During the last three decades a new idea for producing fructose has been developed: the enzymatic hydrolysis of inulin catalyzed by the enzyme inulinase. This seems to be a promising process, since it brings the final concentration of fructose up to 95% (Rhee and Kim, 1989). This review collects the main advancements in both the catalyst basic research and the applications proposed in the literature until now.

INULIN, THE REACTANT

Information about inulin characteristics and its availability is necessary to understand what kind of reactant is treated in a process for producing fructose via inulinase action.

Inulin is a fructan found in many plants as a storage carbohydrate. From a chemical point of view, it is a polydisperse carbohydrate consisting of beta(2[arrow right]1) fructosyl-fructose links. At one end of the molecule, a glucose residue may be present. In vegetable origin inulin, the number of fructose units linked to a terminal glucose can vary from few units to about 70, which means that inulin is a mixture of oligomers and polymers (Franck and De Leenheer, 2004). It must be pointed out that there is no complete agreement in the literature about an inulin definition; Fleming et al. (1979), for instance, define inulin as a polyfructan with a degree of polymerization-number of monomelic units-, DP, of at least 30.

The inulin DP and the presence of branches are important properties that influence the way inulin reacts, as shown in a forthcoming section, and the product purity, assuming that every inulin molecule contains one glucose residue. Complete hydrolysis of inulin brings the formation of fructose and glucose, whose concentration ratio is proportional to the initial inulin DP. The degrees of polymerization and branching depend on the origin of inulin; in this context a strict distinction must be made between inulin of plant origin and that of bacterial origin. Commercially available inulin, derived from dahlia, Jerusalem artichoke or chicory, has an average DP of about 27-29. Even though it is often thought that inulin has a straight-chain structure, De Leenheer and Hoebregs (1994) pointed out that inulin always contains small amounts of branched molecules; they showed that native inulin from chicory (average DP 6-12) has a very small degree of branching , i.e. 1-2%, and dahlia inulin 4-5%. In contrast to plant inulin, bacterial inulin has a very high DP, ranging from 10,000 to over 100,000; moreover, this inulin is highly branched (>15%) (Franck and De Leenheer, 2004). Ultimately, not only different plants but also different ways of growing give different inulin properties, as extensively reported by Fleming and GrootWassink (1979), who treated agronomic and storage influences on carbohydrate content of Jerusalem artichoke tubers. Of course, the raw material for any possible future large-scale fructose production must be inulin of plant origin, since huge quantities of it will be required; thus a poorly branched, almost linear molecule will need to be treated.

Another important physical property of inulin, which may constitute a limiting factor for its usage as a reactant and, therefore, one of the crucial points of the process, is its low solubility in water. Experimental points from Phelps (1965) are reported as shown in Figure 1.

Figure 1 shows that water solubility of inulin depends on the way it has been recrystallized. Obviously, since a not-well-defined raw material is to be treated in an industrial process, a dispersion of data due to different inulin provenience is to be expected and the Phelps data should be taken as an indication. More recent solubility data show values of about 6% at 10[degrees]C and 35% at 90[degrees]C (Leiten et al., 2004).

As far as inulin availability is concerned, it is important to know that fructans are among the most abundant nonstructural polysaccharides found in nature (Franck and De Leenheer, 2004). They are present in a wide variety of plants and can be obtained from some bacteria and fungi. Moreover, plant inulin can be easily extracted in hot water and the subsequent separation from water is achieved by lowering the temperature (Percival, 1976) and taking advantage of its scarce solubility in water. Rhee and Kim (1989) described the procedure for obtaining juices containing inulin from Jerusalem artichoke and used as feed to the process for producing fructose; they sliced and crushed artichoke tubers, then they adjusted the pH to 4.5, adding phosphoric acid, sterilized at 121[degrees]C for 15 min, and filtered the obtained juice with tissue paper. Wenling et al. (1999) used a similar procedure, but starting from artichoke tuber dry powder and adding water. The entire process from chicory seeds sowing to inulin extraction was reported by Toneli et al. (2007); after the extraction step, a solution with approximately 8[degrees] Brix was obtained. Fleming and GrootWassink(1979) reported different techniques such as squeezing juice out of Jerusalem artichoke tubers, the subsequent addition of water to the tubers prior to extraction, grinding or counter-current diffusion. FIGURE 1 Inulin solubility In water vs temperature; filled symbols = water-recrystalllzed Inulin, empty symbols = ethanol -recrystallized inulin. Adapted from (Phelps, 1965).

Inulin and oligofructose separation and detection can be performed in different ways; a description of these ways has been reported by Bancal et al. (1993).

Inulin-containing plants, which are commonly used for human nutrition, belong mainly to either the Liliaceae, e.g., leek, onion, garlic and asparagus, or the Compositeie, e.g., Jerusalem artichoke, dahlia, chicory (Zittan, 1981; Franck and De Leenheer, 2004); van Loo eted. (1995) reported the measured content of inulin in common plants (Table 1).

TABLE 1 Inulin Content (% of Fresh Weight) of Some Plants; NA = Not Available, *= Estimated Value. Readapted from Franck and De Leenheer [2004]

The van Loo data about chicory roots were confirmed by Monti et ed. (2002), who studied eleven varieties of chicory (Cichorium intybus, L.) cultivated in Italy; inulin content was about 13% of fresh weight and the dry solids contents ranged from 22% to 25% for any chicory variety analyzed.

Many existing research works refer to inulin derived from Cotnpositae: Nakamura et al. (1995) used inulin from dahlia tubers, Derycke et al. (Derycke and Vandamme 1984) used chicory inulin, while Fleming and GrootWassink (1979), Uhm et al. (1982), Remize et al. (1998), Rhee and Kim (1989), Wenling et al. (1999) reported the production of fructose from Jerusalem artichoke tubers inulin. Particularly, Fleming and Groot-Wassink (1979) reported a comparison among yield data of carbohydrates from Jerusalem artichoke and those for corn and sugar beet (the last two can be considered as Jerusalem artichoke competitors in sweetener production, since they are used to produce conventional sugars), showing that Jerusalem artichoke is the richest in carbohydrates. Most of the inulin used in the food industry today is obtained from chicory (Niness, 1999).

The performances of an industrial process for inulin treatment can be strongly affected by the rheological properties of its solutions; these have been determined for inulin concentrations ranging between 2 and 40% w/w in deionized water at room temperature (Zimeri et al., 2003).

INULINASE: THE ENZYME AND ITS CATALYTIC REACTION

The enzyme that hydrolyses beta-1,2-fructan links in inulin is referred to as inulase or beta-1,2-fructan fructanohydrolase (E.C. 3.2.1.7) (Barman, 1969) and it is commonly known as inulinase.

Inulinase Production

Inulinase can be derived from plants and many micro-organisms. Dandelion, chicory and Jerusalem artichoke have been proven to contain inulinase that can be extracted and purified, but according to Kochhar et al. (1999) these plant sources are not as productive as the microbial ones, which seem to be the only source capable of producing enough enzymes for industrial applications. For this reason, in the last three decades, significant efforts have been made to find the best microbial source for the extraction of inulinase.

What distinguishes enzymes produced by different micro-organisms is the yield (units of activity/ml culture broth) of enzyme obtained and the enzyme properties which stricdy depend not only on the kind of micro-organism used, but also on the way the fermentation is carried out. Fungi, yeasts and bacteria are all capable of producing inulinase and many of them have been successfully cultured and subjected to enzyme purification, as reported by Pandey et al. (1999) and by Vandamme and Derycke (1983). These works indicate that the most commonly employed micro-organisms are fungal strains belonging to Aspergillus sp. and yeast strains belonging to Kluyveromyces sp. A list of microorganisms used for the production of inulinases is reported in Table 2.

Ohta eted. (2004) reported the main advances in production, purification and molecular characterization of fungal inulinases, showing their molecular evolution, while Tsujimoto et al. (2003) reported multiple sequence alignment of inulinases from Aspergillus and Penicillium species.

TABLE 2 Inulinase Yield from Different Microorganisms. Yields are Expressed in U/ml if not Otherwise Specified; Data Marked by the Symbol “*” are Expressed in U/g

Enzyme yield and location of enzyme activity seem to be determined by the kind of micro-organism used as source and the substrate used during fermentation. Regarding inulinase yield, among fungi the best results are given by Aspergillus niger (75 U/ml) and A.niger A42 (4600 U/g); some mutant strains of yeasts such as K. marxianus var. marxianus, CBS 6556 and C. pseudotropicalis, IP513 are very interesting, exhibiting yields of 3000 U/ml and 25,000 U/ g, respectively; bacteria do not show an inulinase yield comparable to that of filamentous fungi and yeasts, but their thermophilic nature could turn out to be a great advantage since a higher temperature of fermentation media would be allowed and, therefore, higher solubility of substrates (Pandey eted., 1999). Derycke and Vandamme (1984) compared yields from different micro-organisms (Rhodotoruh sp., Kluyveromyces fragilis, Schizosaccharomyces pombe, Penicillium sp., Rbizopus sp., A. niger, Aspergillus sp.) and observed that A. niger gave the maximum yield; Pandey eted. (1999) reported a vast list of data, shown in Table 2.

A screening among different mutants of Aspergillus niger was performed and some mutants showed an inulinase yield 1.2- to 4.5- fold higher than that of the parent strain (Skowronek and Fiedurek, 2003).

Maximal yields are observed when inulin is used as the substrate, while sucrose has sometimes been shown to exhibit an inhibitory effect on inulinase production; but diis holds true only if the enzyme has no invertase activity; otherwise the presence of sucrose can improve the yield of enzyme in the culture (Vandamme and Derycke, 1983; Pandey et al., 1999). A Kluyveromyces marxianus var. bulgaricus grown in different media showed maximal enzyme production when using inulin as the C-source (170 U/g), instead of fructose (95 U/g), glucose (60 U/g) or sucrose (35 U/g) (Kushi et al., 2000). From an industrial point of view, it is important to note that sometimes a crude plant inulin extract has been reported to be a better carbon source than pure inulin. A comparison among different media used to culture the same strain (A. niger) was made by Derycke and Vandamme (1984); it was found that medium consisting of maltose and corn steep liquor in tap water was better than the media based on inulin or sucrose. They even proved that the substrate has a certain effect on the extra-cellular or intra-cellular nature of the enzymes, but from the literature this property seems to depend mainly on the kind of micro-organism employed; particularly, most of the fungi produce extracellular enzymes, while yeasts-produced enzymes are present both in the culture broth and within the cell walls (Pandey etal., 1999). A comparison among different C-sources in the cultivation of Streptomyces sp. has also been performed; crude inulin was as good as pure inulin, except garlic extract, which showed a 60% inulinase yield enhancement with respect to pure inulin (Sharma et al., 2006).

GrootWassink and Fleming (1980) found no influence of pH on enzyme yield in their work on fermentation of K. fragilis in the range 3.5

A factorial design was used to optimize inulinase production by Kluyveromyces marxianus and the following conditions were established: 14 g/1 of sucrose, 10 g/1 of yeast extract, 20 g/1 of peptone and 1 g/1 of dipotassium hydrogen phosphate (Kalil et al., 2001) and a yield of 127 U/ml (six times larger than before optimization) was found. Separately from the former, another study was conducted in which aeration and agitation conditions in the culture of Kluyveromyces marxianus were investigated; an agitation of 450 rpm and a volumetric air flow rate to broth volume ratio of 1 min^sup -1^ were suggested as optimal (Silva-Santisteban and Filho, 2005).

Purification of extra-cellular inulinases is usually made by the conventional metiiods of centrifugation, ultrafiltration, salt or solvent precipitation, column chromatography, while intra-cellular inulinases require cell wall disruption before practicing the usual metiiodology reported for extra-cellular enzymes (Pandey et al., 1999).

Inulinase Properties and Kinetic Characterization

The process of inulinase extraction does not only determine enzyme yield and whether the enzyme is extraor intra-cellular, but also many other important characteristics, summarized in the following paragraphs.

Inulinase Molecular Weight

Inulinase molecular weight (MW) is strictly related to the producing micro-organism. Kaur etal. (1994) reported a MW of 250 kDa for an inulinase from K. fragilis. MW data for inulinase from four different strains of Aspergillus have been published: A. niger (300 kDa), A. ficuum (53 kDa), A. candidus (54 +- 4 kDa) (Kochhar et al., 1999), and A. awamori var. 2250 (69 kDa) (Arand et ed., 2002); their values range in more than one order of magnitude. But the microbial source is not the only cause of inulinase MW variation, as reported in the next section. Exo and Endo Action

It is clear from the literature (Vandamme and Derycke, 1983; Zittan, 1981; Kochhar et ed., 1999; Derycke and Vandamme, 1984; Pandey, 1999; Arand et al., 2002; Ettalibi and Baratti, 1990; Baratti and Ettalibi, 1987) that two different actions can be exerted by inulinases on inulin molecules: an endwise action and an internal action; the corresponding two kinds of inulinases are called exo-inulinase and endo-inulinase, respectively. The exo- inulinase action begins with the separation of the first D-fructose molecule and goes on until the last linkage within the molecule of inulin is broken and a molecule of D-glucose is released; on the otJier hand, endo-inulinase acts on the internal linkages and yields a set of inulo-oligosaccharides (Bailey and Ollis, 1986). The property of having an exo- or an endoaction also depends on the microbial origin of the enzyme. According to Pandey et al. (1999) inulinases from fungi are generally exo-acting; additional data from different literature sources are reported in Table 3.

One of the most interesting strains in this context appears to be A.ficuum, since it produces both endoand exo-inulinases. Preparations from various strains of Aspergillus spp. were studied by Zittan (1981), who separated two fractions, one containing pure endo-inulinase and the other one containing pure exo-inulinase. Each fraction was characterized separately; the two inulinases showed similar but not identical properties. The audior concluded that a mixture of endo- and exo-inulinase could result in better conversion of inulin to fructose than the isolated pure enzymes. An optimization study was carried out at different endo/exo ratios, from which the optimal ratio resulted to be unity. The reason for this conversion enhancement may be found in the different actions exerted by the two different inulinases: endoinulinases break inulin molecules in many oligosaccharides, thus multiplying the attack points for exoinulinase, with a subsequent increase of the reaction rate of inulin to fructose.

TABLE 3 Modes of Action of Inulinase of Different Microbial Sources

A commercial inulinase from A.ficuum , Novozym 230,(TM) was studied by Ettalibi and Baratti in two works (1990; 1987). They separated the original preparation into five exo-inulinases, three endo-inulinases and an invertase and characterized them separately from kinetic, molecular, stability and activity standpoints. They also evaluated the activities of a pure exo-inulinase, of a pure endo-inulinase and of a mixture of the two inulinases and observed that the mixture exhibited a much higher activity than that given by the sum of the activities of the pure inulinases independently of inulin concentration. Differences in molecular properties between exo-inulinases and endo-inulinases were also highlighted, the former showing MWs of about 74 kDa and the latter 64 kDa, while no deep differences were detected as far as temperature and pH optima and enzyme stability were concerned.

Azhari et al. (1989) found that an exo-inulinase and an endo- inulinase coming from the same strain (Aspergillus) had profoundly different MWs, 81 and 53 kDa, respectively.

An interesting point about the mode of action of exo-inulinases has been outlined by Snyder and Phaff (1962), They proposed that, depending mainly on pH, exo-inulinase could exhibit two kinds of action: a single chain or a multi-chain action. Particularly they noted that at low pH values (3.0) the enzyme attacked the ends of different polysaccharides randomly (multi-chain), while at higher pH (5.1 and 7.0)-and independently of temperature- the enzyme attacked one inulin molecule at time, i.e. its action on an inulin molecule finished when the molecule had been completely degraded into fructose and glucose.

Recently, the X-ray structure of an exo-inulinase from Aspergillus awamori was proposed showing the catalytically relevant residues (Nagem et al., 2004).

A possible mechanism of action of an inulinase from Bacillus polymyxa 722 on inulin was proposed and the presence of imidazole and sulfhydryl groups in its active center was proved (Zherebtsov et al., 2003).

Inulinase Hydrolytic Activity Towards Sucrose

Enzymes capable of hydrolyzing beta-1,2-fructan links and known as inulinases often show a certain activity towards beta-2,6- fructan links, which means that they can hydrolyze sucrose into fructose and glucose. Sucrose hydrolytic enzymes are called invertases (E.C. 3.2.1.26, beta-D-fructo-furanoside-fructo- hydrolase) and present specific characteristics. Starting from this point, a distinction between inulinases and invertases, which constitute the group of beta-fructosidases, is necessary, but it appears difficult and controversial. It is a common practice to distinguish inulinases from invertases according to the ratio alpha = inulinase activity/invertase activity; if alpha > 10^sup -2^ the enzyme is referred to as an inulinase, if alpha

TABLE 4 Invertase vs Inulinase Activity; T = Type of Enzyme

It is important to point out that there are few cases of true inulinases or true invertases; generally an enzyme shows both the activities, although to a different extent.

Clearly an invertase activity of inulinase is desirable, since it is necessary to hydrolyze the ultimate link between glucose and fructose residues remaining after the completion of inulinase action on an inulin molecule.

Ettalibi and Baratti (1990; 1987), as mentioned above, separated invertases and inulinases in a preparation from A. ficuum. Due to the similarities of the responses to inhibitors, pH and temperature, in terms of inulinase activity and invertase activity, the authors postulated that inulinases possess common catalytic sites, but different binding sites for inulin and sucrose hydrolysis.

Enzyme Activity and Stability: Response to Changes in Temperature and pH

Many works in the literature deal with the effects of temperature and pH on inulinase activity and stability. It is certain that the response of the enzyme to these variables depends mainly on the strain used as a source for enzyme production. The results of these researchers are usually expressed in terms of optimal values of T and pH within specified ranges. Some of these are reported in Table 5.

Pandey et al. (1999) noted that fungal inulinases exhibited an optimum pH between 4.5 and 7.0, yeast inulinases between 4.4 and 6.5 and bacterial inulinases between 4.8 and 7.0. Optimal temperature values were generally higher for bacteria and yeasts tiian for fungi.

Information about the shapes of the curves of activity vs. T and pH, rather than only optimal values, is also very important from an industrial point of view, since large ranges mean great flexibility in operating conditions. Some authors have reported the entire curve of activity vs temperature and pH from which ranges reported in Table 5 are extracted.

TABLE 5 Optimal Temperature (T^sub opt^) and pH (pH^sub opt^) for Inulinases from Different Strains; *T Range and pH Range Represent the Ranges of T and pH in Which Inulinases Exhibited 95% of Maximum Activity

However, optimal T and pH values are not necessarily those that would be used in an industrial process, because many other factors, such as enzyme stability, the risk of contamination, inulin solubility and color formation, contribute to define the optimal values of operating conditions. pH values as low as the optimal ones seem to be suggested because they avoid coloring formation, while temperature values which maximize enzyme activity may endanger its stability, due to thermal deactivation; in this regard the need for a high temperature stable enzyme is obvious because it would permit thermal conditions favoring inulin solubility, which may be a limiting factor as shown next, and contrasting contamination, without massive deactivation of the catalyst. Unfortunately, only few works in the literature report information on both activity and stability versus temperature. Data about denaturation of inulinase from A. ficuum (Novozym 230(TM)) were reported by Focher etal. (1991). They conducted a study on thermal deactivation in which they determined the parameters for the Arrhenius equation describing the kinetics of inulinase deactivation, and these were described by 1st order rate equation. Enzyme capability of hydrolyzing inulin into reducing sugars (RS) and fructose-t-glucose (F+G), as well as invertase activity, were taken into account and results are reported in Table 6. Further information about inulinase deactivation is reported in the literature, but the estimation of both Arrhenius parameters was never achieved. Arand et al. (2002) stuthed an inulinase from A. awamori var. 2250 and reported T^sub opt^ = 60[degrees]C, but pointed out that inactivation started at 45[degrees]C and after 24 h incubation at 50[degrees]C the retained activity was 90%, and at 60-70[degrees]C, enzyme activity was retained for only 1-2 h. Vullo et ed. (1991) studied inulinase from Bacillus subtilis 430 A and reported a decrease as high as 20% at 45[degrees]C after 7 h incubation in the presence of inulin, while at 55[degrees]C activity was rapidly lost. Kochhar et al. (1999) reported the loss of inulinase and invertase activity measured at T^sub opt^ after 1 h incubation in the absence of substrate at different temperatures (Table 7). TABLE 6 Arrhenius Parameters: k = Ae^sub -Ea/RT^ for Deactivation of Inulinase from A. ficuum (A [=] min) (Focher et al., 1991)

Remarkably, an inulinase from Streptomyces sp. has been produced with a T^sub opt^ of 70[degrees]C and no activity loss after 6 h incubation at that temperature (Sharma and Gill, 2007).

An inulinase from Aspergillus Jumigatus was obtained and compared to a commercial inulinase produced from Aspergillus niger; the former was proven to be more stable and to have a T^sub opt^ of 60[degrees]C with an appreciable stability even at higher temperatures (Gill et al., 2006).

Other data taken from Beluche et al. (1980) are reported in Table 8.

Many other data about T^sub opt^, pH^sub opt^ and deactivation of immobilized inulinases have been published and are reported in the next sections.

Kinetic Properties

Kinetic properties of inulinases coming from different micro- organisms are reported in the literature. A point oudined by some authors (Derycke and Vandamme, 1984) is fundamental in this context: the different inulin DP and its variation during the reaction progress do not allow a precise estimation of the kinetic parameters and, in general, prevent inulinase kinetics from being described by a Michaelis-Menten type rate equation (Michaelis and Menten, 1913). A complete kinetic study was conducted by Focher et al. (1991) on inulinase from A.ficuum; they found the dependence of k^sub 2^ and K^sub m^ with respect to temperature and gave it under an analytical form (some of the data are reported in Table 9). They found a Michaelis-Menten behavior for both inulin and sucrose hydrolysis activity. Other authors evaluated some kinetic parameters of different strains’ inulinases, but only at one temperature; observed values in terms of Michaelis constant (K^sub m^) and rate coefficient (k^sub 2^) are reported in Table 9.

TABLE 7 Thermal Deactivation of Inulinase from Bacillus subtilis (Kochhar, Gupta and Kaur, 1999)

TABLE 8 Thermal Deactivation of Inulinase from Debaromyces Cantarellil (Beluche, Guiraud and Galzy, 1980)

Azhari et al. (1989) pointed out the necessity to express kinetic parameters of exo- and endo-inulinase on a different basis; for endo- inulinase separated from Novozym 230(TM) they found K^sub m^ = 570 mM and v^sub max^ = 8.3 mM/min, both referring to glycosidic bonds, while for the exo-inulinase from the same preparation they found K^sub m^ = 60 mM and v^sub max^ =3.3 mM/min referring to inulin chain ends.

Effect of Substrate Concentration and Properties

The substrate concentration influences the rate of hydrolysis by inulinase for many reasons. From a kinetic point of view, a high concentration of substrate determines a high rate of reaction; in fact different authors (Zittan, 1981 ; Focher et al., 1991 ; Uhm et al., 1982) found that the rate of reaction increases with inulin concentration and no inhibition effect was observed at any concentration, even though Zittan (1981) noticed that inulin concentration values higher than 12.5% no longer affected the rate of hydrolysis. A significant inulin concentration is also desirable because the presence of substrate has been proven to stabilize the enzyme (Zittan, 1981; Uhm et al., 1982); particularly Uhm et al. (1982) reported that storage at 55[degrees]C for 4 h of an inulinase from K. fragilis, in the presence of inulin, resulted in no loss of activity, while deactivation occurred in the same interval of time at T > 10[degrees]C in the absence of substrate and, in particular, the enzyme retained only 20% of its maximum activity when stored for 4 h at 55[degrees]C without inulin. In any case, high inulin concentrations are limited by its low solubility. Figure 1 shows that solubility of pure inulin at 50[degrees]C is about 1% (w/w), which is very low; fortunately, as mentioned before, in an industrial process, plant inulin characterized by a lower average DP, but higher solubility, will most likely be used. Zittan (1981) suggested an inulin concentration value of 13% (w/w), and Uhm et al. (1982) reported inulin solubilization up to 16%, but any concentration above 10% rapidly led to precipitation. Another important property of inulin influencing the rate of hydrolysis is the DP. Derycke and Vandamme (1984) demonstrated that hydrolysis was much faster when a less refined inulin substrate extracted from chicory (DP = 8) was used than when a pure inulin (DP = 24) was processed. The reason for this evidence could be that, since exoinulinases act on one end of inulin molecule, the higher the DP is, the less available ends are within the reacting mixture.

TABLE 9 Kinetic Parameters; *k^sup cat^ [sec^sup -1^]

TABLE 10 Inhibitors and Effectors of Some Inulinases

Effect of Some Inhibitors and Effectors

Inulinase activity is inhibited by some metal ions and chemicals; data are reported in Table 10.

IMMOBILIZED INULINASES

In the late 1970s, inulinase enzymes started to be immobilized for use in continuous systems. One of the first works on inulinase immobilization was by Nahm et al. (1979), who immobilized inulinases from K. fragilis on Tygon tubes by silanation in chloroform with 10% glutaraldehyde. Since then the enhanced properties of immobilized inulinases have been appreciated and many works have followed. Of course, immobilized enzymes differ from native ones as far as their activity, stability and kinetic properties are concerned. Some characteristics of immobilized inulinases are reported below and compared to those of the same enzyme in its native state.

Rhee and Kim (1989) immobilized inulinase from A. ficuum on chitin and analyzed enzyme activity and stability with respect to glutaraldehyde concentration, temperature and pH. They found that a 3% glutaraldehyde concentration was optimal and that native enzyme treated for 48 h at 4[degrees]C with such a concentration did not show any activity decay. The values of pH^sub opt^ (4.5) and T^sub opt^ (60[degrees]C) were found almost coincident with those characterizing the native enzyme behavior, while the immobilized enzyme was a little more stable as compared to the native enzyme. At 40[degrees]C the immobilized inulinase showed a good stability, exhibiting a half-life of 561 h.

TABLE 11 Kinetic and Activity Data on Immobilized Inulinases

Nakamura et al. (1995) immobilized an inulinase from A. niger on Amino-Cellulofine, a support chosen among different materials (CNBr- activated Sepharose 4B, cellulose carbonate, bromoacetyl-cellulose, Carboxy-Cellulofine), for immobilization and the activity yields achieved were 96% and 15%, respectively, with an enzyme concentration of 9 mg/ml. T^sub opt^ and pH^sub opt^ are shown in Table 11.

Wenling et al. (1999) immobilized inulinase from Kluyveromyces sp. Y-85 on polystyrene beads; they added 0.5 g beads to different amounts of an enzyme solution of 50 U/ml and found that the optimal enzyme solution amount was 10 ml. Glutaraldehyde concentration was optimal at 0.03%. T^sub opt^ and pH^sub opt^ were 55[degrees]C and 5.0, respectively, higher than those of the free enzyme (50[degrees]C and 4.5). Immobilization did not seem to bring any stability enhancement with respect to changes of pH, while improved enzyme thermal stability, activity retained being equal to 95% and 67% for immobilized and native enzyme, respectively (in both cases incubation was carried out for 24 h, at 50[degrees]C and pH = 5.0)

TABLE 12 Half-Life of Inulinase from K. fragilis [Kaur et al., 1994]

Uhm et al. (1982) immobilized inulinase from K. fragilis on aminoethylcellulose with an optimal glutaraldehyde concentration of 2%. pH^sub opt^ was 5.5, the same as for free enzyme, while T^sub opt^ (45[degrees]C) was anomalously lower than that observed for native inulinase (55[degrees]C). The authors evaluated immobilized enzyme deactivation in a 7% inulin solution and noted that no loss of activity occurred witiiin the first 4 days; after this period of time a first order deactivation kinetic was observed with a deactivation constant, K^sub d^, equal to 0.05 days^sup -1^.

Gupta et al. (1992) made a comparison among inulinases immobilized on different supports. They immobilized inulinase from Fusarium oxysporum on soybean, mungbean and hen egg white derivatives and on DEAE-cellulose; DEAE-cellulose proved to be the best support in terms of inulinase activity retention (40%). On the other hand, temperature optimal values were higher for soybean and mungbean derivatives (45[degrees]C) than for DEAE-cellulose (37[degrees]C, the same as native enzyme). pH^sub opt^ was also dependent on the support used (see Table 11), while for native enzyme it was equal to 6.2. DEAE-cellulose was the support that gave the best thermal stability (half-life of 45 min at 50[degrees]C). K^sub m^ values were also estimated and are reported in Table 11.

TABLE 13 Batch Reactor Performance with Free Enzyme (Zittan, 1981)

Kochhar et al. (1999) made a similar work comparing inulinase from Aspergillus candidus immobilized on chitin, casein and cellulose. Using cellulose as support gave a maximal immobilization yield (45.8%) and the best thermal stability (after 1 h heating at 55[degrees]C immobilized inulinase retained 76% of its activity), while temperature optimal values were equal for the three cases (55[degrees]C) and higher than that of free enzyme (45[degrees]C). K^sub m^ values are reported in Table 11.

Kaur et al. (1994) proved that the same enzyme was more resistant to heat when immobilized on cellulose than in its free state after purification, while the crude preparation was the most stable of all; data are reported in Table 12.

Factorial design and surface response methodology were also used to determine optimal operating conditions for the commercial inulinase Fructozyme L(TM) immobilized onto Amberlite; values of pH^sub opt^ 5.5 and T^sub opt^ 50[degrees]C were found experimentally and accurately predicted by the model proposed (Rocha et al., 2006). APPLICATIONS OF INULINASES TO FRUCTOSE PRODUCTION

In the literature, several biochemical reactors have been described for the hydrolysis of inulin to fructose. Many authors obviously report about batch operations, which are used to characterize the enzymes, but more complex systems are also used, such as packed bed reactors (PBR) with immobilized enzymes and reactors using whole cells containing inulinase.

Batch Operations with Free Enzymes

Data from batch operations may give an idea of the efficacy of certain inulinases in hydrolyzing inulin. Zittan (1981), in his study on Aspergillus inulinase, suggested the best conditions to be used to run an enzymatic batch reactor; those indications are summarized in Table 13.

Derycke and Vandamme (1984) reported results on inulin batch hydrolysis by 1 ml of inulinase solution from A. niger in 49 ml of a 5% inulin solution, at T = 55[degrees]C and pH = 4 for two substrates (Tables 14a and 14b).

TABLE 14 (a) Hydrolysis Performance with Refined Inulin; (b) Hydrolysis Performance with Chicory Extract; Data from Derycke and Vandamme (1984)

Immobilized Enzymes

Reactions carried out by immobilized enzymes seem to be more attractive for process proposals and many works are present in the literature. Some authors reported on immobilized inulinases used in batch reactors (Kochhar et al., 1999; Rhee and Kim, 1989; Uhm et al., 1982), while others evaluated the performances of continuous reactors packed with immobilized inulinases on different supports (Gupta et al., 1992; Rhee and Kim, 1989; Wenling et al., 1999; Nakamura et al., 1995; Uhm et al., 1982).

Kochhar et al. (1999) found that a 1 g preparation of cellulose immobilized inulinase from Aspergillus candidus hydrolyzed 89% of 10 ml of 1% inulin in 6 h at 37[degrees]C. Rhee and Kim (1989) stuthed the hydrolysis of Jerusalem artichoke tuber juice by inulinase from A. ficuum (Novozym 230(TM)) in both batch and continuous reactors. They incubated 400 ml of juice containing 100 g/l of total carbohydrate in a batch reactor with 1536 units of inulinase immobilized on 20 g of chitin at 40[degrees]C; the percentage of hydrolysis was 80% after 4 h run and 90% after 10 h. In the PBR continuous process, they used the same conditions as for the batch system (immobilized enzyme dosage, temperature, feed concentration); at a residence time of 4.5 h almost 90% hydrolysis was achieved.

Uhm et al. (1982) conducted the hydrolysis of inulin in a PBR after proving that, with other conditions unchanged, reactors with different height/diameter (H/D) ratios had different steady state conversions; particularly, among three columns characterized by the H/D ratios 3.9, 10.3, 21.3, respectively, the one with H/D = 10.3 showed the best performance. In such a column they packed 28 ml of aminoethylcellulose, on which 167.6 units of inulinase from K. fragilis had been immobilized. An inulin solution (7%) was almost completely converted at 40[degrees]C with a space time of 6 h and the half-life of the immobilized enzyme under these conditions was 13.9 days.

TABLE 15 PBR Performance (Gupta et al., 1992)

Gill et al. (2006) performed PBR tests with an exoinulinase from Aspergillus fumigatus immobilized on chitin, QAE-Sephadex and ConA linked-amino activated silica beads at 60[degrees]C, 2.5% inulin concentration, for 50 days, observing 35-, 22-, 45-day half lives and 2.7, 2.3, 3.4 g/(l*h) productivities, respectively.

Another experience on a packed column was reported by Gupta et al. (1992); data from their work are summarized in Table 15.

Nakamura et al. (1995) investigated the performance of a PBR in different conditions in order to find operating conditions that would permit complete hydrolysis of inulin at pH 5.0. The following ranges were considered: inulin solution concentration from 2.5 to 10% (w/v), flow rates from 0.5 to 8.0 ml/min and temperatures from 30[degrees] to 60[degrees]C. From a purely kinetic point of view, higher temperatures were advisable, since they allowed complete hydrolysis of more concentrated solutions pumped at higher flow rates. When taking into account deactivation aspects it was found that complete hydrolysis could be carried on for 45 days at 40[degrees]C and only for 2 days at 60[degrees]C; in the latter case the enzyme showed a half-life of 16 days.

Wenling et al. (1999) analyzed different operating conditions. A 4.5% (w/v) inulin solution was completely hydrolyzed at 50[degrees]C by inulinase obtained from Kluyveromyces sp. Y-85 and immobilized on macroporous ionic polystyrene beads (D201-GM resin); the reactor packed with those beads and fed at a flow rate of 30 ml/h showed a half-life of 32 days.

The commercial enzyme Fructzyme L(TM), immobilized by Rocha et al. (2006) on Amberlite, was washed and reused at 50[degrees]C in consecutive reaction cycles, but it revealed a significant loss of activity even after the first usage.

A shell-tube configuration membrane bioreactor with immobilized enzymes has recently been tested, showing that 50% activity was retained after five reaction cycles of 5 h each. Best performances were obtained with shell-side immobilization, while fiber diameter was found crucial to prevent clogging. However, as the authors pointed out, further improvement of the immobilization technique should be made in order for the membrane reactor to gain industrial importance (Diaz et al., 2006).

Whole Cells

Some applications using immobilized whole cells have been also reported. Workman and Day (1984) immobilized inulinase from K . fragilis on yeast cells by using glutaraldehyde. This treatment prevented the enzyme from being easily disrupted by physical or chemical action and reportedly did not affect enzyme activity. Experiments proved that a 0.24% (w/v) fixed enzyme suspension completely converted a Jerusalem artichoke extract in 3.5 h.

Results from batch and continuous reactors using entire cells have been reported by Bajpai and Margaritis (1985). They immobilized K. marxianus in an open pore gelatin matrix and they observed a 93% hydrolysis of an extract from Jerusalem artichoke treated for 3 h in a batch reactor. In addition, the same immobilized cells were used repeatedly in ten batch cycles without showing any decrease of the hydrolysis yield at the end of each cycle. The immobilized cells were also used in a PBR. At a dilution rate of about 2 h^sup -1^ and a conversion of 80%, the volumetric productivity was found to attain its maximum (100 g l^sup -1^ h^sup -1^). Cell loss through leakage was low, proving that cells immobilized within the gelatin matrix were quite stable.

CONCLUSIONS

In this review, the main aspects of the fructose production process by inulin enzymatic hydrolysis of inulin have been reported.

In particular, information about inulin seems to be complete, since many sources are known and so are the characteristics of inulin as the process substrate. On the other hand, as far as the properties of the enzyme inulinase are concerned, even though many works have been published on the subject, much work is still necessary due to enzyme complexity. Many micro-organisms capable of producing inulinase are known, but the search for an economically interesting source is still under way. Moreover, much information about inulinase properties exists, but there is an almost complete lack of systematic studies on reaction kinetics and enzyme deactivation; this is particularly true for immobilized inulinases. Regarding the use of enzymatic reactors, only a few experimental data are present in the literature, especially for reactors packed with immobilized inulinases (PBR). Neither are there significant approaches to process modelling and simulation that could provide a complete understanding and prediction of the phenomena that take place in these reactors. This certainly poses a limit in order for the process of fructose production from inulin enzymatic hydrolysis to become an industrial reality.

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Emanuele Ricca, Vincenza Calabro, Stefano Curcio, and Gabriele Iorio

Department of Chemical

Engineering and Materials,

University of Calabria,

Arcavacata di Rende, Italy

Address correspondence to Gabriele Iorio, Department of Chemical Engineering and Materials, University of Calabria, 1-87030 Arcavacata di Rende (CS), Italy. E-mail: gabriele.iorio@unical.it

Copyright Taylor & Francis Ltd. Jul-Sep 2007

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