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Good afternoon ladies and gentlemen, I am proud to present to you the results of my research entitled Determination of the optimum conditions for the activity of crude lipase from germinating Artocarpus heterophyllus (Moraceae) seeds using response surface methodology. As the presentation goes on, the following will be discussed. As an introduction, let us start to know what lipases are. Lipases are enzymes that are water soluble which catalyzes the hydrolysis of ester bonds in water insoluble subtrates such as fats and oils at waterlipid interface and which are systematically known as triacylglycerol hydrolases.

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The use of biocatalysts in industrial synthetic chemistry is on the verge of significant growth. And biocatalysts are preferred over the use of the conventional catalysts such as the use of inorganic materials. Why? Researches showed that biocatalysts, especially lipases are of high value because of its high specificity, high activity under mild conditions and high biodegradability. For such reason, an increase in interest on biocatalysts is in consonance to the promotion of green chemistry. As what Ive mentioned, one of these biocatalysts are lipases. Another reason is that the lipases have already established many novel biotechnological applications in the industry. Synthesis of products in Food Industry and Nutraceuticals Pharmaceutical, Manufacturing Detergents, Fine Chemistry, Biodiesel Production, and even used in treating fat containing effluents from this industries. Of the known lipases only 11% are from plant sources. Why have I chosen plant lipases over other lipases? It is because plant lipases have unique and high specificity, stereospecificity, and enantioselectivity to substrate. Techniques for isolation are simple. Unlike mammalian cell systems, which can sometimes be a source of pathogenic viral agents, plants are intrinsically free from diseases and pathogens. Thus, plant systems may offer advantages over bacterial and mammalian cell cuture systems. The jackfruits seeds are suspected to contain lipases because seeds mostly contain the most of food reserves, like oils and fats, which are converted using lipases to support the germinating seed. I choose jackfruit seeds because in Asia, these seeds are considered to be an agroindustrial waste.

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This research general aimed to determine the optimum conditions for the activity of the crude lipase extract from germinating seeds of Artocarpus heterophyllus. Specifically it aimed to:

Germinate seeds of Artocarpus heterophyllus at various germination periods;

Extract crude lipase from defatted germinating seeds of Artocarpus heterophyllus at various germination periods; Determine the specific activity of the crude lipase extracts; Compare the specific activities of the crude lipase from Artocarpus heterophyllus seeds at various periods; and Determine the optimum conditions (pH, temperature, and incubation time)for the activity of the crude lipase extract having biggest specific activity among seeds at germinated at various periods. Provides information about additional possible source of plant lipases.

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Gives insights for further studies such as lipase purification and subsequent possible applications into its potential for industrial and medical utilization. The fitted second-order model will be the basis for the future mass production of the lipase Further promotes green chemistry through agro-industrial waste utilization and extraction of a biocatalyst with economic value from waste materials.

9th Slide: This is the process flow of the research Sample Collection using the method described by Elevitch and Manner (2006) Sample preparation (including germination) and Extraction of Lipase using the method described by Yesiloglu and Demirkan (2010) Titrimetric assay for the activity as described by Fudalej et al. (2000) as cited by Tejano (2008) Protein Content Determination using Bradford assay Specific Activity Determination The Statistical Analysis used in this is followed as shown. 2 level- 3 variable face-centered central composite design response surface methodology is used. Values for the variables (pH, temperature, and incubation time) are selected for the 20 treatments. The volumetric and specific activity is then determined for each treatment. Analysis of Variance is used to determine whether the experimental design is adequate or not. And if it is adequate, a model is then fit. Optimization of the activity is then done using response surface methodology using the same set of treatments. 11th Slide: These are the treatments used for the experimental design. Showing Coded variables. Those set with -1 are the lower settings or the lower

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bound. Those set with +1 are higher setting or the higher bound. And those set at the 0 are the central points. 12th Slide: This figure shows he images of A. heterophyllus seeds germinated at various periods. There were observable structural developments during the germination period. A. heterophyllus seeds germinated at 24 hours did not show any visible physical changes but those germinated at 48 hours started sprouting. This table shows the differences in the volumetric and specific activities of crude lipase extract from seeds germinated at various period. This shows that those germinated at 48 hours gave the highest volumetric and specific activity with a value of 8.41 U/mL and 24.73 U/mg, respectively. The protein concentration is also low which contributes to the high value of specific activity and this means that at this period, the lipase concentration is high. The figure shows the summary of what Ive been talking about. That at germination period at 48 hours shows highest specific activity. In this data, it can be speculated that the initial decrease in the protein content after 24 hours of germination can be attributed to the cell regulation via ubiquitin-proteasome degradation pathway. Proteins in the seeds degraded via such pathways when cells are under stressed situation, such as germination. This simultaneously increases the activation of lipase to sustain amount of ATP needed for the ubiquitinproteasome pathway to proceed. It can also be speculated that the decrease in protein and increase in specific activity from 24 to 48 hours of seed germination occurred through ubiquitin-proteasome pathway. It can be observed that the protein content increased while the volumetric decreased. According to M. Barros et al (2010) grains or seeds use proteins, starch and triacylglycerols, depending on the species, as reserve sources of energy. During the mobilization or germination, the seeds may activate or deactivate enzymes generally proteases, amylases and lipases depending on the major nutrient used by the seed to sustain its need as it germinates. However, if the triacylglycerols are used up as source of energy as the germination proceeds and partly for ubiquitination its concentration in the seed decreases. The decrease in the triacylglycerol concentration decreases the energy for ubiquitination is no longer sustained thus, the degradation of proteins does not proceed rather the formation of a more useful enzymes. As the concentration of triacylglycerol in the seed decreases, the signal perceived by the seed is to lessen the production of lipase and activating the production of other enzymes involved in the activated metabolic pathway (such as gluconeogenesis and subjected to the formation of cellulose and other tissues as can be seen in its structural developments)

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15th and 16th Slide: The table shows the actual/ experimental values and this values were used in the ANOVA and fitting of the model. The model, if found adequate, is used for the determination of the predicted values that is fitted to the model. In this table the actual and predicted values are found to have variations. 17th Slide: In the analysis of variance, the lack of fit is insignificant which means that the model is really adequate and the experimental design used is also adequate to obtain the model. The model consists of terms that develops the activity. In this model, all the terms ( the linear response A, B, C are the linear responses, A2, B2, C2 are the quadratic responses and the AB, AC and BC are the interaction responses) are significant. A pure error of 2.03 and determination coefficient of 0.9941 shows that the design is satisfactory and thus the model approximates the true surface response to locate optimum conditions. In summary the model is significant because of the F-value of 188.76 and Prob>F value shows that there is only 0.01% that this value could occur due to noise. The lack of fit F- value of 4.93 implies that it is not significant relative to the pure error. There is a 5.23% chance that a Lack of Fit F- value could be due to noise or unexplained variation of unpleasant data. The analysis of variance also revealed a low pure error of 2.03 which indicates that the design is reproducible. The pure quadratic coefficient estimates are each highly significant, indicating that surface curvature is present in the observed specific activity. 18th Slide: In the analysis of the second order model, the effect of each variable on the specific activity can also be represented graphically. The perturbation plot shown in Figure 9 shows the trend of specific activity (y-axis) as each of the variables (coded, x-axis) changes from its lower setting (-1) to its higher setting (-1) while the other two factors are held constant at its central points. This provides a silhouette view of the response surface. It can be seen that each factors optimum value fall within the given range. The negative coefficients of the square of the factors means that the parabola that it will form will face downward and showing maxima as reflected in the figure. three dimensional plots and contour plots for the interaction of the two variables while setting the other variable as constant. Interpret the

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The variables used in Face-Centered Central Composite Design showed a significant linear, quadratic and interaction effect on the specific activity shown in the second order model represented by a polynomial equation.

Using numerical optimization, the optimum conditions of pH, temperature and incubation time determined are 7.43, 46.190C and 14.46 minutes, respectively.

There is a corresponding linear increase in the specific activity for every unit increase of pH, temperature and incubation time by about 25.89044, 22.13028, and 10.44402, respectively. However, there is a corresponding decrease in specific activity for square unit increase of pH, temperature and incubation time by 1.40962, 0.22124 and 0.23104, respectively. There is also a corresponding decrease of specific activity by 0.077513, 0.093016, and 0.0617178 for every unit increase of the interaction of pHtemperature, pH incubation time and temperature-incubation time, respectively. Its insignificant lack of fit (0.0523), determination coefficient (R2=0.9941) and inclusion of all the model terms in the second order model equation clearly demonstrate the efficiency of the model for the optimization.