Parameter optimization for lipase production by Bacillus megaterium using response surface methodology

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1 Indian Journal of Biochemistry & Biophysics Vol. 52, October-December 2015, pp Parameter optimization for lipase production by Bacillus megaterium using response surface methodology Sakshi Rani* & Manjeet Kaur* University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana , India Received 22 July 2014; revised 03 March 2015 Lipases have diverse commercial applications and microorganisms serve as potential source of production. In this study, we carried out lipase production in shake flask culture using Bacillus megaterium, isolated and characterized earlier in our laboratory. Optimization of batch culture conditions of incubation time, ph and temperature was done by conventional approach. Response surface methodology (RSM) was used in this study for Optimization of three variables using central composite design (CCD). The variables optimized for lipase activity were incubation time (24-96 h), ph (6-8) and temperature (30-60 C). The analysis of variance revealed that the established model was significant at (P <0.01). Maximum lipase activity 0.92 U/mL was observed at 45 C temperature, ph 5.6 after 48 h of incubation time. This study will form basis for media optimization and higher production of lipase at pilot scale. Keywords: Factorial central composite design (FCCD), Optimization, RSM Lipases from microbial sources have considerable importance due to their diverse commercial applications, both in aqueous and non-aqueous systems 1. Bacterial lipases are generally extracellular and are greatly influenced by media composition and physico-chemical parameters, such as temperature, ph, agitation and dissolved oxygen concentration 2,3. The optimum conditions for lipase production can be selected by assaying lipase activity 4. Maximization of lipase activity is generally done by varying one factor at a time while keeping others at constant levels. The conventional methodology does not describe the interactive effects among the variables and it does not establish optimal conditions for the processes. However, the response surface methodology (RSM), an alternative approach in many biotechnological processes 5-8, reveals correlations between the variables and response as well as the optimum levels of each variable engaged 9. Factorial central composite design (FCCD) approach of RSM was used to optimize the medium composition and process variables such as temperature, ph, salts, incubation period, for lipase production 7, The present research study is focused on optimization of three process parameters (ph, temperature and incubation time) for maximal lipase activity. *Correspondence: Chabra.sakshi89@gmail.com (SR); manjeet.kundu@gmail.com (MK) Materials and Methods Micro organism and inoculum preparation Bacillus megaterium (de Bary) pure culture used in present study was isolated and characterized by earlier researchers from our laboratory 13. Culture was assayed for lipase activity and maintained at 4 C on Tributyrin agar plates and slants. The inoculum was prepared by aseptically transferring a colony from the agar slant into Tributyrin broth medium. The cultivation was carried out at 37 C on an orbital shaker at 150 rpm for 24 h. Determination of lipase activity Lipase was assayed by a standard titrimetric method using olive oil as substrate. Olive oil (10% v/v) was emulsified with gum Arabic (5% w/v) in 100 mm potassium phosphate buffer (ph 7.0). An aliquot (100 μl) of the enzyme solution was added to the emulsion and incubated for 15 min at 37 C. After this incubation, the reaction mixture was cooled quickly in an ice-water bath and the reaction was terminated with the addition of 1 ml of acetone: ethanol solution (1:1) to extract the fatty acids. The amounts of fatty acids liberated were estimated by titrating with 0.05 N NaOH (ph 10.5) using phenophathelin as indicator 14. One unit of enzyme is defined as the amount of enzyme required to hydrolyze 1 μm of fatty acids from triglycerides. Vol. of alkali consumed Normality of NaOH (g/ml/min) Lipase activity (in U/mL) = Time of incubation (min) Vol. of enzyme solution (μl)

2 312 INDIAN J. BIOCHEM. BIOPHYS., VOL. 52, OCTOBER-DECEMBER 2015 Optimization by conventional approach Production of lipase was carried at different temperature, ph and time period in shake flask and activity assay was done by titrimetric method. Effect of these parameters was studied by measuring the enzyme activity at temperature range C, ph values , and different intervals of incubation time. Statistical optimization by RSM Three independent variables such as temperature, ph and Incubation period have significant effect on lipase activity. The response function of interest was lipase activity. Optimization of these variables was done by RSM. The statistical approach using CCD developed by Design Expert Software Dx 6.0 was used to generate a set of 15 experiments. All experiments were carried out in triplicates by taking different combination of variables each time. Variables were studied between selected minimum and maximum range given in Table 1. results were reported with Bacillus strain 18 and B. megaterium AKG Most of the bacterial lipases have neutral or alkaline ph optima 20. Most of the Bacillus sp. are reported to produce lipase after 24 h of incubation time 16,20,21. During the growth of B. megaterium, maximum lipase activity was found at 24 h and minimum at 96 h. of incubation period. This indicates that the enzyme is secreted in active log phase of growth and its titer decreases in stationary phase due to depletion of nutrients in media and accumulation of undesired by products. The optimization of temperature, ph and incubation time by conventional approach indicated that lipase production by B. megaterium was highest at 40 C, ph 7.0 and after 24 h. of incubation time. Results and Discussion Optimization studies Parameter optimization by conventional approach Fig. 1 (A-D) depicts the effect of incubation time, ph and temperature on lipase activity. As observed from Fig. 1C, optimum temperature for lipase activity was found to be 40 C (1.13 U/mL). As shown in Fig. 1D, the enzyme activity declined with the increasing temperature.our results coincides with those reported for Bacillus sp. earlier 15. Thermophillic nature of present strain for optimum temperature for lipase production is similar to the earlier study 16. Temperature plays a significant role in maintenance of activity and structural stability of enzymes. Optimum temperature range of C was noticed for lipase from Rhizopus oryzae 17. The ph of media plays critical role for optimal physiological activities of bacterial cells and transport of various nutrients across the cell membrane resulting in maximum yield of enzyme. Lipase activity was found to be highest at ph 7.0. Similar Table 1 List of three variables, codes of variables and their experimental range Variables Codes Experimental Range Minimum Maximum Incubation time A ph B 6 8 Temperature C Fig. 1 Effect of temperature, ph and incubation time on lipase activity at (A) 30ºC; (B) 37ºC; (C) 40ºC; and (D) 50ºC

3 RANI & KAUR: MEDIA OPTIMIZATION FOR ENHANCED LIPASE PRODUCTION 313 Table 2 Showing observed and predicted lipase activity obtained by RSM experimental design Std. Runs Time (h) ph Temp.( C) Observed enzyme Predicted Order activity(u/ml) activity(u/ml) 7 Block Block Block Block Block Block Block Block Block Block Block Block Block Block Block Statistical optimization by RSM Table 2 provides the observed experimental data. It reveals that the values of lipase activity varied from 0.28 U/mL to 0.92 U/mL for the interaction among three variables. The maximum lipase activity was obtained under optimum conditions of 48 h incubation time, ph 5.6 and at temperature of 45 C. Maximum predicted lipase activity in standard order 7 was U/mL and observed activity was 0.92 U/mL. The adequate prediction of lipase activity by model was proved by achievement of 100% validity. According to response surface quardratic model equation, lipase activity was expressed as the function of incubation time (A), ph (B) and temperature (C) and results obtained are subjected to ANOVA. Y= β 0 + β 1A + β 2 B+ β 3 C +β 12 AB + β 13 AC + β 23 BC + β 11 A 2 + β 22 B 2 + β 33 C 2. (Eq. I) where Y is predicted response (lipase activity); A, B and C are the independents variables (incubation time, ph and temperature); β 0 is the intercept, β 1, β 2 and β 3 are the linear coefficient; β 12, β 13 and β 23 are the interaction coefficient; β 11, β 22 and β 33 are the quadratic coefficients. Analysis of Variance (ANOVA) is presented in Table 3 and lipase activity is well described by this model as the model generated statistically significant value of probability > F less than The non significant lack of fit (0.0654) indicated that the model was significant 22. The value of R 2 was showing high correlation between the predicted and the observed values, and % of variability in the response could be explained by the model described earlier 23. The predicted R 2 value was and a negative "Pred R-Squared" implies that the overall enzyme Table 3 Analysis of Variance (ANOVA) Data Source Sum of square Degree of freedom Mean squares F-value Prob>F Model Residual Error Lack of fit Pure error Total R 2 =0.9725, R 2 pred=nil, R 2 adj=0.923, adequate precision=14.47 mean is a better predictor of response than the current model. The adequate precision that measures the signal to noise ratio was observed as ; a ratio >4 further indicated an adequate signal This model can be used to navigate the design space. RSM 3D graphs Figures 2-4 depicts the three dimensional (3D) response surface curves and contour plots represented interaction among Incubation time, ph and temperature on lipase activity. Two variables were studied at one time keeping the third one fixed. Fig. 2 shows the effect of ph and incubation time on lipase activity. At 48 h of incubation time, it can be observed that with the increase in ph, lipase activity decreased. Minimum lipase activity was at ph 6.0 after 72 h of incubation time. Our results are comparable with the earlier research 10. Fig. 3 shows elliptical contour, depicting the cumulative effect of temperature and incubation time on lipase activity. After 48 h of incubation time, there was increase in lipase activity up to 45 C and with further increase in temperature a significant decrease in activity was observed, which could be due to denaturation of protein structure. Fig. 4 shows the effect of temperature and ph on lipase activity. At 45 C, decrease in enzyme activity with increased ph

4 314 INDIAN J. BIOCHEM. BIOPHYS., VOL. 52, OCTOBER-DECEMBER 2015 Fig. 2 Interaction between ph and time on lipase activity. (A) Response surface curves; and (B) contour plots Fig. 3 Interaction between temperature and time on lipase activity. (A) Response surface curves; and (B) contour plots Fig. 4 Interaction between temperature and ph on lipase activity. (A) Response surface curves; and (B) contour plots

5 RANI & KAUR: MEDIA OPTIMIZATION FOR ENHANCED LIPASE PRODUCTION 315 in alkaline range was observed. Both increase in temperature and ph showed synergistic effect on lipase activity. Present observations are similar to the earlier works 10. Most of the enzymes show maximum activity at their optimum conditions and slight change in these conditions lead to change of protein structure thus affecting the enzyme activity. Conclusion In this study, we tried to optimize parameters for cultural conditions and their effect on lipase production by B. megaterium using response surface methodology. The 15 experimental sets generated by CCD were performed by varying two parameters at one time to identify the effect of interaction of variables on lipase activity. The R 2 value of showed a good model for the experimental data. The maximum lipase activity of 0.92 U/mL was obtained under optimum conditions of 48 h incubation time, ph 5.6 and at temperature of 45 C. 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