Kluyveromyces Marxianus NRRL Y-7571 Research Articles

<b>Agave syrup as a substrate for inulinase production by <i>Kluyveromyces marxianus</i> NRRL Y-7571

The factorial planning was used to plan and optimize inulinase production by the yeast Kluyveromyces marxianus NRRL Y-7571. The experiments were conducted using a Central Composite Design (CCD) 22, at different concentrations of agave syrup (3.6 to 6.4%) and yeast extract (2.2 to 3.0%). After 96 hours of fermentation, the best condition for the inulinase production was 5% agave syrup and 2.5% yeast extract, which yielded an average of 129.21 U mL-1 of inulinase. Partial characterization of the crude enzyme showed that the optimal pH and temperature were 4.0 and 60°C, respectively. The enzyme showed thermal stability at 55°C for 4 hours.

Modelación matemática del efecto de la temperatura en la actividad y la estabilidad térmica de la inulinasa de Kluyveromyces marxianus NRRL Y-7571

A mathematical model was used in this study to describe the effect of temperature on the activity and thermal stability (non-reactive conditions) of the inulinase of Kluyveromyces marxianus NRRL Y-7571. The model was taken from the literature, which was developed using the Arrhenius equation and experimental data. A first-order kinetic equation was takenfor enzyme inactivation. The model parameters were determined by linear and nonlinear regression, reporting confidence intervals. The experimental data showed that the maximum activity acting on sucrose and inulin was reached at 55 °C and that these activities were highly sensitive to higher temperatures. Furthermore, inulolytica activity was more thermally stable than the invertase activity in the range from 48 to 60 °C. Using the model, the process temperature for the hydrolysis of sucrose and inulin was determined at the intersection of the curves of the relative activity and relative half-life, resulting 49 °C for both processes. Although this model was used with reference to the sucrose and inulin hydrolysis, the approach is a useful tool that can be applied to other enzymatic processes for determining the operating temperature, which is ultimately determined by an economic evaluation.

Yacon Flour and Corn Steep Liquor as Substrate for Inulinase and Biomass Production by Kluyveromyces Marxianus NRRL Y-7571

Yacon is a perennial plant native of the Andesthat stores inulin-type polyfructans as reserve carbohydrates in its tubers. The enzymes that hydrolyze the inulin β-2,1 fructose  linkages are called inulinases and are much used to produce high fructose syrups (HFS) and ethanol. The present study investigated inulinase and biomass production by Kluyveromyces marxianus NRRL Y-7571 using yacon flour as carbon source and corn steep liquor as nitrogen source by a Central Composite Design (CCD) 22 to carry out experiments and Surface Response Methodology (RSM) to analyze the results. The maximum enzyme production was 36.1 U.ml-1 and biomass production was 8.95 g.l-1 under the condition of 7% yacon flour and 2% corn steep liquor. The crude enzyme partial characterization showed that inulinase had optimal pH at 4.0 and optimal temperature at60°C. The enzyme maintained over 90% of its activity for four hours at45°C and50°C.

Fructooligosacharides production in aqueous medium with inulinase from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 immobilized and treated in pressurized CO2

This work investigated the influence of compressed CO2 treatment on the enzymatic activity of immobilized inulinases, and the production of fructooligosacharides in aqueous medium using these enzymes. The effects of system pressure, exposure time and depressurization rate on the enzymatic activity were evaluated through central composite designs (CCD) 23. Inulinase from Kluyveromyces marxianus NRRL Y-7571 presented an increase of 104% in the residual activity using CO2 at 275bar submitted to 6h treatment, at a depressurization rate of 10kgm−3min−1. For Aspergillus niger commercial inulinase, a decrease in enzyme activity was observed (residual activity of 39%) using CO2 treatment at 75bar for 6h exposure at the highest depressurization rate (200kgm−3min−1). Enzymatic activities changed significantly depending on the enzyme source and the experimental treatment conditions investigated. The values of FOS obtained using inulinases from A. niger were 30.64% of GF2; 13.90% of GF3 and 2.88% of GF4 in the medium containing inulin as substrate. Results demonstrate that the use of compressed CO2 might be of technological importance as a preceding, preparation step, to improve enzyme activity, hence helping the development of new biotransformation processes.

Enzymatic synthesis of fructooligosaccharides by inulinases from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 in aqueous–organic medium

This work is focused on the synthesis of the fructooligosaccharides (FOS) from sucrose and inulin, using free, immobilized and pre-treated immobilized inulinase from Kluyveromyces marxianus NRRL Y 7571 and Aspergillus niger in an aqueous–organic system. Initially, the influence of pre-treatment using four different gases, propane, n-butane, CO2 and liquefied petroleum gas (LPG), was investigated towards FOS production and best results were found when both enzymes were previously treated with LPG. The best reaction yields were obtained when the immobilized enzymes were treated with LPG. Considering FOS synthesis using the enzyme from A. niger, yields of 26.62% of GF2 (kestose), 30.62% of GF3 (nystose) and 8.47% of GF4 (fructosyl nystose) were achieved using sucrose as substrate. Using inulinases from K. marxianus NRRL Y 7571, 11.89% of GF2 and 20.83% of GF3 were obtained, using inulin as substrate. However, promising results were achieved using the free form of inulinase from A. niger (77.19% of GF2; 14.03% of GF3 and 0.07% of GF4) using inulin as substrate.

Natural montmorillonite as support for the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571

Abstract The objective of this study was to investigate the process of immobilization of inulinases using natural montmorillonite as inorganic support. The enzyme to buffer ratio of 3:10 and 10 min of immobilization led to the highest specific activity, 375.07 U/mg protein. The immobilized inulinase kept its activity after 1968 h under storage at low temperatures and after 456–1826 h at high temperatures. The pH value of 3.5 led to the highest specific activity. Km values of 1.46 and 0.38 mM, and vmax of 0.2487 and 0.2396 mol/L min, were obtained, respectively, for sucrose and inulin.

Assessment of fructooligosaccharides production from sucrose in aqueous and aqueous-organic systems using immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571

This work investigated the fructooligosaccharides (FOS) synthesis by immobilized inulinase obtained from Kluyveromyces marxianus NRRL Y-7571 in aqueous and aqueous-organic systems using sucrose as substrate. The sequential strategy of experimental design was used to optimize the FOS conversion in both systems. For the aqueous-organic system, a 2(6-2) fractional design was carried out to evaluate the effects of temperature, sucrose concentration, pH, aqueous/organic ratio, enzyme activity, and polyethylene glycol concentration. For the aqueous system, a central composite design for the enzyme activity and the sucrose concentration was carried out. The highest fructooligosaccharides yield (Y FOS) for the aqueous-organic system was 18.2 ± S0.9 wt%, at 40 ºC, pH 5.0, sucrose concentration of 60% (w/w), enzyme activity of 4 U.mL-1, and aqueous/organic ratio of 25/75 wt%. The highest Y FOS for the aqueous system was 14.6 ± 0.9 wt% at 40 ºC, pH 5.0, sucrose concentration of 60 wt%, and enzyme activity of 4.0 U.mL-1.

Effect of compressed fluids treatment on the activity of inulinase from Kluyveromyces marxianus NRRL Y-7571 immobilized in montmorillonite

This work is aimed at assessing the influence of pressurized fluids treatment on the enzymatic activity of inulinases from Kluyveromyces marxianus NRRL Y-7571 immobilized in natural montmorillonite. The effects of system pressure, exposure time and depressurization rate, using propane and liquefied petroleum gas (LPG) on the enzymatic activity were evaluated through central composite designs (CCD) 23. In general, results showed that the enzyme activity changes significantly depending on the experimental conditions investigated, allowing the selection of proper operating conditions for advantageous application of this biocatalyst in hydrolysis reactions. Further, the stability of the enzyme after high-pressure pre-treatment was also experimentally monitored, and results demonstrated that the activity of the biocatalyst was always higher than the non-treated one. Results obtained here might be useful as a basis for the selection of appropriate process conditions, so that the catalyst can be applied in biotransformation reactions.

Immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571 using modified sodium alginate beads

An experimental design was carried out to evaluate the effect of the concentrations of sodium alginate, glutaraldehyde and activated coal on the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571. The experimental condition of 20g/L of sodium alginate, 50mL/L of glutaraldehyde and 30g/L of activated coal led to the highest specific activity (2,063.5U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also evaluated, showing optimal activities at pH of 5.5 and 55°C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43days of storage at 40 and 50°C and after 138days of storage either at 4 or 25°C.

Ethanol precipitation and ultrafiltration of inulinases from Kluyveromyces marxianus

Inulinase (EC 3.2.1.7) produced by Kluyveromyces marxianus NRRL Y-7571 by solid state fermentation of sugarcane bagasse was purified from culture broth by ethanol precipitation and ultrafiltration. The effects of the concentration of ethanol and the rate of addition of ethanol to the crude extract on the purification yield and purification factor were assessed using the experimental design technique. Precipitation caused an activation of enzyme and allowed purification factor up to 2-fold. After precipitation with ethanol 55% (v/v) and a flow rate of 10 mL/min, followed by ultrafiltration with a 100 kDa membrane, the inulinase could be purified by a factor of 5.5-fold with 86.1% yield.

Comparative studies of the stability of free and immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571 in aqueous-organic solutions

Enzymes have been extensively used in organic solvents to catalyze a variety of reactions of biological and industrial significance. In this work, the characteristics of free and immobilized inulinase were investigated in buffered solutions of butyl acetate. The influences of the organic solvent content on the optimal temperature and pH, the stabilities to temperature and pH and the kinetic parameters were systematically evaluated. The results showed that the organic solvent content had no effect on the optimal pH, either in the free or immobilized inulinase. For the immobilized enzyme, the optimal temperatures ranged from 55ºC to 60ºC, depending on the content of butyl acetate. At higher butyl acetate content, the stability of the immobilized enzyme increased for both pH and temperature. The organic solvent showed the tendency to increase the values of the kinetic parameters Km and v max for both free and immobilized inulinase.

Mathematical modeling of thin-layer drying of fermented and non-fermented sugarcane bagasse

This work reports hot-air convective drying of thin-layer fermented and non-fermented sugarcane bagasse. For this purpose, experiments were carried out in a laboratory-scale dryer assessing the effects of solid-state fermentation (SSF) on the drying kinetics of the processing material. The fermented sugarcane bagasse in SSF was obtained with the use of Kluyveromyces marxianus NRRL Y-7571. Drying experiments were carried out at 30, 35, 40 and 45 °C, at volumetric air flow rates of 2 and 3 m3 h−1. The ability of ten different thin-layer mathematical models was evaluated towards representing the experimental drying profiles obtained. Results showed that the fermented sugarcane bagasse presents a distinct, faster drying, behavior from that verified for the non-fermented material at the same conditions of temperature and volumetric air flow rate. It is shown that the fermented sugarcane bagasse presented effective diffusion coefficient values of about 1.3 times higher than the non-fermented material. A satisfactory agreement between experimental data and model results of the thin-layer drying of fermented and non-fermented sugarcane bagasse was achieved at the evaluated experimental conditions.

Mathematical modeling of Kluyveromyces marxianus growth in solid-state fermentation using a packed-bed bioreactor

This work investigated the growth of Kluyveromyces marxianus NRRL Y-7571 in solid-state fermentation in a medium composed of sugarcane bagasse, molasses, corn steep liquor and soybean meal within a packed-bed bioreactor. Seven experimental runs were carried out to evaluate the effects of flow rate and inlet air temperature on the following microbial rates: cell mass production, total reducing sugar and oxygen consumption, carbon dioxide and ethanol production, metabolic heat and water generation. A mathematical model based on an artificial neural network was developed to predict the above-mentioned microbial rates as a function of the fermentation time, initial total reducing sugar concentration, inlet and outlet air temperatures. The results showed that the microbial rates were temperature dependent for the range 27-50 degrees C. The proposed model efficiently predicted the microbial rates, indicating that the neural network approach could be used to simulate the microbial growth in SSF.

Kinetics of inulinase production by solid-state fermentation in a packed-bed bioreactor

In this work the cell growth and inulinase production by Kluyveromyces marxianus NRRL Y-7571 were investigated in a packed-bed bioreactor. For this purpose, seven experimental runs were carried out in order to verify the influence of the inlet air temperature and volumetric air flow rate on the process dynamics. The results showed that the manipulated variables affected significantly the process performance. The best condition for enzyme production was an inlet air temperature and volumetric flow rate of 30 °C and 3 m 3 h −1, respectively, reaching an activity of around 463 U gds −1. The results obtained here can be useful in the scale-up and optimisation of packed-bed bioreactors configuration for inulinase production.

Comparison Between Systems for Synthesis of Fructooligosaccharides from Sucrose Using Free Inulinase from Kluyveromyces marxianus NRRL Y-7571

This work is focused on the synthesis of the fructooligosaccharides (FOS) from sucrose using free inulinase from Kluyveromyces marxianus NRRL Y-7571 in aqueous and aqueous–organic systems. The most significant variables for the aqueous–organic system were identified using a fractional factorial design. The evaluated variables were the temperature, pH, sucrose concentration, inulinase activity, aqueous/organic ratio, and the polyethylene glycol concentration. The use of sequential experimental design methodology was shown to be very useful in the optimization of the FOS synthesis by inulinase either in aqueous or aqueous–organic systems. For the aqueous–organic system, the maximum Y FOS reached was 16.7 ± 1.1 wt.% with the following operational conditions: temperature of 40 °C, enzyme activity of 4 U mL−1, organic solvent/total system ratio of 25/100, pH of 6.0, and sucrose concentration of 55%. In the aqueous system, the maximum conversion obtained was 12.8 ± 1.0 wt.% under the following conditions: 40 °C, pH 5.0, 55% sucrose, and inulinase activity 4 U mL−1.

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett-Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 +/- 65 U/ml.

Inulinase production in a batch bioreactor using agroindustrial residues as the substrate: experimental data and modeling

The production of inulinase employing agroindustrial residues as the substrate is a good alternative to reduce production costs and to minimize the environmental impact of disposing these residues in the environment. This study focused on the use of a phenomenological model and an artificial neural network (ANN) to simulate the inulinase production during the batch cultivation of the yeast Kluyveromyces marxianus NRRL Y-7571, employing a medium containing agroindustrial residues such as molasses, corn steep liquor and yeast extract. It was concluded that due to the complexity of the medium composition it was rather difficult to use a phenomenological model with sufficient accuracy. For this reason, an alternative and more cost-effective methodology based on ANN was adopted. The predictive capacity of the ANN was superior to that of the phenomenological model, indicating that the neural network approach could be used as an alternative in the predictive modeling of complex batch cultivations.

Extraction of Inulinase Obtained by Solid State Fermentation of Sugarcane Bagasse by Kluyveromyces marxianus NRRL Y-7571

Production of inulinase by solid state fermentation always involves an extraction step, which dictates enzyme recovery yield and is related to cultivation conditions and control of process parameters. This work is focused on the study of extraction conditions aiming to maximize yield of an inulinase obtained by solid state fermentation of sugar cane bagasse and Kluyveromyces marxianus NRRL Y-7571. Kinetics of extraction was followed varying the kind of solvent used. After determining the best solvent, an experimental design was carried out to study the effect of the solid/liquid ratio (1:10-1:20), extraction temperature (20-53 degrees C), and stirring rate (50-177 rpm). Results showed that maximum yield was obtained when sodium acetate buffer 0.1 M pH 4.8 was used, using a solid/liquid ratio of 1:10, at 53 degrees C and 150 rpm for 40 min.

Inulinase Production by <i>Kluyveromyces marxianus</i> NRRL Y-7571 Using Solid State Fermentation

Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.

Adsorption of the inulinase from Kluyveromyces marxianus NRRL Y-7571 on Streamline® DEAE resin

The enzyme inulinase is used to produce oligosaccharides and fructose, with up to 95% fructose in a single stage of inulina hydrolysis. With in the aim to purify the enzyme, studies on the conditions of enzyme adsorption in an expanded-bed column were conducted using phosphate and tris-HCl buffers. The inulinase used in this work was obtained from Kluyveromyces marxianus NRRL Y-7571 by fermentation in an industrial medium. Using the anionic resin Streamline DEAE, the adsorption equilibrium time was determined. It was observed that the adsorption isotherm follows the Langmuir model; the parameters for the maximum amount of adsorbed inulinase (Qm) and the dissociation constant (k d) were determined. With 0.05 M sodium phosphate buffer at pH 6.0, the parameter values 1428 UI/mL and 2 UI/mL with a correlation coefficient of 0.96 were obtained. For 0.02 M tris-HCl buffer at pH 7.5, the same parameters were 5000 UI/mL and 0.05 UI/mL with a correlation coefficient of 0.99. The best purification conditions for the fixed bed were shown to be a 0.4 M phosphate buffer with NaCl as eluter, a purification factor of 11.4, and a recovery yield of up to 79%.