Statistical Optimization of Parameters for the Enhancement of Lipase Activity

In this present study, lipase producing bacteria were isolated and screened from an indigenous soil sample and was used for lipase production with high enzyme activity. Different production media were screened and lipase production was found to be induced by olive oil, 14 mL/L, in the production medium. It was observed from Luedeking and Piret model that the lipase production was mixed growth associated with its maximum activity at 37 °C and pH 7. To understand the interaction of different parameters, statistical optimization using Response Surface Methodology was performed and the standardized conditions obtained were as follows: Peptone 10 g/L, yeast extract 7.5 g/L and olive oil 14 mL/L. The predicted data were validated and the model predicted was signicant with maximum specic activity of 1.1 µmole/min/mg proteins. The lipase specic activity was found to be enhanced by 10% and 23% after a single parameter and statistical optimization respectively.


Introduction
Enzymes are used as biological catalysts due to their high speci city and economic advantages without any adverse environmental impact. Lipases (triacylglycerol acyl-hydrolases, EC 3.1.1.3) are important enzymes due to their various industrial applications (Salihu et al., 2012). Lipase produced from bacteria is called glycoproteins (Bajpai, 1999;Eugene, 1974; Nakamura and Nasu, 1990; Noureddini et al., 2005) and it hydrolyze triacylglycerol ester linkage that in turn used in industries for the processing of fats and oils, cosmetics, paper manufacturing degreasing of leather and pharmaceutical industries (Hasan- Lipase can be extracted from different sources like bacteria, fungi, plant or animal. Microbial lipase production is preferred due to its higher yield over plant or animal. Different substrates and different sources viz. agricultural soil, oil spilled soil, coconut industry soil, natural hot spring, salt pan, residual grease, mucus layer and gill of sh, rock lobster, wastewater, oil mill waste, marine fungus etc. were reported for isolation of lipase-producing bacteria ( Optimization of various physico-chemical conditions for lipase production was classically carried out by single parameter optimization method. In this process, values for one parameter were changed keeping all other parameters constant. Though this method is simple, interaction or in uence of one parameter to another cannot be analyzed (Liu et al., 2012). To overcome this problem, statistical optimization such as The major challenge is to produce lipase with a high speci c activity and single parameter optimization alone cannot able to increase activity signi cantly. Thus, the present study aims to optimize different physicochemical parameters statistically to enhance lipase activity produced by bacteria isolated from the native soil sample.

Materials
The Bradford protein estimation kit was purchased from Bangalore Genei Pvt. Ltd. (Bangalore, India).
Tributyrin was obtained from Himedia (Mumbai, India). All other chemicals were procured from SRL (Mumbai, India) and were analytical grade.

Isolation and screening of lipolytic bacteria
Lipase producing microbial cultures was isolated from indigenous soil and screened on an LB agar plate supplemented with Rhodamine B (0.9% w/v) and olive oil (1% v/v). The colonies were visualized under UV-transilluminator at 310 nm and positive colonies with shiny orange halos were used for further studies.

Screening of different production medium
In this study, different media were used for the production of lipase. The media that provide lipase with maximum activity was selected and all subsequent studies were performed using that production medium.

Study of growth association using Luedeking and Piret Model
Speci c growth rate and product formation rate was measured in selected production media and Luedeking-Piret Model was tted to determine the model constants to predict the association between speci c growth rate and product formation rate. The model equation is: Where, P = product concentration, x = cell concentration, µ = speci c growth rate, t = time, α and β are coe cients.

Enzyme Assay
Lipase activity was measured by the method described in earlier reports (Chattopadhyay and Sen, 2012).
In brief, One mL of tributyrin was added to 9 mL of 20 g/L polyvinyl alcohol (PVA) solution. The reaction mixture composed of 500 µl of the emulsion, 400 µl of phosphate buffer (0.1 M, pH 7.0) and 100 µl of the enzyme solution was incubated at 37 °C for 1 h in an incubator shaker at 120 rpm. The reaction was terminated by addition of 2 mL of chilled acetone − ethanol mixture (1:1) and the liberated fatty acids were titrated with 0.01 N NaOH using phenolphthalein as indicator. The activity was expressed in terms of micromoles of free fatty acids liberated by enzyme per min under assay condition. Total protein was estimated using Bradford reagent where BSA (Bovine Serum Albumin) was used as a standard. The speci c activity was calculated by dividing enzyme activity with total protein and expressed in µmole/min/mg proteins unit.  Table 1. Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA). The experimental data obtained were tted into the model equation and analysed. The data obtained for speci c activity after optimization of media was compared with the predicted value of Response Surface Methodology (RSM).

Lipase activity in different production media
The lipase activity in different production media is presented in Fig. 1. From the results, it is clear that media C (Kulkarni et al., 2002) is found to be the best in terms of lipase activity and it was used in the further experiment for production of lipase and optimization studies.

Association of speci c growth rate and product formation rate
Lipase production was started after 90 h of growth as shown in Fig. 2a, which indicate that the product formation is not fully associated with growth. The relation between speci c growth rate and product formation rate was shown in Fig. 2b. From the model equation it can be observed that the product formation is mixed growth associated with the values of model constants are α = 3.9198 and β = 0.0106.

Effect of pH
It was observed from the results that the bacterium is capable of producing lipase from initial pH 6.5 to pH 8.5. The enzyme production varied considerably from 1000 to 3300 µmole/min. The bacteria showed optimum lipase production in between the pH 7.0 to 7.5 (3300 µmole/min) as shown in Fig. 3(I). When the pH is optimized the active site is fully ionized while in low or high in pH some charge is neutralized that is re ected by the decreasing of enzyme activity. Golani et al. (2016) also found that optimum lipase production at pH 7.0. However, it was noted that the lipase production was declined with an increase in pH from 7.0 to 10.0 but was able to produce lipase towards alkaline pH which shows its alkalitolerant nature. Chaturvedi and Khare (2016) optimized pH for lipase production and the optimum pH for lipase production was found to be 6.0.

Effect of temperature
Lipase activity was determined at a range of 30 to 50 °C which is showed in Fig. 3

Effect of peptone concentration
Among the ve different concentration of peptone, 10 g/L peptone in media showed maximum lipase production (3300 µmole/min) and enzyme activity found deceased with the increase of peptone dose which is showed in Fig. 3

Effect of different oils
From the result, the maximum lipase production was found in olive oil followed by rice bran oil, sun ower oil, mustard oil and coconut oil (Fig. 3(V)). The reason might be due to the in uence of the higher amount of unsaturated fatty acids present in olive oil (86%) as compared to the other oils used. Paul et al. (2015) shows the effect of different oils as the lipid source in production media. Comparing Lipase production, determined by quantitative lipase activity assay, the production media using olive oil as lipid source shows maximum lipase production compared to sun ower oil and coconut oil.
3.4.6. Effect of concentration of olive oil The Activity of lipase was found to be increased gradually from 0 to 15 mL/L and decrease with further increase in concentration (Fig. 3(VI)). This might be due to the fact that higher amount of olive oil hinders the microbial growth by restricting oxygen transfer and in turn reduces the lipase production. Paul et al. (2015) optimize the olive oil concentration and found to be approximately 10 mL/L as an optimum value.

Statistical optimization using RSM
The design layout with the response after the experiment is shown in Table 2. It is observed from the table that the speci c activity was varied from 0.16 to 1.05 µmole/min/mg protein. After ANOVA of the quadratic model, the p value (< 0.05) was found to be signi cant with adjusted R 2 (0.9701) and predicted R 2 (0.8796) are in good agreement with each other (Table 3). Multiple regression analysis of experimental data gave the following equation where 'Y' represents enzyme speci c activity:  The model was further processed to understand the interactions among variables followed by optimization to get the maximum speci c activity. Effect of peptone and yeast extract, the values were converging and the contour and 3D plot are shown in Fig. 4a. Similarly, in Fig. 4b and Fig. 4c, the effect of peptone and olive oil and yeast extract and olive oil are shown respectively. From Table 4, the optimized values were found to be 10.0 g/L peptone, yeast 7.5 g/L extract and 14 mL/L olive oil and the model predicted maximum speci c activity at these optimized conditions as 1.05 µmole/min/mg protein. After the experimental run with optimized value, speci c activity was obtained as 1.1 µmole/min/mg proteins, which is very close to the predicted value, indicating the validity of RSM model.

Conclusion
From the results obtained in this present study, it can be concluded that lipase was successfully produced from the indigenously isolated bacteria. The incubation temperature, pH, peptone concentration and yeast extract concentration were optimized. It was observed that lipase production was enhanced in presence of unsaturated oil such as olive oil. After optimization, a 10% increase in lipase speci c activity was observed by using single parameter optimization whereas 23% activity was increased by a statistical optimization method. With the optimum conditions, large scale production of lipase can be possible using the isolated bacteria and the product after puri cation can be used for various hydrolysis and methanolysis reactions and other industrial applications. Screening of different media for the production of lipase    Lipase speci c activity before optimization, after single parameter optimization and after statistical optimization using RSM