Optimisation of Immobilisation Conditions of β-galactosidase onto Chitosan Beads Using Response Surface Methodology

Response Surface Methodology (RSM) was advantageously used to optimize production conditions of immobilization of β-galactosidase onto chitosan beads. The influence of immobilization conditions on the immobilized enzyme activity was studied by using Box–Behnken experimental design, resulting in the appropriate average values of OD420 and the maximum value of immobilized enzyme activity. This illustrated that the experimental model was reliable and the experimental results were of good stability. Analysis of variance was performed to determine the adequacy and significance of the quadratic model. Various model parameters also could be seen from F value that the interaction between three factors was not particularly obvious, but effects of X1, X1 , X2 2 and X3 2 on the activity of immobilized enzyme were very obvious. The optimisation parameters studied in accordance with the results were: the appropriate average values of OD420 loaded in the range from 0.713 to 0.721. Furthermore the immobilized enzyme activity reached about 162U/g when the adsorption time, adsorption pH and cross-linking pH were 4h, 7 and 7, respectively. Thus, the relative activity of β-galactosidase immobilized increased more than 37% compared with the previous single factor research.


INTRODUCTION
Lactose intolerance is a clinical syndrome that can cause lactose maldigestion, the symptoms of the disease are diarrhea, pain, nausea and flatulence, this will lead to lots of people to avoid milk or milk products (Devesh and Arvind, 2012;Kocin, 1988) due to the inability to digest lactose into its constituents, glucose and galactose, because of low levels of lactase (Valentina et al., 2011).Thus, it is necessary to remove the lactose through the use of enzymes.One of the more common ways to accomplish this is through the use of enzymes.
Response surface methodology (RSM) is one of the popular statistical methods (Neelesh et al., 2014) commonly applied for optimization of immobilization of enzymes and cells (Piyushkumar et al., 2007;Park and Chang, 2000;Ariga et al., 1997).This method involves various statistical and mathematical techniques based on the multivariate non-linear model that has been widely used method of modeling and analyzing the relationships between several independent variables and response variable(s) (Neelesh et al., 2014;Alessandro et al., 2013;Piyushkumar et al., 2007).
Chitosan and its derivatives are known as a swollen bead support for preparation of immobilized enzyme (Juang et al., 2002;Muzzarelli, 1980).Chitosan has very good biocompatibility, low toxicity, chemically inert and high hydrophilicity (Alka and Arvind, 2009).
As far as can be ascertained, the present literatures mainly contain studies relating to the optimisation of immobilization of β-galactosidase onto epoxy-activated (Pedro and Francisco, 2012), alkylamine glass (Devesh and Arvind, 2012), Sephadex (Alka and Arvind, 2009), even cell immobilization (Piyushkumar et al., 2007) and so on.Unfortunately, the main disadvantages do not like particularly high immobilization efficiency and heavy leakage of enzyme have not been desirably improved (Qiuyun et al., 2011).
The present work reported that various factors implicated in immobilization of β-galactosidase onto chitosan beads were optimized using the response surface methodology.Furthermore, a comparison study involving the values of OD 420 and immobilized enzyme activity in the previous single factor research (He et al., 2013) were carried out.Finally, optimum production conditions were determined when higher OD 420 value and immobilized enzyme activity.

Preparation of chitosan beads:
With 20 g/L acetic acid solution dissolved 30 g/L chitosan.Using 1mL medical needle tube added chitosan into 1mol/L NaOH solution, the chitosan beads were rinsed several times until neutral with distilled water after coagulating.Finally, the chitosan beads were filtered and air-dried (Adriano et al., 2008).

Preparation of immobilized lactase:
The βgalactosidase was immobilized on chitosan beads by using glutaraldehyde.Added 1 g chitosan beads into 10 mL 0.5% glutaraldehyde and cross-linked at 25°C for 1 h, then washed chitosan beads with distilled water until there was no residual glutaraldehyde solution.Chitosan beads were placed into 10 mL 1g/mL lactase solution and soaked at 25°C for 2 h and then washed the immobilized enzyme with distilled water and filtered by suction until enzyme activity could not be detected in the distilled water, finally the immobilized lactase activity was measured.
Enzyme activity assays: Using ONPG as substrate, the activity of β-galactosidase could be assayed by colorimetric test.The β-galactosidase can catalyze ONPG to ONP and galactose, The ONP in alkaline medium was yellow, which has the absorbance value at the wavelength of 420 nm in the solution.A standard curve was constructed by using ONP at various concentrations (Cavaille and Combes, 1995).
Dissolved 100 mg β-galactosidase into phosphate buffer (pH 6.5) and diluted to 100mLwith distilled water to prepare enzyme solution.Then 1mL the enzyme solution was diluted by using 100mLphosphate buffer (pH 6.5).The 3 mL ONPG solution prepared with phosphate buffer (4 mg/mL) was added to test tube and set at 38°C for 7 min, the ONPG solution was well mixed with the 1mLdiluted enzyme solution and kept at 38°C for 10 min.Added 2 mL1 mol/ L Na 2 CO 3 solution to terminate the reaction, then the absorbance value was measured at 420 nm.
Under the measurement conditions (38°C, pH6.5, for 10 min), an enzyme activity Unit (U) was the amount of enzyme that catalyses to generate 1µmol ONP per min under standard assay conditions.The immobilized enzyme activity was measured by the same method, However, we had to consider the immobilized protein content (He et al., 2013).

Determination of immobilization parameters:
"Immobilized lactase activity" was defined as the unit of the amount of immobilized lactase was required when 1 µg ONP was generated per hour under the conditions (38°C, pH 6.5, reaction time of 20 min).This was calculated as: where, Y = The immobilized lactase activity unit E (OD) = The absorbance value, 1.8436 is the conversion ratio T(h) = The reaction time M(g) = The amount of immobilized lactase

RESULTS AND DISCUSSION
According to the results of P-B test and the test results to determine the adsorption time (X 1 ), the adsorption of pH (X 2 ) and cross linked pH (X 3 ), the response surface analysis method regarded the three factors as variables.And the response value was the value of OD 420 for optimization of component of lactase immobilization; the Y value was the immobilized enzyme activity.As shown in Table 1, three factors were set to three gradients, respectively.And there were more reasonable range between each factor gradient.Analysis of Table 2 experimental data using SAS software was also done to determine the adequacy and significance of quadratic model and then could find the optimal response factor.The response surface model was provided in Table 3.The final equation in terms of actual factors, which governed the response, was as follows: As shown in Table 3 regression analysis revealed a coefficient of determination (R-squared) value of 0.9452, indicating that the model was able to explain total variations.Various model parameters also can be seen that the interaction of the F value was very small and the interaction between these 3 factors indicated very little effect.Effects of X 1 X 2 , X 1 X 3 , X 2 X 3 on the activity of immobilized enzyme were not significant, the interaction between the effects of each other included no statistical significance, but effects of X 1 , X 1 2 , X 2 2 and X 3 2 on the activity of immobilized enzyme were more obvious.As Fig. 1 showed variation tendency of each factor and response value of each factor level affect the activity of immobilized lactase.Figure 2 to 4 directly reflected the influence of each factor on the response value.When one factor was under optimal conditions, the relationship between other 2 factors and response value was performed with three-dimensional coordinate graph.
As Fig. 2 showed when the value of cross linking pH was in the center level, the activity of immobilized enzyme would firstly increase and then decrease with prolonging adsorption time and increasing adsorption pH.And as the contour plot displayed that it approximated to roundness, the interaction between each other was not obvious.
As Fig. 3 showed when the value of adsorption pH was in the center level, the activity of immobilized enzyme would firstly increase and then decrease with prolonging adsorption time and increasing cross linked pH.And as the contour plot displayed that it approximated to an oval, the interaction existed between each other, but the effect is not significant.
As Fig. 4 showed when the value of adsorption time was in the center level, the activity of immobilized enzyme would firstly increase and then decrease with prolonging adsorption pH and increasing cross linked pH.And as the contour plot displayed that it approximated to an oval, the interaction existed between each other, but the effect is not signi Analysis of regression by SAS software was done to obtain the partial derivatives of X1, X2, X3 respectively and the required partial derivatives were easily worked out.Then the maximum point (0, 0, 0) was worked out and achieved.Under conditions prediction of activity of the immobilized Response surface plot and contour plot of influence of X 1 , X 2 on the response of Y1 : Response surface plot and contour plot of influence of X 1 , X 3 on the response of Y1 Response surface plot and contour plot of influence of X 2 , X 3 on the response of Y1 pH.And as the contour plot displayed that it approximated to an oval, the interaction existed between each other, but the effect is not significant.
Analysis of regression by SAS software was done to obtain the partial derivatives of X1, X2, X3 respectively and the required partial derivatives were easily worked out.Then the maximum point (0, 0, 0) was worked out and achieved.Under the above conditions prediction of activity of the immobilized lactase would reach 0.712.On second thoughts the repeated experiments were conducted.To be specific, added accurately 1 g chitosan beads into 10 mL 0.3% glutaraldehyde and cross-linked at 2.5 h, then washed chitosan beads with distilled water until there was no residual glutaraldehyde solution.Chitosan beads were placed into 10 lactase solution and soaked at 30°C and pH7.0 for 4 h and then washed the immobilized enzyme with distilled lactase would reach 0.712.On second thoughts the repeated experiments were conducted.To be specific, g chitosan beads into 10 mL 0.3% linked at 25°C and pH7.0 for 2.5 h, then washed chitosan beads with distilled water until there was no residual glutaraldehyde solution.Chitosan beads were placed into 10 mL 7.5 g/mL C and pH7.0 for 4 h zed enzyme with distilled water and filtered by suction until enzyme activity could not be detected in the distilled water, finally the immobilized lactase activity was measured.The above experiment was performed 3 times in parallel.Ultimately, the test results of OD 420 values were 0.721, 0.718 and 0.713 and the average is 0.717±0.004.The results increased about 0.7% than the predictive value (predictive value was 0.712).At this point, the immobilized enzyme activity reached the maximum value of 162 U/g.This illustrated that the experimental model was reliable and the experimental results were of good stability.

CONCLUSION
Statistically designed experimentation for optimum immobilization of β-galactosidase onto chitosan beads exhibited best production conditions due to the optimal activity of the immobilized enzyme achieved.The optimisation parameters studied in accordance with the results were: the immobilized enzyme activity reached about 162 U/g when the adsorption time, adsorption pH and cross-linking pH were 4h, 7 and 7, respectively.Thus, the relative activity of β-galactosidase immobilized increased more than 37% compared with the previous single factor research.

Fig. 1 :
Fig. 1: The variation tendency of activity of immobilized lactase with each factors

Fig. 2 :
Fig. 2: Response surface plot and contour plot of influence of X Table 1 the factor level coding table of Box-Behnken experiment design.Table 2 the experimental design and results of Box-Behnken test.

Table 1 :
The factor level coding table of Box-

Table 3 :
Analysis of variance for response surface model pertaining to percent immobilization