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Inulinase or Î²-fructan fructanohydrolase hydrolyzes inulin, a polysaccharide of Î²-(2,1)-linked fructose residues attached to a terminal glucose molecule, to fructose and fructo-oligosaccharides, both of which are important ingredients in food and pharmaceutical industry (Vandamme and Derycke, 1983). It is mainly used in confectionery industry where fructose is preferred over sucrose because it is sweeter and does not easily crystallize (Rubio and Navarro, 2006). Inulinases are encountered in plants and many microorganisms. Microorganisms are the best source for commercial production of inulinases because of their easy cultivation and high yields of the enzyme. It has been found that the microorganisms which can produce high level of inulinases include Aspergillus spp., Penicilium spp., Arthrobacter spp., Bacillus spp, Clostridium spp, Pseudomonas spp, Arthrobacter spp, Staphylococcus spp, Xanthomonas spp, Kluyveromyces spp, Cryptococcus spp, Pichia spp, Sporotrichum spp, and Candida spp (Chi et al., 2009). It also has been confirmed that yeast strains can produce more inulinase than fungal and bacterial strains. Among the yeasts, Pichia sp., two strains of Kluyveromyces fragilis, Cryptococcus aureus, and Kluyveromyces marxianus have high potential for producing commercially acceptable yields of the enzyme (Gong et al., 2007; Sheng et al., 2008b). As many enzymes of industrial significance are regulated by medium compositions and fermentation condition, the study of this regulation is important in the commercial production of such enzymes. Optimization of fermentation condition and media compositions to have a balanced proportion is very important to get optimum microbial growth and enzyme yield.
Major improvements in the productivity of a fermentation process can be achieved by modifying the parameters like physicochemical and nutritional parameters, to which organism are exposed. The increasing potential of inulinase applications prompts screening for new inulinase producing microorganisms that can meet the conditions favorable for industrial applications. Therefore, the present study was undertaken to explore the inulinase production ability of a newly isolated strain of Kluyveromyces marxianus YS-1.
To study the optimum conditions, such as carbon source, nitrogen source,
different trace element,surfactant, pH, temperature, inoculums age, inoculums size and incubation time for a newly isolated strain of Kluyveromyces marxianus YS-1.
Inulin consists of linear Î²-2,1-linked polyfructose chains displaying a terminal glucose unit (Vandamme and Derycke, 1983). The fructose units in this mixture of linear fructose polymers and oligomers are each linked by Î² -2,1 bonds. A glucose molecule typically resides at the end of each fructose chain and is linked by an Î±-1,2 bond, as in sucrose (Molina et al., 2005). Fructans serve as storage polymers in many members of the Compositae such as Cichorium intybus (chicory), Inula helenium (elecampane), Taraxacum officinalis (dandelion), and Helianthus tuberosus (Jerusalem artichoke). All fructans found in the dicotyledons, as well as some monocotyledons, are of this type. By comparison, fructans composed predominately of linear fructose units bound by a Î²(2 6) glycosidic bond are typically levans that are produced by many soil and oral bacteria, yeasts and fungi. The molecular formula of inulin is GFn, with G being a terminal glucosyl unit, F representing the fructosyl units and "n" representing the number of fructosyl units. The basic GF2 trimer in inulin and the shortest fructan of the inulin type is 1-kestose. The same bonds link the ensuing fructosyl units, i.e. Î²(2 l) as that in 1-kestose, Figure 1. Short chain fractions of fructooligosaccharides such as 1-kestose, the major GF, compound in chicory roots or Jerusalem artichoke, and neokestose in onion do not differ analytically (Van Loo et al., 1995). Further, fructan chains linked to either of these naturally occurring trisaccharides have the Î²(2 1) configuration, implying that with the exception of one glycosidic linkage within the basic trisaccharide there is no difference between a fructan molecule based on 1-kestose or neokestose (Crow, 2000).
Figure 1 Chemical Structure of sucrose, inulin and oligofructose
Source: Crow (2000)
Inulin is a mixture of oligomers and polymers of fructose having varying degrees of polymerization (DP), but typically having a DP range from three (corresponding to GF2) to about 60 with a modal chain length of approximately nine. Native inulin from Compositae often is made up of about 1 - 2 percent Î²(2 6) branching with short side-chains (Van Loo et al., 1995). In addition to having predominately linear chains of the GFn-type, native inulin extracted from fresh chicory roots also has been shown to contain about 1- 1.5% (Fn) compounds on dry solids, those composed of homopolymers of fructose bound by a Î²(2 1) linkage (Van Loo et al., 1995). The n represents the number of fructosyl moieties in the homopolymers. Both GFn and Fm have very similar physicochernical properties except that Fm type products are reducing, due to the presence of a reducing fructose-end group, whereas GFn compounds are not. An understanding of different terms used to describe fructose-containing polymers is important as more commercial products become available. Oligofructose was introduced as a synonym for fructo-oligosaccharides (FOS). The oligofructose product is a partial enzymatic hydrozlyate of inulin containing predominately molecules of the Fm-type (homopolymers of fructose bound by a Î²(2 1) glycosidic linkage having no terminal glucose), Figure 1. Oligofructose has been defined by the IUB-IUPAC Joint Commission on Biochemical Nomenclature and the AOAC as fructose oligosaccharides containing 2-10 monosaccharide residues connected by glycosidic linkages (Niness, 1999). As previously stated, inulin, as extracted from Compositae root, is a polydisperse fructan with chain lengths ranging from 2 to 60 units with a modal DP of ± 9. Commercial chicory inulin is composed of approximately 2% monosaccharides, 5% disaccharides and 93% inulin.
Neosugar is a FOS mixture of kestose (n=2), nystose (n=3) and IF-B-fructofuranosyl nystose (n=4). Basically these are sucrose molecules to which one to three additional fructose units have been added. The development and isolation of neosugar was first reported in the Japanese literature in 1983 (Oku et al., 1984). Neosugar can be isolated from brans of triticale, wheat and rye or artificially by the action of the fungal enzyme Î²-fructofuranisidase on sucrose, the enzyme being a product of the fungus Aspergillus niger (Fishbein et al., 1988). Using the most widely available and accepted nomenclature, all FOS and inulins are fructans. Those inulin molecules having a degree of polymerization of < 10 fructose units generally are considered to represent FOS.
Fructooligosaccharides (FOS) have been accepted as functional sweeteners similar to other microbial oligosaccharides. Recent interest in the process development for the production of Fructooligosaccharides has concentrated on high content commercial products. Inulin, being a polyfructan, has been widely investigated as a useful source for production of ultra- high fructose syrup. However, the effort to utilize inulin for production of functional sweeteners is a more recent approach (Cho et al., 2001).
Inulin is hydrolyzed by enzyme known as inulinases. The inulinases are classified among the hydrolyzes and target on the Î²-2,1-linked of inulin and hydrolyze it into fructose and glucose. They can be divided into exoinulinases and endoinulinase. The exoinulinases catalyze removal of the terminal fructose residues from the non-reducing end of the inulin molecule while the endoinulinases hydrolyze the internal linkages in inulin to yield inulotriose, inulotetraose, and inulopentaose.
Type of inulinases
Endo-inulinases are specific for inulin. It hydrolyzes inulin by breaking bonds between fructose units that are located away from the ends of the polymer network, to produce oligosaccharide.
Exo-inulinases split terminal fructose units in sucrose, rafffinose and inulin to liberate the fructose.
Figure 2 Mechanism of inulinase
Source: Worawuthiyanun (2005)
Production of inulinase
Inulinase is found in filamentous fungi, yeasts, bacteria. The production of inulinases is affected by type of the organism medium components and process parameters used for fermentation.
Pichia guilliermondii strain 1 (the collection number: 2E00048 at
Marine Microorganisms Culture Collection of China), isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium (Gong et al., 2007). Under the optimal conditions, over 61.5± 0.4 U/ml of inulinase activity is produced within 48 h of fermentation in the shaking flasks. The results indicate that 2.0% (w/v) of added NaCl is the most suitable for the inulinase production by the marine yeast strain 1 (Gong et al., 2007). Especially when the marine yeast strain is grown in the medium prepared with seawater, the inulinase activity reaches the highest activity (61.5±0.4 U/ml).
In order to isolate the inulinase overproducers of the P. guilliermondii
strain 1, its haploid cells were treated by using UV light and LiCl. One mutant (M-30) that produces 115±1.1 U/ml of inulinase activity was obtained (Guo et al., 2008). It found that glucose repression on the inulinase production by the mutant M- 30 was relieved in some degree compared to that by its parent strain when added glucose concentration was more than 20.0 g/l (Yu et al., 2008).
Response surface methodology (RSM) was used to optimize the
medium compositions and cultivation conditions for the inulinase production by the mutant M-30 in the submerged fermentation (Yu et al., 2008). After the optimization, 127.7±0.6 U/ml of inulinase activity is reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml is derived from RSM regression. Under the same conditions, its parent strain only produces 48.1±0.4 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. The results demonstrate that the inulinase production can be greatly improved by the genetic modification of the natural producers.
RSM was also used to optimize the medium compositions and
cultivation conditions for the inulinase production by the inulinase overproducer (the mutant M-30) from P. guilliermondii strain 1 in solid-state fermentation (SSF) (Guo et al., 2008). Under the optimized conditions, 455.9± 1.2 U/g of dry substrate (gds) of inulinase activity is reached in the solid-state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds is derived from RSM regression. Under the same conditions, its parent strain only produces 291.0± 0.7 U/gds of inulinase activity. This also is the highest inulinase activity in the culture of solid-state fermentation produced by the yeast strains reported so far.
The marine yeast strain C. aureus G7a (the collection number:
2E00002 at Marine Microorganisms Culture Collection of China) isolated from sediment of South China Sea was found to secrete a large amount of inulinase into the medium (Sheng et al., 2007). Under the optimal conditions, over 85.0±1.1 U/ml of inulinase activity is produced within 42 h of fermentation at shake flask level. It is worthy to observe that 4.0% (w/v) of added NaCl and 0.6% (w/v) of added MgCl2·6H2O is the most suitable for the inulinase production (84.1±0.6 U/ml) by the marine yeast (Sheng et al., 2007). The optimization of process parameters for the high inulinase production by the marine yeast strain C. aureus G7a in SSF is carried out using RSM (Sheng et al., 2008a). Under the optimized conditions, 420.9±1.3 U/g of dry substrate of inulinase activity is reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U/g of dry weight is derived from RSM regression.
Two strains of K. marxianus (A1 and A2) isolated from "aguamiel"
(agave sap) and one strain of Kluyveromyces lactis var. lactis (P7) isolated from "pulque" (its fermented product) show good inulinase producers (less than 32 U/ml) in the liquid medium containing different concentrations of inulin and they have low susceptibility to catabolic repression (Cruz-Guerrero et al., 2006).
It has been shown that the inulinase production by K. marxianus
ATCC 16045 is strongly influenced by mixing conditions in the liquid medium. The disk impeller at 450 rpm and aeration at 1.0 volume/volume per minute led to an activity of 121 U/ml, while the pitched blade was shown to be the best impeller for this process, leading to the best production. The maximum shear stress for the inulinase production was about 0.22 Pa (Silva-Santisteban and Filho, 2005).
The maximum inulinase activity of 59.5 U/ml is produced by
Kluyveromyces sp. Y-85 within 24 h of fermentation when extract of Jerusalem artichoke, urea, beef extract, and corn steep liquor concentrations in the liquid medium are 8.0%, 2.0%, 0.2%, and 4.0%, respectively (Wei et al., 1998). The fermentation in 15-l fermentor and scaling-up in 1,000-l tower fermentor were carried out and the inulinase activity in the scaling-up is 68.9 U/ml (Wei et al., 1999b). After the cells of Kluyveromyces sp. Y- 85 were treated by ethyl methane sulfonate, one mutant named Kluyveromyces sp.Y-85 K6 which is resistant to catabolite repression of glucose was obtained (Wei et al., 1999a). Unfortunately, no further work on the mutant has been reported since then.
Maximum inulinase production (55.4 U/ml) by a newly isolated strain
of K. marxianus YS-1 was obtained at an agitation rate of 200 rpm and aeration of 0.75 volume/ volume per minute in a stirred tank reactor (1.5 l) with a fermentation time of 60 h (Singh et al., 2007). In another study, inulinase activity of 50.2 U/ml was obtained from the same yeast strain under agitation (200 rpm) and aeration (0.75 volume/volume per minute) at 30°C after 60 h of fermentation in 1.5 l of fermentor when raw inulin (4.0%) from root tubers of Asparagus officinalis was used as the production medium. Inulinase yield in the bioreactor is almost six times higher than the basal medium used initially in shake flask (Singh and Bhermi, 2008).
It was found that the yeast K. marxianus var. bulgaricu produces large
amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose, and glucose as carbon sources. The levels of inulinase in sucrose-limited chemostat cultures are strongly dependent on the dilution rate. When the dilution rate D is 0.05 per hour, a maximum of 107 U/ml is obtained, indicating that the inulinase production by this yeast strain is regulated by the residual sugar in the continuous cultures (Kushi et al., 2000).
When K. marxianus NRRL Y-7571 is grown in the culture containing
sugarcane bagasse as support and carbon source and corn steep liquor as nitrogen supplement, the extracellular inulinase concentration reaches 391.9 U/g of dry fermented bagasse (Bender et al., 2006). The use of soybean bran decreases the time to reach the maximum activity from 96 to 24 h and the maximum productivity achieved K. marxianus NRRL Y-7571 is 8.87 U/gds per hour (Mazutti et al., 2006). The average inulinase activity produced by a newly isolated strain Kluyveromyces S120 in SSF is 409.8 U/g initial dry substrate in the optimal medium (Chen et al., 2007).
The inulinolytic fungi Penicillium sp. TN-88 showed a high
endoinulinase productivity of 9.9 U/ml in the culture filtrate when grown in a liquid medium containing inulin as the carbon source at 30°C for 4 days (Nakamura et al.,
The fungus Aspergillus niger NK-126 shows good growth on a
medium containing 40% (v/v) of dandelion tap root extract composed of 50-g tap roots blended with 200-ml water and 2% yeast extract medium and produces 55 U/ml of inulinase activity in 96 h at 30°C and 150 rpm during the liquid fermentation. The results also suggest that the dandelion tap root extract in the liquid medium induces the endoinulinase synthesis in A. niger NK-126 (Kango, 2008).
Although it has been reported that yeasts can produce more inulinase than
bacteria and filamentous fungi, Streptomyces sp. GNDU 1 was still used to produce high levels of extracellular inulinase (0.552 U/ml) after 24 h at pH 7.5, temperature 46°C, in the presence of 1.0% inulin in the liquid medium (Gill et al., 2006).
Solid-state fermentation for the synthesis of inulinase from the
bacterium Staphylococcus sp. was also carried out using the medium containing wheat bran, rice bran, coconut oil cake, and corn flour, individually or in combinations. Under the optimized conditions, the extracellular inulinase concentration reaches a peak in 48 h with Staphylococcus sp. (107.6 U of inulinase per gram dry fermented substrate) while the yeast K. marxianus ATCC 52466 produces the highest level of the extracellular inulinase in 72 h (122.9 U/gds). The results again demonstrate that the yeasts can produce more inulinase than bacteria (Selvakumar and Pandey, 1999).
Table 1 summarizes the inulinase microorganisms and their inulinase
activity. It can be clearly seen from the results in Table 1 that most of the inulinase microorganisms that are being used are yeasts, especially the strains of Kluyveromyces, Cryptococcus, and Pichia. They also produce much higher inulinase activity than any other microorganisms.
Table 1 Inulinase microorganisms and Inulinase activity
Type of cultivation