A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method

Transcription

1 Journal of Oleo Science Copyright 2016 by Japan Oil Chemists Society doi : /jos.ess16066 A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method Ahmad Mustafa 1, 2, Amin Karmali 2* and Wael Abdelmoez 1* 1 Department of Chemical Engineering, Faculty of Engineering, Minia University, Minia, EGYPT 2 Chemical Engineering and Biotechnology Research Center and Departmental Area of Chemical Engineering of Instituto Superior de Engenharia de Lisboa, R. Conselheiro Emídio Navarro, 1, , Lisboa, PORTUGAL Abstract: The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR ph was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37 and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in ph 8) and sodium phosphate buffer of ph 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in ph 8) and sodium phosphate buffer of ph 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4 compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations. Key words: 96-well microplate, colorimetric assay, olive oil, oleic acid, Candida sp. lipase, Lipozyme RMIM 1 Introduction Lipases have versatile applications, importance and significance in several fields. The physiologic role of lipases is to hydrolyze triglycerides into di-glycerides, mono-glycerides, fatty acids, and glycerol. Also lipases catalyze the reactions of esterification, transestrification and intraesterification of lipids 1 5. Enzyme assays are analytical methods to visualize enzymes activities 6 8. Free fatty acids are released during the enzymatic hydrolysis of the triacylglycerols by lipases. Measurement of lipase activity requires a method for determining the amount of these released fatty acids. As can be seen in the literature, there are many procedures available for assaying the hydrolytic activity of lipase 9, 10. The most widely used assays for measuring the free fatty acids librated during the enzymatic hydrolysis of the triglycerides are the titrimetric method and the colori- metric copper soap method. In the titrimetric assay, the fatty acids librated during the course of reaction can be determined quantitatively by titration using a standard alkaline titrant and an appropriate end point 11. It should be mentioned that the titrimetric protocol is considered as a benchmark method, as some newly developed assay methods are usually validated using this method. However, from the point of view of routine usage and speed, the titrimetric method is not a good choice due to its labor-intensive nature. The colorimetric copper soap method is similar to titrimetry in that released fatty acids are being measured; however it is more specific for fatty acids. In the colorimetric method, free fatty acids released from the hydrolysis reaction of the emulsified olive oil substrate are combined with a biphasic mixture of cupric acetate pyridine and * Correspondence to: Amin Karmali: Chemical Engineering and Biotechnology Research Center and Departmental Area of Chemical Engineering of Instituto Superior de Engenharia de Lisboa, R. Conselheiro Emídio Navarro, 1, Lisboa, PORTUGAL. akarmali@deq.isel.ipl.pt; Wael Abdelmoez: Department of Chemical Engineering, Faculty of Engineering, Minia University, Minia, Egypt. drengwael2003@yahoo.com Accepted April 18, 2016 (received for review March 12, 2016) Journal of Oleo Science ISSN print / ISSN online

2 A. Mustafa, A. Karmali and W. Abdelmoez benzene. The cupric salts of fatty acids formed in the solvent yield green color thereby allowing for the measurement of the absorbance. In order to validate the specific colorimetric method as a routinely reliable used protocol, several drawbacks had to be solved, such as increasing its sensitivity, replacing the toxic benzene with another more acceptable solvent, reducing both the procedure steps and the unit operations required, and using lower quantities of materials including lipase. The colorimetric copper soap method has been examined and altered by many authors. Duncombe used both Cu NO 3 23H 2 O and triethanolamine as copper reagent and color reagent, respectively 12. Lowry and Tinsely used cupric acetate-pyridine instead as the copper reagent 13. Further modifications have conducted by Kwon and Rhee by replacing benzene with isooctane and solvent centrifugation step was avoided 14. Other significant improvement was using the microplate format. Zheng developed a model reaction of lipase-catalyzed transestrification between vinyl acetate and n-butanol in n-hexane 8. The content of acetaldehyde was quantified in a 96-well Microplate. Gomes optimized a colorimetric assay for measuring yeast lipase activity in complex systems. Samples were analyzed in a microplate reader 15. In this paper a significant increase in the sensitivity of the colorimetric copper soap assay was achieved. Up to the authors knowledge there are no papers in the literature optimized a microtiter plate based colorimetric lipase assay using factorial design methodology to increase the sensitivity. In the present paper, three factors two levels factorial design methodology was applied aimed to study the interactive effects of different assay variables such as type of solvent, CAPR concentration and CAPR ph on the assay sensitivity. In addition, the proposed assay was performed in a microtiter plate using heptane for the first time instead of traditional benzene and iso-octane with a modified concentration of CAPR which gave an additional sensitivity for the assay. The significance of the developed mathematical model was analyzed by ANOVA. Afterward, to study the efficiency of the proposed assay method, two lipase assays were performed utilizing free and immobilized lipases. Finally the developed assay method was validated with the well established conventional copper soap method. 2 Material and Methods 2.1 Chemicals Candida sp. lipase LPL1F was used as a free lipase and was supplied by Biozyme international, UK. Immobilized Rhizomucor miehei lipase, commercially known Lipozyme RMIM was granted by Novozymes, Denmark. Oleic acid, cupric acetate and pyridine were purchased by merck. Olive oil was purchased from the local market in Portugal. All other chemicals were of analytical grade. Cupric acetate pyridine reagent CAPR was prepared by placing different amounts of cupric acetate 2.5 g, 5 g, and 7.5 g into a 100-mL volumetric flask and bringing to volume with water. Then, the content was swirled to dissolve the cupric acetate, and then it was filtered through Whatman no. 1 filter paper. Finally, the ph of the solution was adjusted to different values of 6.0, 7.0 and 8.0 using pyridine Conditions optimization Before we use the proposed assay for measuring the activity of lipase, it was very important to maximize its sensitivity to obtain a high throughput assay. A systematic study was thus carried out to evaluate the effect of a three different parameters on the sensitivity of the proposed copper soap assay. These parameters were ph value of CAPR, type of solvent and concentration of CAPR. In order to construct the calibration curves, mixtures containing different concentrations of 50 mm oleic acid stock solution were prepared with a total volume of 300 µl per each well in a 96 microtiter plate. 50 µl samples were taken and allowed to react with 60 µl of CAPR previously contained in 250 µl of solvent. Wells were subjected to agitation at room temperature for 60 seconds by means of multichannel pipette. Subsequently, fatty acids soaps were produced from the reaction and dissolved in the 250 µl of solvent. 200 µl of the upper layers were taken out to other wells. Absorbance was measured at 655 nm. 2.3 Effect of reaction conditions on the sensitivity of the colorimetric assay Three different parameters were studied against the slope/sensitivity of the calibration curves. All experiments in this paper were carried out in triplicate. 2.4 Effect of hydronium ion concentration To find out the optimum ph of CAPR, three different ph preparations were screened. The ph of each preparation was adjusted by pyridine to 6, 7 and 8. Calibration curves were constructed using these three different ph preparations. Oleic acid was allowed to react with CAPR. Absorbance of the upper layer was measured at 655 nm. 2.5 Effect of solvent type Hexane, heptane and octane were tested in our microplate based assay. The main purpose of adding solvent is to dissolve the formed fatty acid soap to generate green color that could be measured at 655 nm by the microplate reader. 2.6 Effect of cupric acetate pyridine reagent concentration CAPR was prepared in three different concentrations of 776

3 A sensitive microplate assay for lipase activity measurement 2.5, 5 and 7.5 in distilled water. Oleic acid was reacted with these different concentrations to yield different colors intensities for the resulted upper layers. Colors intensities were measured at 655 nm by the microplate reader. 2.7 Experimental design A factorial design of 2 levels 3 factors was employed in this study, requiring 8 experiments. The response was the value of the slopes/sensitivity of the calibration curves. The variables selected were ph 6 and 8 and cupric acetate pyridine reagent concentration 2.5 and 7.5 as numeric variables, and type of solvent heptane and octane as a categoric variable. Selection of the levels was carried out on the basis of the results obtained in the preliminary study. The lower level indicated a negative symbol while the higher level indicated a positive symbol. Based on this a design matrix has been developed. The data obtained were fitted to the following equation Y B 0 B a x a B b x b B c x c B ac x ac. 1 Where Y is the response slope/sensitivity of the calibration curves, B 0 is the mean of all responses, B a, B b and B c, are the regression coefficients of ph, solvent type and CAPR concentration respectively. x a, x b and x c are the uncoded independent variables. The variable B is a categoric one, therefore the term B b x b equal zero. Finally a statistical analysis of the model was performed to evaluate the analysis of variance ANOVA. 2.8 Lipase assay After maximizing the assay sensitivity, two enzymatic reactions were performed to investigate the efficiency of the proposed method. Candida sp. lipase was used as a free enzyme. The liquid preparation of Candida sp. lipase was prepared by dissolving a known quantity of lipase in a sodium phosphate buffer of ph 8. One Candida sp. lipase unit U is defined as the amount of enzyme that produces 1 µmol of fatty acid per minute under the specified assay conditions. In the other hand, lipozyme RMIM was used as an immobilized enzyme. Since triton X-100 is considered as a suitable emulsifier for lipase activity determination, it was tested in the assay at concentrations of 2.5, 5, 7.5 and 10 v/v in sodium phosphate buffer of ph8 to find out the best emulsification. Based on this, the triglyceride emulsion substrate was formulated by using olive oil, triton X v/v in ph 8 and sodium phosphate buffer of ph 8, in ratio of 1:1:1 volume basis for Candida sp. lipase case. For lipozyme RMIM case, the emulsion substrate was formulated by using olive oil, triton X v/v in ph 8 and sodium phosphate buffer of ph 8 in ratio of 2:1:1 volume basis. Then the mixture was emulsified by sonication. 2.9 Constructing the progress curves of the enzymatic reactions To construct the enzymatic reaction progress curves, the reaction mixtures were prepared in the same manner with different amounts of Candida sp. lipase from 0.15 U to 1 U and lipozyme RMIM from 10 mg to 40 mg. The total volume inside each well was kept at 300 µl. The microtiter plate was incubated inside an incubated shaker at 37 and 150 rpm for 40 min. 50 µl samples were taken every 10 min and allowed to react with a 60 µl of cupric acetate pyridine reagent which contained in 250 µl of solvent. Shaking was carried out using multichannel pipette for 60 seconds at room temperature. During the shaking process, pipette tips were placed at the top of the wells and solvent was taken from the top and pushed at the bottom of the wells by the pipette to allow good mixing between the solvent and the formed fatty acids soap. Time-dependent absorbance variation under the optimized assay conditions was monitored at A655 nm Protein content Protein concentrations were determined by Coomassie blue dye method by using bovine serum albumin BSA as the protein standard Linearity evaluation For evaluation of the enzymatic reaction linearity, Candida sp. lipase amounts as units U were converted to protein content µg using the calibration curve equation previously obtained by Coomassie blue dye method using BSA 16. Then protein content was plotted against reaction initial velocity. For Lipozyme RMIM, amounts in milligrams were directly plotted against reaction initial velocity Colors stability The stability of the proposed assay was checked by following the value of the colored solvent phase absorbance readings through one complete hour and then after 24 hours. The microplate reader was adjusted to work in the kinetics mode and the absorbance measurements were carried out every 10 min within one complete hour at room temperature Method validation In order to prove that the proposed colorimetric assay method can be implemented as a routine analyses assay for lipase activity determination, it was very important validating it with other well established traditional method. The method herein proposed was compared with the well established colorimetric copper soap method

4 A. Mustafa, A. Karmali and W. Abdelmoez 3 Results and discussion Preliminary experiments were carried out to study the effect of different levels of the three factors on the formation of cupric soaps through the reaction between oleic acid and CAPR inside the microtiter plate wells. During the preparation process of the CAPR, ph values were adjusted using pyridine to values of 6, 7 and 8. Decreasing the ph value less than 6 was not preferred, as the ph of the cupric acetate reagent solution before adding pyridine was 5.7. Furthermore, inclusion of pyridine in the assay cocktail increases the assay sensitivity by enhancing the solubility of the formed copper soap in the polar phase solvent 11. In the other hand, utilizing a concentration more than 7.5 wasn't possible due to reaching the saturation point. Effect of solvent type on the assay sensitivity was studied. Three different solvents of hexane, heptane and octane were screened in this assay. It was seen that the taken samples based hexane were not stable before measuring their absorbance, due to the rapid volatilization of hexane which has a boiling point of 68. Therefore hexane was excluded from this work. The third studied parameter was the CAPR concentration, where three concentrations of 2.5, 5 and 7.5 v/v were prepared. 3.1 Analysis of Variance ANOVA The conventional method of optimization includes varying one parameter at a time and keeping the other constant, therefore leads to a large number of experiments. Being single-dimensional, this method often does not guarantee determination of optimal conditions 17. Factorial designs are effective techniques for the investigation of complex processes. By carrying out just a few selected experiments. Such designs explain the reaction completely bringing out the finer details 18. Experimental data used for optimizing the assay sensitivity slope of the calibration curve are shown in Table 1 along with the predicted results. The predicted values were obtained using the Design-Expert software version and found to be sufficiently correlated to the experimental values. As shown in Table 2, the model F-value of implies the model was significant and there was only a 2.16 chance that an F-value could due to noise. In ad- Table 1 Run No. Source Experimental design of three independent variables along with the experimental and predicted values. ph Type of solvent CAPR concentration (%v/v) Slope/sensitivity of the calibration curves Experimental responses Predicted responses 1 6 Heptane Octane Heptane Octane Octane Heptane Heptane Octane Table 2 Analysis of variance ANOVA of the factorial design model. Sum of Squares Degree of freedom Mean Square F Value p-value Prob > F Model A-pH B-Solvent Type C-CAPR Conc AC Residual Corrected Total R 2 =0.9571, C.V.=11.72%, Adequate precision=11.06, Contribution of A=74.31%, Contribution of B=11.55%, Contribution of C=2.19%. 778

5 A sensitive microplate assay for lipase activity measurement dition high coefficient of determination R indicated that there was a statistical relation between the response and the selected variables at confidence and only 4.29 of the total variation is not explained by the model. Therefore, other minor variables associated in the reaction between oleic acid and CAPR inside the microplate wells may have influenced the assay sensitivity. 3.2 Regression Analysis Subsequent regression analysis of each set of data then generated corresponding sets of coefficients for developing model equations, such as the adequate precision that measures the signal to noise ratio and a value 4 is considered appropriate for desirable models. The adequate precision value of for the proposed model indicates that the model can be used to navigate the design space. Also, the coefficient of variation C.V. indicates the degree of precision with which the treatments are compared, and the low value of C.V. showed the reliability of experiment. In this study, a relatively lower value of the coefficient of variation C.V suggested a good precision and reliability of the experiments. Values of Prob F less than indicate model terms are significant. Table 2 shows that among the independent variables, the Prob F for variable A ph was followed by for variable B solvent type. Therefore ph value was considered as the most important variable affected the sensitivity of the proposed microplate assay followed by the type of solvent. Based on the above A mentioned values, two empirical equations were obtained to estimate the value of the response slope/sensitivity of the calibration curves and the screened coded variables in any point. Equation 1: when octane is the used solvent in the assay, while equation 2: when heptane is the used solvent. Octane case -1 Y A C E 003AC 2 Heptane case 1 Y A C E 003AC 3 Where the slope value is Y and A, B and C are the values of ph, type of solvent and CAPR concentration. 3.3 Response surface analyses Equations derived from the regression analysis were then used to facilitate plotting the response surfaces. Two parameters of A and C CAPR ph and CAPR Concentration were plotted twice against the response, once with octane, while the other with heptane as shown in Fig. 1a and 1b respectively. B Fig.1 Three-dimensional graph for interaction of two parameters affecting the slope of the calibration curves. The fixed conditions are: temperature, room; shaking time, 60 seconds by multichannel pipette; microplate samples volume dissolved fatty acids soap, 200 µl; absorbance, 655 nm. a The response surface plot as functions of ph value versus CAPR concentration using octane 50 µl of fatty acid soap was dissolved in 200 µl of octance. b The response surface plot as functions of ph value versus CAPR concentration using heptane 50 µl of fatty acid soap was dissolved in 200 µl of heptane. 779

6 A. Mustafa, A. Karmali and W. Abdelmoez 3.4 Interactive effect of parameters on response with octane and heptane Figure 1a and 1b show the response surface plots as functions of CAPR ph versus CAPR concentration using octane and heptane respectively for estimating the slope of the calibration curves. As can be seen from the figures, using CAPR with concentration of 7.5 and ph of 6 with both octane and heptane exhibited the highest effect on the slope of the calibration curves. However the slope obtained using heptane was 20 higher than the slope obtained using octane at the same parameters of CAPR ph and concentration for CAPR, which explaines that type of solvent is an important parameter in the proposed assay method. Replacing the CAPR ph to be 8 instead of 6 at 7.5 CAPR concentration exhibited the lowest sensitivity in both octane and heptane cases. The slope value decreased more than 2 fold in the case of octane to be instead of 0.14, while decrease more than 2.25 fold in the case of heptane to be instead of Finally it was noted that the effect of CAPR concentration is less significant on the value of the slope. Thus, we conclude that the most sensitive parameter in our microtiter plate was CAPR ph with a contribution of followed by type of solvent and CAPR concentrations with contributions of and 2.19 respectively as shown in Table 2. Figure 2 shows the calibration curve under the optimized conditions. The calibration curve was performed under other fixed parameters namely, room temperature, absorbance of 655 nm, and fixed shaking time of 60 seconds by means of multichannel microplate pipette. It can be seen from the figure that the value of the slope of the calibration curve is with a correlation coefficient R 2 of In addition, a limit of detection 19 LOD was found to be µmoles of oleic acid/well which was obtained according to the following equation: S y LD a Where S y denotes the standard deviation of blank responses, and a is the slope of the calibration curve. The standard deviation of blank responses obtained in this research was , while the slope of the calibration curve was In the other side, the slope of the calibration curve of the conventional method was found to be which gives a LOD of µmoles of oleic acid/well. This finding led to that the developed microtiter plate assay could detect lower oleic acid concentration than that of conventional method by more than 78. Kwon and Rhee 14 developed a colorimetric assay method for rapid measurement of lipase activity using cupric acetate pyridine reagent. The slope of the constructed calibration curve was at 715 nm using oleic acid dissolved in isooctane. Ravikumar 20 constructed a calibration curve to quantify the lipid content inside an oleaginous plant by colorimetric assay; a slope of at 715 nm using oleic acid dissolved in isooctane could be achieved. In this research, we must stress that after optimization we could increase the sensitivity by a factor of 7.7 compared with Ravikumar 20 and a factor of 8.5 compared with Kwon and Rhee 14 which indicates the effectiveness of the proposed method. Therefore, the novelty of the proposed assay method was based on applying the factorial design methodology to optimize the sensitivity of the colorimetric copper soap assay, using high concentration of CAPR 7.5 w/v, using heptane, and using a 96-wells microtiter plate format to increase both simplicity and throughput. For the LOD comparative purposes, it should be mentioned that there are no enough data either of the conventional method or of other methods for lipase assay in the literature. Fig. 2 Assay calibration curve under the optimized conditions: CAPR concentration, 7.5 w/v; CAPR ph, 6; solvent, heptane; temperature, room; shaking time, 60 seconds by multichannel pipette; microplate samples volume dissolved fatty acid soaps, 200 µl; absorbance, 655 nm. Reaction mixture contained different concentration of 50 mm of oleic acid stock solution to a total volume of 300 µl/well. Each data point represents the mean from triplicate determinations, and the error bars represents the standard deviation. 3.5 Lipase assay and influence of triton X-100 concentration on the sensitivity of the lipase assay method After maximizing the assay sensitivity by optimizing the conditions affecting the value of the slope of the calibration curves, the enzymatic hydrolyses of olive oil emulsion substrate was performed under the optimized conditions involved CAPR ph of 6, CAPR concentration of 7.5 and with using heptane. Triton X-100 was selected in this enzymatic reaction as it has been considered as an appropriate emulsifier in the enzymatic reactions, because it would ease the substrate access to the active sites of lipase 21. In the present assay, it was important to screen different concentrations of triton X-100 to find out the most adequate concentration for 780

7 A sensitive microplate assay for lipase activity measurement more accurate lipase activity measurement. Different concentrations of triton X-100 were used to emulsify the olive oil. The substrate was incubated 15 min at 37. Then equal volumes of substrates were put into the microplate wells. In the case of Candida sp. lipase, the hydrolysis reaction was initiated by adding 0.5 U of lipase for each concentration. Figure 3 shows the effect of using four different triton X-100 concentrations of 2.5, 5, 7.5 and 10 v/ v on the performance of Candida sp. Lipase. It is clear from the figure that by increasing the triton X-100 concentration, the hydrolytic activity increased and this is obvious by increasing the absorbance which led to sensitive results in the lipase activity measurement. This achievement was not as same as the immobilized lipase case. Since by emulsify the olive oil by 10 of triton, lipase RMIM exhibited lower hydrolytic activity. This may be due to the high concentration of triton may affect the kinetic behavior of Lipozyme RMIM. Wang 22 indicated that use of 1 of triton concentration instead of 10 would enhance some enzymes stability. Therefore concentration of 1 Triton was used in this research. Regarding the substrate formation, using the same substrate formation of the free lipase case led to less contact between enzyme and substrate due to that the immobilized lipase settled down at the bottom of the microplate wells and thereby no good activity. For further enhancing the hydrolytic activity of lipozyme RMIM, a modification on the substrate formation was carried out aimed to increase the substrate viscosity so the immobilized lipase could be suspended inside the substrate, thus good contact was achieved. The emulsion substrate used in the case of lipozyme RMIM was formulated using olive oil, Triton X v/v in ph 8 and sodium phosphate buffer of ph 8 in ratio of 2:1:1 volume basis. 3.6 Constructing the progress curves and studying the linearity evaluation of the enzymatic reactions In order to prove the efficiency of the proposed colorimetric assay method, two forms of free and immobilized lipase based enzymatic reactions were tested. Two progress curves using different amounts of lipases were constructed using the optimum assay conditions previously obtained. Figure 4a and 4b show the time-dependent absorbance a b Fig. 3 The influence of triton X-100 concentration on the sensitivity of the lipase assay method. The triglyceride emulsion substrate was formulated by using olive oil, Triton X-100 and sodium phosphate buffer of ph 8 in ratio of 1:1:1 volume basis utilizing Candida sp. lipase. Each data point represents the mean from triplicate determinations, and the error bars represents the standard deviation. Fig. 4 Time-dependent absorbance changes using different amounts of lipases. Reaction mixtures were prepared with different amount of lipase ranged from a 0.15 U to 1 U per well in the case of Candida sp. lipase. b 10 mg to 40 mg in the case of lipozyme RMIM. The triglyceride emulsion substrate was formulated by using olive oil, triton X v/v in ph 8 and sodium phosphate buffer of ph 8, in ratio of 1:1:1 and 2:1:1 volume basis utilizing Candida sp. lipase and lipozyme RMIM respectively. The microtiter plate was incubated inside an incubated shaker at 37 and 150 rpm for 40 min. Each data point represents the mean from triplicate determinations, and the error bars represents the standard deviation. 781

8 A. Mustafa, A. Karmali and W. Abdelmoez change. It can be seen from the figures that the initial reaction rates increased with increasing the lipase amounts. The maximum absorbance obtained in the case of using Candida sp. lipase was about 0.8 nm when amount of 1 U was added to the reaction mixture. While using an amount of 1.5 U of lipozyme RMIM gave a maximum absorbance of 2.5 nm after 30 min of reaction. For the evolution of the lipase assay method linearity, different amounts of Candida sp. lipase ranged from 2.2 µg to 22 µg and 10 mg to 40 mg from lipozyme RMIM were plotted against their reactions initial velocities. Figure 5a a b and 5b show that the reaction initial velocities correlate well with the amount of enzymes with R and R in cases of using Candida sp. lipase and lipozyme RMIM respectively. 4 Assay stability After maximizing the sensitivity of the proposed assay and after proving its efficiency, it was necessary to study its stability and reproducibility. The assay stability was determined by studying the variation in the absorbance readings for the colored heptane phase against time. Figure 6 shows that, when keeping the microtiter plate at room temperature for 1 h, the absorbance readings lost about 22 compared with what obtained at zero time. However, when keeping the microtiter plate at 4, there was no any change observed in the readings until 1 h. Rather, up to 24 h, the colored heptane phase stability kept more than 92.5 of the absorbance readings compared to what measured at zero time. When benzene was used as a solvent in this assay, the formed colored benzene phase was not stable, as after 1 h at 25 there was more than 54 loss in the stability. This was due to the relativity lower boiling point and higher vapor pressure of benzene as demonstrated in Table 3. Furthermore, it was not possible keeping benzene at 4 as its melting point is 5.5. Therefore, using heptane in our Fig. 5 Linearity evaluation: the initial velocity of reaction was correlated with the amount of enzymes. a Protein content against reaction initial velocity, in the case of lipase from Candida sp. amounts as units U were converted to protein content µg. b Amounts in milligrams of lipozyme RMIM against reaction initial velocity. The triglyceride emulsion substrate was formulated by using olive oil, Triton X v/v in ph 8 and sodium phosphate buffer of ph 8, in ratio of 1:1:1 and 2:1:1 volume basis utilizing Candida sp. lipase and lipozyme RMIM respectively. Each data point represents the mean from triplicate determinations, and the error bars represents the standard deviation. Fig. 6 Time dependent assay stability using heptane at 4, 25 and benzene at 25 under the optimized conditions CAPR concentration, 7.5 w/v; CAPR ph, 6; solvent, heptane; absorbance, 655 nm. The microplate reader was adjusted to work in the kinetics mode and the absorbance measurements were carried out every 10 min within one complete hour at room temperature. Reaction mixture contained different concentration of 50 mm of oleic acid stock solution to a total volume of 300 µl/well. Microplate samples volume dissolved fatty acids soap, 200 µl/well. Each data point represents the mean from triplicate determinations, and the error. Each data point represents the mean from triplicate determinations, and the error bars represents the standard deviation. 782

9 A sensitive microplate assay for lipase activity measurement Table 3 Comparison between our new copper soap colorimetric method and the conventional copper soap method. Needs Conventional assay Our Colorimetric assay Instrument Spectrophotometer Microtiter plate reader Tools Eppendorf tubes and glass cuvettes Microtiter plates Unit operation Vortex mixer, centrifuge, homogenizer, water bath and magnetic stirrer Sonicator and incubated shaker Materials required for substrate preparation Olive oil, triton X-100, Chloroform and buffer Olive oil, triton X-100 and buffer. CAPR concentration 5%( v/v) 7.5%( v/v) CAPR ph 6 6 Solvent boiling point, vapor pressure at room temperature and melting point Limit of detection Benzene (80.1 & 12.7 kpa & 5.5 ) µmoles of oleic acid/well Heptane (98.4 & 5.3 kpa & -91 ) µmoles of oleic acid/well Estimated time 3.5 hours 20 min microtiter plate assay method permitted measuring the absorbance of a given samples during a period of at least 1 h, which gives a flexibility and reliability for the proposed method. Table 3 shows also the difference between assays in terms of used solvent, CAPR concentration, instruments, time required etc. It was worth noting that the required time for preparing the calibration curve, preparing the samples and measuring the lipase activity for assays were 3.5 h and 20 min for conventional assay and developed assay respectively. The reason of such wide range was due to requiring further processes steps when the conventional method was utilized. Some of them are centrifugation centrifuge, mixing vortex mixer and measuring the absorbance spectrophotometer for every sample individually. In the other hand, the developed assay uses a 96 wells microtiter plate reader which could analyze a lot of sample in a short time. In addition, centrifugation and mixing steps were avoided in the proposed developed assay. Because of these findings, this assay could be both sensitive and reproducible and it could be easily used with both free and immobilized lipases based enzymatic reactions for measuring lipase activity. In addition, the simplicity and flexibility offered by using the proposed assay, made it suitable for serving those who use lipase assays routinely in the industry and/or in the research. 5 Validation To validate the proposed colorimetric assay, the well-established colorimetric assay method was used to measure the lipase activity. The results obtained from both assays Fig. 7 Assay validity: a comparison between our new assay and the well established colorimetric copper soap assay. New assay conditions are CAPR concentration, 7.5 w/v; CAPR ph, 6; solvent, heptane; absorbance, 655 nm, conventional assay conditions are CAPR concentration, 5 w/v; CAPR ph, 6; solvent, Benzene; absorbance, 655 nm. The triglyceride emulsion substrate was formulated by using olive oil, triton X v/v in ph 8 and sodium phosphate buffer of ph 8, in ratio of 1:1:1. Enzyme used was Candida sp. lipase. are compared. The results in Fig. 7 show that the specific activity of Candida sp. lipase using our developed assay was U/mg, while by using the conventional method the specific activity obtained was 49.1 U/mg. The higher value obtained using our method could be due to using heptane instead of benzene and using a concentration of 7.5 v/v of CAPR instead of 5. This indicates that, concentration of 5 v/v of CAPR in the old method led to no 783

10 A. Mustafa, A. Karmali and W. Abdelmoez enough substrate saturation. Thus, by using 7.5 CAPR instead of 5, more substrate saturation was achieved thereby more specific activity obtained. 6 Conclusion A simple and high throughput colorimetric assay method has been developed for measuring the hydrolytic activity of lipase. The activity of both free and immobilized lipases could be successfully measured by applying our developed colorimetric 96 wells microtiter plate assay. The stability of this assay in terms of colored heptane phase absorbance readings kept more than 92.5 after 24 h at 4 compared to the absorbance readings measured at zero time. This indicated the reproducibility and the effectiveness of the proposed assay over the conventional methods. Beside the sensitivity of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations. References 1 Abdelmoez, W.; Mostafa, N. A.; Mustafa, A. Utilization of oleochemical industry residues as substrates for lipase production for enzymatic sunflower oil splitting. J. Clean Prod. 59, Abdelmoez, W.; Mustafa, A. Oleochemical industry future through biotechnology. J. Oleo Sci. 63, Abdelmoez, W.; Tayeb, A. M.; Mustafa, A.; Abdelhamid, M. Green approach for biodiesel production from jojoba oil supported by process modeling and simulation. Int. J. Chem. React. Eng. 14, Abdel Fattah, R.; Mostafa, N. A.; Mahmoud, M. S.; Abdelmoez, W. Recovery of oil and free fatty acids from spent bleaching earth using sub-critical water technology supported with kinetic and thermodynamic study. Adv. Biosci. Biotechnol. 5, Stoytcheva, M.; Zlatev, R.; Behar, S.; Bois, J. A spectrophotometric lipase assay based on substrate nanoparticleassembly degradation. Anal. Methods 5, Reymond, J. L.; Fluxa, V. S.; Maillard, N. L. Enzyme assays. Chemcomm. 35, Okyay, T. O.; Rodrigues, D. F. High throughput colorimetric assay for rapid urease activity quantification. J. Microbiol. Methods. 95, Zheng, J.; Fu, X.; Ying, X.; Zhang, Y.; Wang, Z. A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay. Anal. Biochem. 452, Beisson, F.; Tiss, A.; Rivière, C. Methods for lipase detection and assay: a critical review. Eur. J. Lipid Sci. Technol. 102, Wang, D.; Wang, J.; Wang, B.; Yu, H. A new and efficient colorimetric high-throughput screening method for triacylglycerol lipase directed evolution. J. Mol. Catal. B Enzym. 82, Pinsirodom, P.; Parkin, P. Current Protocols in Food Analytical Chemistry. John Wiley & Sond, Inc. Chapter 3, pp Duncombe, W. G. The colorimetric micro-determination of long-chain fatty acids. Biochem. J. 88, Lowry, R. R.; Tinsley, I. J. Rapid colorimetric determination of free fatty acids. J. Am. Oil Chem. Soc. 53, Kwon, D. Y.; Rhee, J. S. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 63, Gomes, N.; Goncalves, C.; Miguel, G. R.; Teixeiraa, J. A.; Belo, I. Optimization of a colorimetric assay for yeast lipase activity in complex systems. Anal. Methods 3, Sedmak, J. J.; Grossberg, S. E. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal. Biochem. 79, Radzi, S. M.; Yunus, N. M.; Othman1, S. S.; Basri, M.; Abd Rahman, M. B.; Noor, H. M.; Muhammad, S. K. Process improvement on the lipase-catalyzed synthesis of oleyl palmitate, a wax ester via response surface methodology RSM. J. Chem. Chem. Eng. 5, Cheng, H. C.; Ju, H. Y.; Ta, W.; Liu, Y. C.; Lee, C. C.; Chang, C.; Chung, Y. L.; Shieh, C. J. Continuous production of lipase-catalyzed biodiesel in a packed-bed reactor: optimization and enzyme reuse study. J. Biomed. Biotechnol. 2011, Article ID , dx.doi.org/ /2011/ Hayashi, Y.; Matsuda, R.; Ito, K.; Nishimura, W.; Imai, K.; Maeda, M. Detection limit estimated from slope of calibration curve: An application to competitive ELISA. Analyt. Sci. 21, Ravikumar, K.; Dakshayini, J.; Girisha, S. T. Biodiesel production from oleaginous fungi. Int. J. Life Sci. 6, Zhang, W.; Qing, W.; Ren, Z.; Li, W.; Chen, J. Lipase immobilized catalytically active membrane for synthesis of lauryl stearate in a pervaporation membrane reactor. Bioresour. Technol. 172, Wang, H.; Zhong, S.; Ma, H.; Zhang, J.; Oi, W. Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1-7 isolated from Bohai Bay in China for detergent formulation. Braz. J. Microbial. 43,