MATERIALS AND METHODS

This study was conducted during the period of May, 2010 to December, 2010.

Procurement of microorganism: Kluyveromyces marxianus NCIM 3465 was procured from the National collection of Industrial Microbiology, National Chemical Laboratory, Pune, India.

Maintenance and cultivation of the culture: The maintenance and cultivation of the culture was carried out by the method of Panesar (2008).

Permeabilization of yeast cells: The permeabilization of yeast cells was carried out by following the method of Joshi et al. (1987) with slight modification. The cells were harvested from 5 mL of fermentation media by centrifugation (5000 rpm for 5 min at 4°C) and washed with potassium phosphate buffer (0.1 M, pH 7.0). Different permeabilization agents such as benzene, n-butanol, n-propanol, isopropanol, toluene, acetone, ethanol and a mixture of permeabilizing agents in 1:1 ratio of ethanol (50%, v/v) and acetone (30%, v/v), ethanol (50%, v/v) and toluene (25%, v/v), ethanol (50%, v/v) and n-propanol (20%, v/v), ethanol (50%, v/v) and iso-propanol (40%, v/v), ethanol (50%, v/v) and n-butanol (10%, v/v) were added to the yeast biomass and the final volume was made 5 mL using the same buffer. The contents were mixed on a vortex mixture and incubated for 10 min, under shaking conditions. After this, the cells was recentrifuged and washed twice with the same buffer and analyzed for the enzyme activity.

Table 1:	Level of different process variables for β-galactosidases

Table 2:	Experimental designs of process variables and values of experimental data for optimization of enzyme activity
aDry weight

Measurement of enzyme activity: β-galactosidase activity assay was carried out by the method of Miller (1972). One unit of enzyme activity is defined as one micromole of o-nitrophenol liberated per min under standard assay conditions.

Experimental design and statistical analysis: Experiments were conducted according to Central Composite Rotatable Design (CCRD) with three variables at five levels each. The design was generated by Design Expert, Trial version 6.0, Stat-Ease INC., Minneapolis, MN, USA statistical software. The variables were toluene: ethanol ratio, treatment time and temperature for the enzyme activity. The low level and high level in actual (un-coded) form were taken as 30:70-50:50 of toluene and ethanol ratio, 10-20 min (treatment time) and 20-30°C (temperature). The relationships between coded to uncoded independent process variables are given in Table 1. The experimental plan in coded and un-coded form of process variables along with result is as given in Table 2. The experiments were conducted randomly.

Statistical analysis and optimization: The second order polynomial equation was fitted to the experimental data of each dependent variable as given:

(1)

where, Y_i is Response {Y₁ is Enzyme activity (IU mg^-1 DW), x_i is Independent variables, (x₁ is Toluene (25%, v/v): Ethanol (50%, v/v), x₂ is Temperature (°C), x₃ is Treatment time (min), β_o is the value of coefficient of fitted response at the central point of design, β_i, β_ij, β_ii are the linear, quadratic and cross product regression coefficients, respectively.

RESULTS AND DISCUSSION

The permeabilization of Kluyveromyces marxianus NCIM 3465 cells was carried out by using various chemical agents (n-butanol, n-propanol, iso-propanol, acetone, ethanol and toluene) for β-galactosidase activity. From the experimentation, it has been observed that permeabilization increase with the chemical concentration up to a critical value, where a maximum enzyme activity can be observed (Fig. 1). At higher concentration of the permeabilizing agent, the enzyme activity decreases which may be due to the leakage of the enzyme from the cells or cell lysis. However, at low concentration, enzyme activity was further decreased due to the insufficient amount of the agent for the permeabilization.

The maximum β-galactsidase activity of 1.378, 1.471, 1.193, 1.432, 1.475 and 1.535 IU mg^-1 DW have been observed with 10% (v/v) of n-butanol, 20% (v/v) n-propanol, 40% (v/v) iso-propanol, 20% (v/v) toluene, 30% acetone and 50% (v/v) ethanol, respectively (Fig. 1). Similarly, Decleire et al. (1987) have observed that the minimum solvent concentrations of 10% n-butanol; 20% propanol; 30% isopropanol, tert-butanol; 40% ethanol and acetone and 70% dimethylsulphoxide were required for maximum enzyme activity. Flores et al. (1994) reported that 40% (v/v) of ethanol was an effective permeabilizing agent for maximum β-galactosidase activity.

The results also revealed that permeabilization of yeast cells further increases by using a mixture of permeabilizing agents (ethanol and acetone, ethanol and toluene, ethanol and n-propanol, ethanol and iso-propanol, ethanol and n-butanol, ethanol and benzene). Among a mixture of different permeabilizing agents (Fig. 2), maximum enzyme activity 1.61 IU mg^-1 DW was found with 1:1 ratio toluene (25%, v/v): ethanol (50%, v/v).

Fig. 1:	Effect of concentration of iso-propanol, n-propanol, acetone, ethanol, n-butanol, toluene on the permeabilization of yeast cells

Fig. 2:	Effect of a mixture of different permeabilizing agents (1:1 ratio) on the permeabilization of yeast cells

Various permeabilizing agents such as iso-propanol, tert-butanol, ethanol: toluene, chloroform, CTAB have been used for the permeabilization of K. lactis NRRL Y-1140 and observed that a small quantity of toluene in ethanol or 70% ethanol was most effective permeabilizing agent for β-galactosidase activity (Siso et al., 1992).

Optimization of permeabilization of yeast cells: From the preliminary experiments to optimize of permeabilization process by using mixture of organic solvents, it was observed that the range of toluene: ethanol ratio, incubation time and temperature for maximum β-galactosidase activity were 30:70-50:50, 10-20 min and 20-40°C, respectively.

Diagnostic checking of fitted model and surface plots: The ANOVA result in Table 3 indicates that the quadratic regression to produce the second order model was significant (p<0.0001). The lack of fit was non significant (p = 1.61) and only 0.29% of the total variation were not explained by model (R² = 99.71%). The model F-value of 383.14 also implies that the model is significant. The value of adjusted determination coefficient (adjusted R² = 99.45%) was high to advocate a high significance of model. The magnitude of p-value in Table 3 indicates that the linear and quadratic terms of all the process variables have significant effects on β-galactosidase activity at 5% level of significance (p<0.05).

The magnitude of β coefficient as given in Table 3 revealed that the linear term of toluene (25%, v/v): ethanol (50%, v/v) had the positive effect (β = +0.039) on enzyme activity. The temperature have negative effect (β = -0.17) followed by treatment time (β = -0.086) on enzyme activity. This indicates that with increase the toluene (25%, v/v): ethanol (25%, v/v) ratio, there is an increase in enzyme activity which indicates that low amount of permeabilizing agent is insufficient for effective permeabilization. However, with an increase in temperature and incubation time enzyme activity decreases which may be due to enzyme inactivation or cell lysis (Joshi et al., 1989).

At lower levels of incubation time, the enzyme activity of yeast cells increased with the increase in toluene: ethanol ratio; but at high levels of incubation time a slight decrease in enzyme activity has been observed with increase in toluene: ethanol ratio (Fig. 3).

Table 3:	Regression summery and ANOVA table for enzyme activity fir coded value of process variable
*Non-significant at 5% level

The decrease in enzyme activity with increase of toluene: ethanol ratio at higher incubation time may be due to the leakage of the enzyme from the cells or cell lysis. The maximum value of enzyme activity has been observed in the range of 45:55 to 47:53 of toluene: ethanol and 12 to 14 min of incubation time. The optimal treatment time of 14 min for the permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 using cetyltrimethylammonium bromide (0.06%, w/v), whereas an optimal time 15 min for the permeabilization of K. lactis NRRL Y-8279 cells using Isoamyl alcohol (20%, v/v) has been observed by previous researchers (Kaur et al., 2009; Dagbagli and Goksungur, 2008).

The variation of enzyme activity as function for interaction of concentration and temperature indicates that there is an increase in enzyme activity with increase of toluene: ethanol ratio (Fig. 4). The increase in temperature decreases the enzyme activity might be because of the inactivation of enzyme or cell lysis at high temperature. The maximum enzyme activity was observed at the temperature range of 20-23°C. An optimal temperature of 25 and 26°C for the permeabilization of Kluyveromyces bulgaricus IRC101 and K. fragilis NRRL Y-1196 cells has been reported, respectively (Decleire et al., 1987; Joshi et al., 1989).

At low levels of temperature, the enzyme activity of yeast cells increased with the increase in incubation time (Fig. 5). However at high incubation time, with increase in temperature a decrease in enzyme activity was observed might be due to the partial inactivation of enzyme or cell lysis at high temperature. The maximum enzyme activity was observed at the temperature range of 20.0-23.0°C and the incubation time of 12.5-15.0 min. Whereas, the optimal treatment time of 18 min and temperature of 23°C using ethanol for the optimal permeabilization of K. marxianus has been observed previously (Panesar, 2008).

The equation of fitted model after neglecting the effect of non-significant term in term of uncoded (actual) terms of process variable is:

(2)

Fig. 3:	Effect of treatment time and toluene (25%, v/v): ethanol (50%, v/v) on β-galactosidase activity

Fig. 4:	Effect of temperature and toluene (25%, v/v): ethanol (50%, v/v) on β-galactosidase activity

Fig. 5:	Effect of increase in temperature and treatment time on β-galactosidase activity

Optimization of permeabilization process: In order to optimize the process conditions for permeabilization by numerical optimization technique, equal importance of ‘3’ was given to all the three process parameters (viz. concentration temperature and time) and response (enzyme activity). The main criterion for constraints optimization was maximum possible enzyme activity. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity were 50:50 toluene (25%, v/v): ethanol (50%, v/v) ratio, 23°C temperature and process duration of 12 min. At these conditions of process variables the predicted value of enzyme activity was found to be 1.68 IU mg^-1 DW. The results of optimization were confirmed by conducting the experiments in triplicate at the above optimized values with±0.2% deviation in the enzyme activity values. During the experimentation, it was observed that the time and temperature are two critical factors for the effective permeabilization of yeast cells.

CONCLUSION

Response surface methodology was effective in optimizing process parameters for the β-galactosidase activity for different conditions in the range of toluene (25%, v/v): ethanol (50%, v/v) 30:70 to 50:50, treatment time 10 to 20 min and temperature 25 to 30°C. The regression equations obtained in this study can be used for optimum conditions for desired response within the range of conditions applied in study. The recommended process variables were 50:50 (toluene, 25% v/v: ethanol, 50% v/v), treatment time 12 min and 23.0°C to get maximum β-galactosidase activity. The use of permeabilized cells can help to overcome the problems/costs associated with enzyme extraction and purification from yeast cells and in the development of a low-cost technology for β-galactosidase production. Beside this, the application of the permeabilized yeast cells can also be explored for hydrolysis of lactose and the synthesis of lactulose and galacto-oligosaccharides which can be used as potential ingredients in functional foods.

ACKNOWLEDGMENTS

The authors acknowledge the financial support given by Council of Scientific and Industrial Research (CSIR), New Delhi, India to carry out this study.