Title of Invention	"AN IMPROVED PROCESS FOR INULINASE PRODUCTION"
Abstract	The present invention relates to a novel isolated yeast strain Kluyveromyces marxianus Y-l deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession no MTCC 5207. The invention further relates to an improved process for the enhanced production of inulinase from microbial cultures of yeast Kluyveromyces marxianus Y-l.

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AN IMPROVED PROCESS FOR INULINASE PRODUCTION FIELD OF INVENTION The present invention relates to a novel isolated yeast strain Kluyveromyces marxianus Y-l deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession no MTCC 5207. The invention also relates to an improved process for the enhanced production of inulinase from microbial cultures of yeast Kluyveromyces marxianus Y-1. BACKGROUND OF THE INVENTION Inulin, a polyfructan, occurs as a reserve carbohydrate in many plant families representing more than 30,000 species. It consists of linear chains of ß-2, 1-linked D-fructofuranose molecules terminated at the reducing end by a glucose residue attached through a sucrose-type linkage by ß- (2, l)-fructofuranosidic bonds. Due to variation in chain length its molecular weight varies between ± 1500-6000 Da. Inulin has received a great interest as a renewable raw material for the production of fructose syrup. Fructose is the sweetest natural sugar, about 1.2 to 2.0 times sweeter than sucrose with low viscosity. It has high solubility and low water activity. Fructose is a low calorie sweetener and organoleptic ally desirable. It is emerging as a safe alternate sweetener than sucrose which causes problems related to corpulence, carcinogenicity, atherosclerosis and diabetes. It increases the absorption of iron as a result of formation of iron-fructose chelate complex which is better absorbed than inorganic iron. Fructose also speeds up the metabolism of ethanol. It is better for diabetic patients, since it is absorbed more slowly than glucose through the intestine. The scope of high fructose syrup extends from food industries like bakeries, biscuits, yoghurt, jams, jellies, breakfast cereals, fruit processing, squashes, lemonades, instant energy drinks to pharmaceuticals. There is much interest in developing processes to produce fructose syrups for use in the food industry. Conventional fructose production from starch needs at least three enzymatic steps using α-amylase, amyloglucosidase and glucose isomerase. The yield of fructose by this method is approximately 45%. The ion-exchange techniques have been developed to give syrups with D-fructose content over 90%, but these techniques add to the cost of production. An alternative method to produce D-fructose could involve the chemical or enzymatic hydrolysis of inulin. The production of fructose by acid hydrolysis of inulin is not recommended because of undesirable coloring of inulin hydrolysate, formation of difructose anhydride which has practically no sweetening properties and appearance of by product hydroxyl methyl furfural. An enzyme known as inulinase or inulinase (EC 3.2.1.7, ß-2, 1-fructan fructanohydrolase), is also used for hydrolysis of inulin, which hydrolyses inulin by splitting terminal D-fructose units by cleaving the glycosidic linkages in polymer moiety. This enzyme acts by liberating fructose from the fructose end of the molecule leaving a reducing sugar residue and glucose appears only when the molecule is completely degraded. The liberation of fructose occurs mostly as a single-chain mechanism under a wide range of pH and temperature conditions. The complete hydrolysis of inulin by this single step enzymatic reaction using inulinase gives a good yield. A number of fungal, yeast and bacterial strains have also been reported for the production of inulinase (Vandamme and Derycke, 1983). Inulinase is also encountered in digestive tracts of some animals and inulin storing tissues of plants. Inulinase from plants and animals have only little commercial value because their quantity is not enough for commercial exploitation. Inulinase only from microbial sources has been used for industrial applications because of many advantages like easy handling, rapid multiplication, easy genetic manipulation and high production yield. The enzyme differs in properties according to their source and cultivation environment. Inulinase producing Aspergillus niger strain was isolated from soil samples by Derycke and Vandamme (1984). Five different mushrooms were screened for extracellular inulinase and invertase activities (Mukherjee and Sengupta, 1987). Pessoni et al. (1999) isolated Penicillium janczewskii from a rhizosphere of Vernonia herbacea for extracellular inulinase. A number of yeast cultures namely Kluyveromyces fragilils, K. marxianus, Hansenula polymorpha, Candida sp., Pichiafermentan, P. polymorpha and P. castellii have been screened for inulinase production (Guiraud and Galzy, 1990; Hensing et al., 1994; Cruz et al., 1995). There are reports on the production of inulinases using different bacterial strains e.g. Acetobacter sp., Achromobactex sp., Arthrobacter sp.. Bacillus sp. and Cladosporium sp. (Elyachioui et al, 1992, Gern et al, 2001). The enzyme has various applications in the production of high fructose syrup, inulo-oligosaccharides, ethanol, acetone and butanol, pullulan, gluconic acid and sorbitol, etc. The new isolate Kluyveromyces marxianus Y-l produces good amount of exoacting extracellular inulinase using simple medium than the existing microorganisms of K. marxianus species, which has a good industrial potential for a variety of applications. The high stability of the preparation over a wide range of pH and temperature are the advantages of the developed technology over the prior art. The high hydrolytic potential and good operational stability for the production of high fructose syrup is another advantage of the developed technology for the production of high fructose syrup. Since, the demand of high fructose syrup is increasing day by day due to its utility in various products and subsequently having several nutritional advantages over other sweeteners. The stable and efficient reactor system developed has the potential of the industrial interest for the preparations of high fructose syrups from inulin extracts of some plants. The enzymatic production of fructose for food and pharmaceutical industry has advantages over chemical processes in terms of purity, yield and reduced pollution. OBJECTS OF THE INVENTION The main object of the invention is to isolate and characterize microorganisms having inulinase activity. Another object of the invention is to develop a process for enhanced production of inulinase from microbial cultures. Yet another object of the invention is to obtain optimum media composition for the production of inulinase from microbial cultures. Still yet another object of the invention is to obtain optimum process conditions for the production of inulinase from microbial cultures. Yet another object of the invention is the use of inulinase from microbial cultures of new isolate of Kluyveromyces marxianus Y-l for preparation of fructose from inulin. SUMMARY OF THE INVENTION The present invention relates to a novel isolated yeast strain Kluyveromyces marxianus Y-l deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession no MTCC 5207. The present invention also relates to a process for the enhanced production of inulinase from microbial cultures. The invention further provides a media composition-containing surfactant for enhanced production and recovery of inulinase from the microbial cultures of Kluyveromyces marxianus Y-1. BRIEF DESCRIPTION OF THE DRAWINGS Figure: 1 shows enzyme activity and growth curve of Kluyveromyces marxianus Y-l as a function of time Figure: 2 shows inulinase production by Kluyveromyces marxianus Y-l with different carbon sources Figure: 3 shows inulinase production by Kluyveromyces marxianus Y-l with different carbon sources and inulin as an inducer Figure: 4 shows effect of concentration of meat extract on Inulinase production Figure: 5 shows effect of trace elements on inulinase production Figure: 6 shows inulinase production by Kluyveromyces marxianus Y-l with surfactants DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel isolated yeast strain Kluyveromyces marxianus Y-l deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession no MTCC 5207. The present invention also relates to an improved process for enhanced production of inulinase from microbial cultures of Kluyveromyces marxianus Y-l. The invention further provides a media composition containing surfactant for enhanced production of inulinase from the microbial cultures of Kluyveromyces marxianus Y-l. In one embodiment the present invention provides an isolated yeast strain Kluyveromyces marxianus Y-l deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession no MTCC 5207. In another embodiment, the present invention provides an improved process for enhanced production of inulinase from yeast Kluyveromyces marxianus, said process comprising steps of: a) growing Kluyveromyces marxianus for 2-4 days in a medium comprising of carbon source, nitrogen source, trace elements and surfactant at an initial pH ranging from 5-7 at a temperature ranging from 25°C to 35°C, and b) recovering the inulinase using conventional methods. Yet another embodiment of the present invention relates to an improved process for enhanced production of inulinase from yeast, wherein said yeast is Kluyveromyces marxianus Y-l (MTCC 5207). Still another embodiment, the invention provides the composition of the medium for growing Kluyveromyces marxianus Y-l (MTCC 5207). Yet still another embodiment, the invention further provides the composition of the medium wherein the medium is consists of inulin, meat extract, CaCl2 and sodium-dodesyl sulphate. Further the invention provides a carbon source which is selected from a group consisting of fructose, glucose, lactose, maltose, starch, sucrose, and inulin or combination thereof. In another embodiment, the present invention also provides the concentration of the carbon source in the growth medium which is in the range of 0.1-3.5% (w/v). In another embodiment, the invention provides inulin as a carbon source in growth medium, wherein the concentration of the inulin ranges from 0.5 to 2.5 % (w/v) preferably 1.5% (w/v). In yet another embodiment, the invention provides a carbon source in combination in growth medium, wherein the concentration of inulin is in the range of 0.1 to 1.0 % (w/v) preferably 0.1 to 0.5% (w/v). In yet another embodiment, the present invention provides nitrogen source selected from a group consisting of peptone, meat extract, ammonium chloride, ammonium nitrate, ammonium sulphate, beef extract, corn steep liquor, sodium nitrate, urea and yeast extract preferably meat extract. In still yet another embodiment, the present invention provides the concentration of meat extract which is in the range of 0.5-5 % (w/v) preferably 2% (w/v). Yet another embodiment of the invention provides the trace elements in the growth medium which is selected from a group consisting of MnCl2, CaCl2, MgSO4, CuSO4, ZnSO4,CoCI2, HBO3, KCl and Na2MoO4. In still yet another embodiment, the invention provides a surfactant selected from a group consisting of sodium-dodecyl sulphate, Brij-35, Tween-20, Tween-60, Tween-80 and Triton X-100 preferably sodium-dodecyl sulphate. The present invention also provides sodium-dodecyl sulphate as surfactant in the growth medium, wherein the concentration of sodium-dodecyl sulphate is in the range of 0.05-2.5 mM. Further the invention provides the temperature for the production of inulinase from new isolate of Kluyveromyces marxianus Y-l, wherein the temperature ranges between 25°C to 35°C. Various inulinase producing cultures were isolated from rotten dahlia rhizosphere and soil samples from chicory fields from Botanical Garden, Punjabi University, Patiala, Punjab, India by growing in enrichment medium followed by pour plate method. The enrichment medium (100 ml) containing inulin (1%, w/v), peptone (0.5%, w/v) and having a pH 5.5 was inoculated with the sample (0.5%, w/v) and incubated at 30°C for 24 h. Thereafter, plating was done on a synthetic agar medium containing (NH4)2SO4 (0.05%, w/v), MgSO4.7H2O (0.02%, w/v), KH2PO4 (0.20%, w/v), inulin (1.00%, w/v), agar (2.00%, w/v), trace elements solution (0.10%, v/v) of FeSO4.7H2O (0.01%, w/v), MnCl2.4H2O (0.01%, w/v) and ZnSO4.7H2O (0.01%, w/v), and adjusted to pH 5.5. The plates were incubated at 30°C for 2-7 days. Only healthy colonies were picked up and maintained for further studies. All the cultures were grown in glucose yeast extract medium (50 ml) containing yeast extract (0.3%, w/v), peptone (0.5%, w/v), dextrose (2.0%, w/v) and having a pH 6.0 for the preparation of inoculum (starter). The flasks were incubated at 30°C for 12 h and inoculum at a concentration of 5% (v/v) was used for yeast fermentations. Screening of cultures for the production of inulinase was done by growing in production media. Production medium (50 ml) containing malt extract (0.3%, w/v), yeast extract (0.3%, w/v), peptone (0.5%o, w/v), inulin (1.0%, w/v) and having a pH 6.0 was sterilized at 121 °C for 20 min. Inulin was sterilized separately at 110°C for 35 min and added aseptically in the medium after cooling. The fermentation media (50 ml) inoculated with their respective cultures was incubated at 30°C at 100 rpm for 72 h. The biomass was harvested by centrifugation at 2500 g for 10 min at 4°C and the supernatant thus obtained was analyzed for enzyme activity. To determine the enzyme activity, the mixture of 0.1 ml of diluted enzyme sample and 0.9 ml sodium acetate buffer (0.1 M, pH 5.0) containing 2.0% inulin was taken in a test tube and incubated at 50°C in a water bath for 15 min. After incubation, the test tube was kept at 100°C for 10 min to inactivate the enzyme. The reaction mixture was assayed for reducing sugars by the DNSA method (Miller, 1959). One IU of enzyme is the amount of enzyme which forms one micromole of reducing sugars per min under standard assay conditions. Many micro-organisms cultures were tested for the production of inulinase of which some cultures produced inulinase. The most efficient inulinase producing microorganism was selected for further studies and it was identified as Kluyveromyces marxianus by Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh. The various characteristics of the isolated microorganism are given in Table. 1. The microorganism was isolated from Botanical Garden, Punjabi university, Patiala, Punjab, and was characterized in the laboratory. This microbial culture was deposited in MTCC-IDA under Budapest Treaty. It has been assigned the MTCC number: 5207. Table: 1 Results of different tests conducted for characterization of microorganism (Table Removed) The grind river sand method was used to break the cells of Kluyveromyces marxianus to test the presence of intracellular enzyme. The cells were centrifuged at 2500 g for 10 min and washed thoroughly with normal saline. The washed cell paste (5 g) was incubated at 4°C for an hour in a mortar. Then, 10g pre-treated and cooled river sand was added in fractions during crushing of yeast cells for 15 min. After grinding, the cells were suspended in sodium acetate buffer (0.1 M, pH 5.0) and centrifuged (2500 g X 10 min, 4°C) to separate the debris and sand. The enzyme activity was determined in both supernatant and pellet. No enzyme activity was observed in supernatant and pellet so no intracellular enzyme was found. Thus, it can be concluded that Kluyveromyces marxianus produces only extracellular enzyme. To investigate the action pattern of extracellular inulinase from Kluyveromyces marxianus, inulin solution (2%, w/v) prepared in sodium acetate buffer (0.1 M, pH 5.0) was incubated with crude enzyme extract at 50°C for 15 min. The reaction was stopped by placing the tubes in a boiling water bath for 10 min. The products of the reaction were analyzed by thin layer chromatography (TLC). The spots (2 µl) of hydrolyzed reaction mixture, inulin (2%, w/v), glucose (2%, w/v) and fructose solution (2%, w/v) were applied to precoated TLC plates. After developing of TLC plates in a solvent mixture, the aniline diphenylamine reagent was sprayed to develop the color of different sugars. There was no sign of fructo-oligosaccharides formation on the TLC plate. Fructo-oligosaccharides are formed by the endo-action of inulinase (endo-inulinase), whereas exoinulinase (exo-action) acts only at fructose terminal end breaking a fructose unit at a time and leaving a glucose unit at the end of reaction. It has been concluded from the results that extracellular enzyme produced by Kluvveromyces marxianus was exo-inulinase. The production medium (50 ml in 250 ml flasks) containing peptone (0.5%, w/v), inulin (1%, w/v) and having a pH 6.5 was inoculated with 5% (v/v) starter culture of Kluyveromyces marxianus MTCC 5207. The flasks were incubated at 30°C for 96 h under shaking conditions at 100 rpm. To study the growth pattern and inulinase production by the microorganism, the samples were withdrawn at 12 h intervals and analyzed for biomass and enzyme activity. The growth curve was presented in Fig. 1. Medium Development Various carbon sources like fructose, glucose, inulin, lactose, maltose, starch and sucrose were supplemented separately at a concentration of 1% (w/v) to study their effect on the production of inulinase (Table 2). The effect of each carbon source was investigated and presented in Fig. 2. The order of enzyme production from various carbon sources was inulin > sucrose > fructose > glucose > lactose > maltose > starch. Inulin was found to be the best carbon source for the production of inulinase from K. marxianus. The effect of each carbon source (1%, w/v) in combination with inulin as an inducer at a concentration of 0.1% and 0.5% (w/v) was also investigated and the results are shown in Fig. 3. Different concentrations (0.5-2.5%, w/v) of inulin were supplemented in the medium to study their effect on inulinase production. The maximum enzyme production was obtained at 1.5 % (w/v) concentration of inulin (Table 3). Table 2: Effect of different carbon sources on Inulinase production (Table Removed) Table 3: Effect of concentration of inulin on Inulinase production (Table Removed) Various nitrogen sources (ammonium chloride, ammonium nitrate, ammonium sulphate, beef extract, corn steep liquor, meat extract, peptone, sodium nitrate, urea and . yeast extract) were supplemented individually in the for inulinase production The maximum inulinase production was supported by meat extract. The order of production of inulinase from various nitrogen sources were meat extract > beef extract > peptone > yeast extract > ammonium chloride > ammonium sulphate > corn steep liquor > ammonium nitrate > sodium nitrate > urea. Various concentrations (0.5-5%, w/v) of meat extract were used in the medium for inulinase production (Fig.4). There was an increase in inulinase production with an increase in the concentration of meat extract upto 2% (w/v). The enzyme activity at 2% of meat extract was 12.1 IU/ml. Effect of trace elements on inulinase production The presence of trace elements in the production medium plays a vital role in the enzyme production. The supplementation of the culture medium with a solution of metal traces improved substantially the enzyme production. The results presented in Fig. 5 clearly revealed that only MnCl2 (0.1 mM) and CaCl2 (0.5 mM) have enhanced the inulinase production. Whereas, MgSO4, CuSO4, ZnSO4, CoCl2, HBO3, KCl and Na2MoO4 did not exert any influence on inulinase production at low concentrations, however, they have exhibited a negative impact on inulinase activity at higher concentration. Further, FeS04, and Ni (CH3COO) 2 resulted in a decrease in inulinase production. Effect of surfactants on inulinase production The effect of various surfactants (Brij-35, sodium dodecyl sulphate (SDS), Tween-20, Tween-60, Tween-80 and Triton X-100) was observed on inulinase production by supplementing individually at 0.002% in medium. Surprising results were obtained when SDS was added in the medium. It was observed that SDS has a positive influence on the inulinase production (Fig. 6). Different concentrations (0.05-2.5 mM) of SDS were supplemented to the medium to investigate their influence on inulinase production and results obtained are presented in Table 4. There was an increase in enzyme production on increasing the SDS concentration upto 0.1 mM. Table: 4 Effect of sodium dodecyl sulphate (SDS) on enzyme production. (Table Removed) The synergetic effect of optimized inulinase enhancing constituents was studied by using their different combinations (Table 5) and it was observed that inulin, meat extract, CaCl2 and SDS in combination resulted in maximum enzyme activity and therefore, selected for further studies. Table: 5 Inulinase production by Kluyveromyces marxianus with different combinations of optimized constituents as a function (Table Removed) It can be concluded from the observations made during the media optimization studies that maximum inulinase was obtained with the following optimized medium and thus was used for further experimentation. Carbon Source : Inulin(1.5%) Nitrogen Source : Meat Extract (2%) CaCl2 : 0.5mMol SDS : 0.1 mMol Process Optimization Different process parameters like pH of the medium, temperature of fermentation, age of inoculum, size of inoculum and incubation time exert a great influence on the enzyme production and therefore, these were optimized to enhance the inulinase production. The initial pH of the fermentation medium was adjusted to 4.0-8.0, to evaluate its effect on enzyme production. The results obtained during the course of experimentation have shown an increase in enzyme production (24.5 IU/ml) with the increase of pH up to 6.5 and thereafter, there was a decline in this function (Table 6). Table: 6 Effect of initial pH of media on enzyme production (Table Removed) Effect of temperature on inulinase production was examined by incubating the inoculated production media at different temperatures (25-40°C). The results obtained are presented in Table. 7 and maximum enzyme production was observed at a temperature of 30°C and at higher temperatures the enzyme activity was appreciably reduced. Table: 7 Effect of temperature on enzyme production. (Table Removed) To investigate the influence of age of inoculum, production medium was inoculated with 6-36 h old cultures of K, marxianus Y-l. inoculum age was observed to be an important parameter and exerted a strong influence on inulinase production. The enzyme production was maximum (24.8 IU/ml) when 12 h old culture was used. Inoculum grown for longer periods over 12 h has resulted in reduction of enzyme production (Table 8). Table 8: The effect of age of inoculum on enzyme production (Table Removed) To study the effect of size of inoculum on enzyme production, the fermentation media was inoculated with 2.5 to 15% (v/v) of 12 h old starter culture. The maximum inulinase production (25.1 IU/ml) was observed by the use of 5% (v/v) inoculum (Table 9). Table 9: The effect of concentration of inoculum on enzyme production (Table Removed) To evaluate the influence of agitation rate enzyme production, fermentation was carried out at different agitation rates (50, 100, 150 and 200 rpm) on a rotary shaker and under stationary conditions (Table 10). The increase in enzyme activity was observed in agitation mode (28.1 IU/ml at 150 rpm) as compared to stationary condition (5.9 IU/ml). Table 10: The effect of agitation on enzyme production (Table Removed) The fermentation was carried out for 96 h under optimized conditions and there was an increase in enzyme production up to 72 h of incubation and thereafter, there is a decline in this function. The maximum enzyme (30.8 IU/ml) was produced at 72 h of incubation (Table 11). Table 11: The effect of incubation time on enzyme production (Table Removed) Effect of Raw inulin extract on inulinase production: Dry powder of dahlia obtained after chopping and drying at 80°C in an oven was subjected to five extraction methods to obtain raw inulin extract for the production of inulinase. The inulin extracts obtained by shaking in cold water for 10 min, boiling water for 10 min, extraction under different pressures (10, 15 & 20 psi) for 10 min in a autoclave were analyzed for inulin (Table 12). The maximum inulin (51.7%, w/w) was extracted by extraction at 15 psi pressure for 10 min. The order of inulin content obtained by different extraction methods was as extraction at 15 psi > extraction at 20 psi > extraction at 10 psi > extraction by boiling in water > extraction in cold water under shaking. Table: 12 Characterization of dahlia extract and their effect on inulinase production by K. marxianus. (Table Removed) The extracts obtained after treatment at different time intervals were analyzed for inulin content (Table 13). The maximum inulin (51.7%, w/w) was extracted by treatment at 15 psi for 10min. Table: 13 Effect of treatment time on dahlia extract and its influence on inulinase production by K. marxianus (Table Removed) Scale-up Studies using Bioreactor: Scale-up Studies using Bioreactor were conducted in a lab scale (1.5 L) bioreactor using a working volume of 1 L. The bioreactor was equipped with automatic control of aeration, agitation and temperature. Exhaust valve was having a fitting of condenser to avoid the losses by evaporation. Since, the temperature of fermentation was 30°C therefore; no need was felt to run the condenser to avoid the evaporation of medium. Various parameters were optimized for the production of inulinase in a batch system. To control the foam formation, sterilized silicon oil (0.002%, w/v) was added two times at the beginning of the fermentation and after 36 h incubation. Fermentation medium developed at flask level was tested at 1.5 L bioreactor level to investigate the effect of scaling up on inulinase production. The aeration and agitation in the bioreactor was kept at 1.0 vvm and 150 rpm, respectively. It was found that there was increase in enzyme production at bioreactor level (45.7 IU/ml) from the enzyme obtained at flask level (30.8 IU/ml). The cost of basic raw material plays a crucial role in all industrial processes. Taking into consideration the high cost of pure inulin, it was replaced by raw inulin extract obtained from dahlia tubers. The different concentrations (1.0-2.5 %, w/v) of raw inulin obtained from dahlia tubers after extraction were supplemented to the fermentation medium. The enzyme production by the use of raw inulin extract at 2.0 % (w/v) inulin concentration was found to be 43.8 IU/ml in bioreactor comparable to 45.7 IU/ml as obtained by use of pure inulin. To study the effect of agitation and aeration rate at lab scale bioreactor on inulinase production by Kluyveromyces marxianus different agitation (100-250 rpm) and aeration (0.5-1.25 vvm) rates were tested. The maximum enzyme production was observed at agitation rates of 200 rpm (Table 14). It is observed that this isolate produced reasonably good inulinase when grown in 0.75 vvm (Table 15). Table 14: The effect of agitation rate on enzyme production in bioreactor (Table Removed) Conditions: Inulin: 2.0, pH: 6.5; Temperature: 30°C; Incubation time: 72h; Agitation: 200 rpm. Table 13: The effect of aeration rate in bioreactor on enzyme production (Table Removed) Conditions: Inulin: 2.0, pH: 6.5; Temperature: 30°C; Incubation time: 72h; aeration: 1VVM A tremendous increase of 7.8 times of enzyme production from K. marxianus Y-l was achieved by optimizing the medium and process parameters at flask and bioreactor level. The optimum fermentation time has been reduced from 72 h to 60 h in bioreactor studies for maximum inulinase production. The fermented broth was subjected to precipitation with different concentrations of ethanol at 4°C. The maximum enzyme precipitation has occurred at 85% (v/v) ethanol. The pellet obtained from ethanol precipitation was dissolved in sodium acetate buffer (0.1 M, pH 5.0) and subjected to ultrafiltration with 100 kDa cut off membrane. The protein sample obtained after ultrafiltration was loaded onto Q-sepharose column pre-equilibrated with phosphate buffer (0.1 M, pH 7.0). Proteins were eluted with a linear gradient of 0.1-0.5 M NaCl over two column volumes before elution. Inulinase was eluted at 220 mM NaCl and the fractions rich in enzyme activity were pooled and used for further purification. The pooled protein sample obtained after Q-sepharose chromatography was loaded onto DEAE-sepharose column pre-equilibrated with phosphate buffer (0.1 M, pH 7.0). Proteins were eluted with a linear gradient of 0.1-0.5 M NaCl over two column volumes before elution. Inulinase was eluted at 100 mM NaCl from column. The fractions containing higher amount of enzyme were pooled and used for further purification. The pooled sample obtained from previous step was concentrated by ultrafiltration. The concentrated sample was loaded onto the sephadex G-100 column (1 X 40 cm) pre-equilibrated with sodium acetate buffer (0.1 M, pH 5.0). The elution was carried out with the same buffer. Fractions rich in inulinase were used as purified enzyme. The enzyme activity studies of purified inulinase were carried out at different pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0). The enzyme was observed to be active over a wide range of pH (5.0-7.0) with maximum activity at pH 5.5. The enzyme activity of purified enzyme was determined at various temperatures (25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80°C). The enzyme exhibited maximum activity at temperature 50°C. It has retained more than 50% of its original activity at 35°C and 65°C, but got rapidly denatured at 70°C and above. The enzyme activity of purified inulinase was studied for various substrates (inulin, sucrose, maltose, lactose, starch, and raffinose). It was observed that the enzyme was active on inulin, sucrose and raffinose, whereas, it did not showed any activity on maltose, lactose and starch. The enzyme has shown maximum relative activity on sucrose (182%) followed by raffinose (67%) with respect to activity on inulin, which was considered as 100%. Km and Vmax for inulin, raffinose and sucrose was determined from Lineweaver-Burk plot. Km and Vmax for inulin was found to be 2.53 mM and 12.78 mM/min, respectively. Km for sucrose and raffinose was found to be 9.15 mM and 13.80 mM, respectively. Whereas Vmax was found to be 21.07 mM/min and 9.18 mM/min for sucrose and raffinose, respectively. The purified enzyme was incubated at different pH for different time intervals (0-180 min) to study its effect on enzyme activity. The enzyme was found to be quite stable over a wide range (4.0-8.0) of pH. Inulinase retained its original activity at pH 5.0, 5.5, 6.0, 6.5 and 7.0 after incubation for 3 h. Only marginal decrease in enzyme activity was observed at pH 7.5, 4.0 and 4.5 after incubation for 3 h. The enzymes which are stable over wide range of pH always have an edge over others and this property of any enzyme is desirable for its industrial exploitation. The thermal stability of purified enzyme was tested at various temperatures (50, 60 and 70°C) for different time periods (15, 30, 45, 60, 90, 120 and 180 min). It was observed that enzyme was stable and retained its 100% activity at 50°C even after 3 h. The effect of metal ions on the enzyme activity of inulinase was tested by using different concentrations (1-10 mM) of various metal salts like AgNO3, Bad:, CaCl2, CoCl2. C11SO4, Fe2(SO4)3, HgCl2, KCl, KF, KI, MgCl2, MnSO4,ZnSO4 and EDTA. The maximum increase in enzyme activity was observed on addition of MnSO4 followed by CaCl2, BaCl2 and CoCl2 (However, KCl, KF and KI did not exert any significant influence on the activity up to the concentration of 7.5 mM. Whereas, AgNO3, CuSO4, Fe2 (SO4)3, HgCl2, MgCl2, ZnSO4 and EDTA had shown inhibitory effect on enzyme activity. The enzyme activity was completely inhibited by HgCl2, CuSO4 and EDTA at concentrations of 1 mM, 7.5mM and 10mM, respectively. Inulin (2.5%, w/v) in sodium acetate buffer (0.1 M, pH 5.5) was incubated with free inulinase (10 IU) under stationary and agitation (100 rpm) conditions at 50°C for different time intervals (30-360 min), to optimize the hydrolysis time of inulin. An increase in inulin hydrolysis was recorded with the increase in time up to 300 min under stationary conditions. The maximum hydrolysis of inulin was achieved after 270 min under agitation. Inulin hydrolysis of 79.51% (w/v) was obtained after 300 min under stationary conditions, whereas, 81% (w/v) hydrolysis was achieved after 270 min under agitation mode. The packed bed reactor (PBR) is the most frequently used immobilized bioreactor. The continuous hydrolysis of inulin was performed at 50°C in a jacketed packed bed column (1 X 20 cm) packed with 10.2 IU of inulinase immobilized on amino-PAN fibers. The upward flow of the inulin solution (2.5%, w/v) was maintained with a peristaltic pump. The packed bed reactors have the advantage of simplicity of operation and low cost. A decrease in inulin hydrolysis was observed with the increase in flow rate of inulin solution. The maximum inulin hydrolysis (approx. 80%, w/v) was obtained at flow rate of 0.5 to 1.5 ml/h. A further increase in flow rate of inulin above 1.5 ml/h has resulted in a decrease of inulin hydrolysis. PBR was continuously operated for 7 days at a flow rate of 1.5 ml/h to test the operational stability of the continuous system and samples were analyzed for inulin and reducing sugars. The hydrolysis of inulin remained in the steady state up to 30 h of operation, thereafter, only a little decrease in hydrolysis was observed up to 66 h of operation. References 1. Vandamme, E.J. and Derycke, D.G. (1983). Microbial inulinases: Fermentation process, properties, and applications. Adv. Appl. Microbiol. 29: 139-176. 2. Derycke, D.G. and Vandamme, E.J. (1984). Production and properties of Aspergillus niger inulinase. J. Chem. Technol. Biotechnol. 34B: 45-51. 3. Mukherjee, K. and Sengupta, S. (1987). Purification and properties of a nonspecific ß-fructofuranosidase inulinase from the mushroom Panaeolus papillonaceus. Can. J.Microbiol. 33:520-524. 4. Pessoni, R.A.B., Figueiredo, R.R.C.L. and Braga, M.R. (1999). Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae). J. Appl. Microbiol. 87: 141-147. 5. Guiraud, J.P., Viard, G.C. and Galzy, P. (1980). Inulinase of Candida salmenticensis. Agric. Biol. Chem. 44: 1245-1252. 6. Hensing, M., Vrouwenvelder, H., Hellinga, C, Baartmans, R. and Dijken, H.V. (1994). Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus. Appl. Microbiol. Biotechnol. 42: 516-521. 7. Cruz, G.A., Gracia, P.I., Barzana, E., Gracia, G.M. and Gomez, R.L. (1995). Kluyveromyces marxianus CDBB-L-278: A wild inulinase hyper producing strain. J. Ferment. Bioengg. 80: 159-163. 8. Elyachioui, M., Hornez, J.P. and Tailliez, R. (1992). General properties of extracellular bacterial inulinase. J. Appl. Bacteriol. 73: 514-519. 9. Gern, R.M.M., Furlan, S.A., Ninow, J.L. and Jonas, R. (2001). Screening for microorganisms that produce only endo-inulinase. Appl. Microbiol. Biotechnol. 55: 632-635. 10. Jonniaux, J.L., Rauw, K., Thonart, P. and Dauvrin, T. (2003). Enzyme or cell preparation with inulinase activity. US Patent No. 6518047. 11. Kerkhoffs, P.L. (1981). Preparation of fructose. US Patent No. 4277563. 12. Nakamura, T., Takizawa, T., Kamo, Y. and Hidaka, H. (1991). Process for the preparation of inulase. US Patent No. 4990451. I/We Claim: 1. An isolated culture of Kluyveromyces marxianus strain having accession number MTCC 5207 capable of producing higher amount of inulinase compared to the wild strain of Kluyveromyces marxianus. 2. A process for enhanced production of inulinase from Kluyveromyces marxianus strain having accession number MTCC 5207, wherein said process comprises: a) growing the Kluyveromyces marxianus strain for 2-4 days in a medium comprising of carbon source, nitrogen source, trace elements and surfactant at an initial pH ranging from 5-7 at a temperature ranging from 25°C to 35°C, and b) recovering the inulinase using conventional methods, wherein said Kluyveromyces marxianus strain is capable of producing higher amount of inulinase compared to the wild type Kluyveromyces marxianus. 3. The process as claimed in claim 2, wherein the medium consists of inulin, meat extract, CaCl2 and sodium-dodecyl sulphate. 4. The process as claimed in claim 2, wherein the carbon source is selected from the group consisting of fructose, glucose, lactose, maltose, starch, sucrose, and inulin and combination thereof. 5. The process as claimed in claim 2, wherein concentration of the carbon source is in the range of 0.1-3.5% (w/v). 6. The process as claimed in claim 2, wherein the carbon source is inulin. 7. The process as claimed in claim 2, wherein the concentration of inulin as a carbon source is in the range of 0.5-2.5 % (w/v), preferably 1.5% (w/v). 8. The process as claimed in claim 2, wherein the concentration of combination of carbon source is in the range of 0.1 to 2.5% comprising inulin is in the range of 0.1 to 1.0 % (w/v), preferably 0.1 to 0.5% (w/v). 9. The process as claimed in claim 2, wherein the nitrogen source is selected from the group consisting of peptone, meat extract, ammonium chloride, ammonium nitrate, ammonium sulphate, beef extract, corn steep liquor, sodium nitrate, urea and yeast extract, preferably meat extract. 10. The process as claimed in claim 9, wherein concentration of the meat extract is in the range of 0.5-5 % (w/v), preferably 2% (w/v). 11. The process as claimed in claim 2, wherein the surfactant is selected from a group consisting of sodium-dodecyl sulphate, Brij-35, Tween-20, Tween-60, Tween-80 and Triton X-100, preferably sodium-dodecyl sulphate. 12. The process as claimed in claim 11, wherein concentration of sodium-dodecyl sulphate is in the range of 0.05 to 2.5 mM, preferably 0.lmM. 13. The process as claimed in claim 2, wherein the Kluyveromyces marxianus strain was grown for a period of 30 to 78 hours, preferably 60 to 72 hours. 14. The process as claimed in claim 2, wherein the temperature is 30°C. 15. The process as claimed in claim 2, wherein said pH of the medium is 6.5.

Documents:

962-DEL-2005-Abstract-(18-06-2012).pdf

962-del-2005-abstract.pdf

962-del-2005-Assignment-(13-01-2015).pdf

962-DEL-2005-Claims-(18-06-2012).pdf

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962-DEL-2005-Correspondence Others-(02-11-2011).pdf

962-del-2005-Correspondence Others-(12-02-2013).pdf

962-del-2005-Correspondence Others-(13-01-2015).pdf

962-del-2005-Correspondence Others-(13-02-2013).pdf

962-DEL-2005-Correspondence Others-(18-06-2012).pdf

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962-del-2005-description (complete).pdf

962-DEL-2005-Drawings-(18-06-2012).pdf

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962-DEL-2005-Form-1-(18-06-2012).pdf

962-del-2005-form-1.pdf

962-del-2005-form-18.pdf

962-del-2005-form-2.pdf

962-DEL-2005-Form-3-(18-06-2012).pdf

962-del-2005-form-3.pdf

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962-DEL-2005-GPA-(02-11-2011).pdf