Keywords

Introduction

Inulin is non‐structural polysaccharide, used as energy‐rich compounds, and also has role in plant metabolism and energy storage. After starch, inulin is one such storage polysaccharide found widely dispersed in many plants. Inulin is made of β-(2 → 1) linked linear poly-fructose units (2–60) terminated by a sucrose residue (Fig. 15.1) (Kango and Jain 2011; Rawat et al. 2015a). Plants that store and synthesize inulin usually do not store other materials as energy reserve. Several temperate and tropical plants, such as dandelion (Taraxacum officinale), chicory (Cichorium intybus), Jerusalem artichoke (Helianthus tuberosus), dahlia (Dahlia pinnata), and asparagus (Asparagus officinale) (Table 15.1), reserve this polymer (Kango 2008; Chi et al. 2011; Rawat et al. 2016).

Fig. 15.1
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Structure of inulin (F—fructose, G—glucose)

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Table 15.1 Inulin content of some plants
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Commercial production of fructose by inulin hydrolysis is more effective than other conventional method which required starch hydrolysis by the action of group of enzymes (α-amylase, amyloglucosidase, and glucose isomerase) liberating less fructose (~45%) in the end product. Inulin hydrolysis using microbial inulinase yields 90–95% fructose solution. Fructose syrup production from inulin-rich material is a major area of inulinase application (Kango 2008; Vijayaraghavan et al. 2009; Liu et al. 2013).

Inulin is utilized by a variety of fungi, bacteria, and yeasts which degrade and modify inulin by enzymes such as endoinulinase , exoinulinase , and invertase . Exoinulinase (EC 3.8.1.80; β‐d‐fructohydrolase) hydrolyzes the terminal unit of fructose which is linked by β-fructofuranosidic bonds and liberates fructose. Endoinulinase (EC 3.2.1.7; β‐d‐fructan fructanohydrolase) breaks the internal glycosidic bonds of inulin and generates inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) as important end products (Fig. 15.2). β‐d‐fructofuranoside fructohydrolase (EC 3.2.1.26; invertase) hydrolyzes β‐2, 1‐fructosidic bond of sucrose to release fructose and glucose. Inulin is also degraded by yeast non-specific β-fructosidases which release fructose units from its reducing end. Relative activities toward sucrose and inulin are represented as inulinase/sucrase (I/S) ratio to differentiate between inulinase and invertase. High I/S ratio indicates predominant inulinase activity (Chi et al. 2009; Kango and Jain 2011; Rawat et al. 2015b; Singh et al. 2016).

Fig. 15.2
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Schematic showing generation of fructose syrup and inulooligosaccharides from inulin-rich plant extract

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Properties and Molecular Biology of Fungal Inulinases

Protein sequence of fungal inulinases has revealed several conserved motifs. Six highly conserved motifs of inulinases are WMND(E)PNGL, EC(V)P, SVEVF, FS(T), RDP, and Q that played an important role in substrate binding and inulin catalysis (Fig. 15.3). SVEVFV and Q are segments common in fungal exo- and endoinulinases, while SVEVFV amino acid segment was not noticed in yeast exoinulinases (Liu et al. 2013; Rawat et al. 2016). Fungal inulinases have been expressed in Yarrowia lipolytica (Liu et al. 2010), S. cerevisiae (Yuan et al. 2013), P. pastoris (Cao et al. 2013; Ma et al. 2015), Kluyveromyces lactis (Yu et al. 2010), and E. coli (Zhou et al. 2015). Such yeasts hydrolyze inulin and ferment fructose into ethanol simultaneously paving the way for consolidated bioprocessing (CBP) (Yuan et al. 2012).

Fig. 15.3
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Amino acid sequence alignment of various fungal exoinulinase using the Clustal Omega software

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Among fungal strains, Aspergillus spp. (Kango 2008; Rawat et al. 2015a) and Penicillium spp. (Rawat et al. 2015b), are common sources of exo- and endoinulinase (Table 15.2). Characteristics of some fungal inulinases are described in Table 15.3. Incidence of both exo- and endo-acting inulinolytic enzymes has been reported in fungi. For instance, exoinulinase gene from A. niger (inuE) encoded protein of 57 kDa (Goosen et al. 2008), while endoinulinase gene from the same strain encoded 54 kDa (Ohta et al. 1998). Another inulinase gene, Inu2 from A. ficuum ATCC 1688L encoded 55.1 kDa protein, while endo1 gene from A. ficuum JNSP5-06 consisted of 1482 bp and was 98% identical to A. niger CBS513.88 and 60% identical to A. fumigatus AF293 inulinase (Chen et al. 2012). Exoinulinase genes, (inuD) and (inuA1), from Penicillium sp. (TN-88) and P. janthinellum (B01) have been isolated and cloned (Moriyama et al. 2002; Wang et al. 2011). Details of various inulinase genes cloned from molds and yeasts are summarized in Table 15.4.

Table 15.2 Fungal sources of inulinases
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Table 15.3 Characteristics of fungal inulinases
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Table 15.4 Characteristics of some inulinase genes from molds and yeasts
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Heterologous Expression of Inulinase Genes of Fungi

Several exo- and endoinulinases encoding genes of filamentous fungi have been cloned in yeasts and characterized (Chi et al. 2011; Liu et al. 2013). The inulinase gene from K. marxianus CBS6556 was expressed in Y. lipolytica ACA-DC50109, and inulinase activity up to 41 U/ml was obtained (Zhao et al. 2010b). Recombinant yeast containing inulinase gene was used in inulin hydrolysis, with production of citric acid and SCP. The K. marxianus (INU 1) gene was expressed in S. cerevisiae, and the recombinant enzyme showed improved thermostability due to hyperglycosylation (Kim et al. 1997).

The endoinulinase gene (inu B) of A. ficuum was expressed in the mutant (Suc Z) S. cerevisiae. The recombinant inulinase was free from sucrase and exoinulinase activity, and the endoinulinase yield was up to 83 U/ml (Park et al. 2001). Yu et al. (2011) isolated inulinase gene from Kluyveromyces cicerisporus and expressed in a hexokinase muted S. cerevisiae strain. The yield of inulinase reached up to 31 U/ml, and recombinant yeast accumulated glucose-free fructose in fermentation broth containing Jesrusalem artichoke tubers. Workers have also expressed inulinase genes in high ethanol producing yeast for direct processing of inulin into ethanol. For instance, inulinase (INU 1) gene was isolated from marine Pichia guilliermondii strain 1 and expressed in Saccharomyces sp. W0 (Zhang et al. 2010). Wang et al. (2011) noticed that INU 1 gene integration into rDNA in Saccharomyces sp. W0 leads to production of more inulinase and ethanol from inulin in less time as compared to Saccharomyces sp. W0 strain carrying INU 1 gene in plasmid. Codon-optimized inulinase gene (INU1Y) from yeast Meyerozyma guilliermondii was expressed in Saccharomyces sp. W0, and recombinant strain (W0 Y13) produced 43 U/ml inulinase which was higher than native gene INU1 containing recombinant yeast (Liu et al. 2014).

Moriyama et al. (2002) have expressed inu E gene from A. niger strain 12 in P. pastoris yielding 16 U/ml inulinase having larger molecular mass (86 kDa) than inulinase produced by wild-type A. niger strain 12. Similarly, Wang et al. (2004) have expressed endoinulinase gene from A. niger 9891 (CGMCC 0991) in P. pastoris and obtained 291 U/ml yield in inulin-containing medium. Wang et al. (2011) obtained 11-fold high exoinulinase production (272 U/ml) when inu A gene from P. janthinellum strain B01 was expressed in P. pastoris X-33. Recombinant P. pastoris containing gene KmInu of K. marxianus produced 6667 U/ml inulinase (Zhang et al. 2012). The recombinant inulinase showed good stability up to 50 ℃ and 5.0 pH as compared to native enzyme. Zhou et al. (2014) disrupted MIG1 gene in K. marxianus and developed a derepressed mutant producing high inulinase (133 U/ml).

Several inulinase genes have been expressed in Kluyvermyces lactis and E. coli (Liu et al. 2013; Rawat et al. 2016). Yu et al. (2010) have isolated inulinase gene (Kcinu) from K. cicerisporus and expressed in mutant K. lactis. They have noticed twofold increase in inulinase activity (391 U/ml) than wild-type strain. Kwon et al. (2000) expressed inulinase gene (inuZ) of Pichia mucidolen in E. coli, and recombinant inulinase was a monomeric protein with MW of 55 kDa. Endoinulinase of A. ficuum was expressed in E. coli expression system (Chen et al. 2012). This endoinulinase was used in IOS production from inulin. Chen et al. (2013) have expressed exoinulinase gene from A. ficuum in E. coli and characterized recombinant enzyme.

Application of Fungal Inulinases

Hydrolysis of inulin can be selectively directed using microbial inulinases (exoinulinases and endoinulinases) for production of fructose-rich syrup and fructooligosaccharides (FOS), and this preparation can also be used as feedstock for production of single-cell protein (SCP), citric acid, ethanol, and other useful products (Chi et al. 2011; Kango and Jain 2011). Hydrolysis of inulin for the fructose bioconversion to ethanol by utilizing fructose from engineered yeast which has a prominent ability of consolidated bioprocessing (CBP) of inulin (Yuan et al. 2012).

Inulin is a renewable and commonly occurring polysaccharide that can be used as fructose feedstock generation. S. cerevisiae can easily utilize and convert fructose into ethanol (Chi et al. 2009; Nevoigt 2008). The same yeast strain was used for SCO production using Jerusalem artichoke (JA) tuber extract. This yeast strain accumulated 48.8% (w/w) and 52.2% (w/w) oil while growing on hydrolyzates of inulin (Zhao et al. 2010a). Y. lipolytica, an oleaginous yeast, accumulated 0.44-0.54 g lipid/g of biomass and produced 9-12 g/l dry biomass (Papanikolaou et al. 2002). An inulin utilizing mutant of this strain, Y. lipolytica ACA-DC 50109 (uracil mutant) containing inulinase gene of K. marxianus CBS 6556, was developed (Papanikolaou and Aggelis 2003).

Inulinase enzyme secreted by this engineered yeast was used to hydrolyze JA juice followed by SCO production from inulin (Zhao et al. 2010b) (Table 15.5).

Table 15.5 Potential uses of inulinase in generation of industrially and nutritionally important end products using fungal inulinases
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Inulinase preparations, therefore, can be used in feed, pharmaceutical, biofuel, and nutraceutical industries. Endoinulinase of Aspergillus niger was immobilized on chitosan and prepared for continuous generation of inulooligosaccharides (IOS) syrup from artichoke juice. This syrup contained IOS with DP 3–7 (Nguyen et al. 2011). Inulinase of A. niger was immobilized on Concanavalin-A (lectins) for the generation of syrup of fructose (Altunbas et al. 2013). IOS are also applicable in animal nutrition for significant change in colonic bacterial populations (Kelly, 2009). IOS also have various applications in food industries like chocolate, ice cream, milk desserts, confectionary, and sauces (Kuntz et al. 2013). Inulin and IOS contribute in improvement of the mineral balance of Ca, Mg, and Fe and show anti-carcinogenic effect by enhancing the bifidogenic flora which improves immunity of the system (Kango and Jain 2011). Endoinulinase inuA gene of A. niger was cloned and expressed in S. cerevisiae. The resultant recombinant enzyme showed maximum activity 3.1 U/ml and ethanol concentrations 55.3 g/L (Yuan et al. 2013).

Conclusions and Future Perspectives

Inulin hydrolysis for generation of fructose and fructooligosaccharides has recently received considerable interest. Some yeasts and fungi have been noticed to produce exoinulinases, while endoinulinase production is limited to few fungal and bacterial strains. Unconventional raw materials with high inulin content are being explored for optimized inulinase production. Cloning and expression of novel inulinases in suitable hosts, mostly yeasts such as Pichia, Saccharomyces, has been useful in consolidated bioprocessing of inulin to bioethanol. Thermostable inulinases from Bacillus smithii T7 and Sphingomonas sp. JB13 indicate possibility of finding robust inulinases among extremophiles. Inulin can be obtained from horticultural crops such as Jerusalem artichoke, chicory and utilized for generation of high-fructose syrup or oligosaccharides. Search for novel inulinase producers, parametric optimization for production and application, enzyme immobilization, and cloning of inulinase gene in suitable hosts are some of the challenges in this area. Efforts for the development of cost-effective bioprocesses suiting to food and nutraceutical industries using inulooligosachrides and fructose in commercial preparations would be helpful in realizing the applications.