CHAPTER 3: MATERIALS AND METHODS. purchased from Himedia Laboratories, Mumbai, India. 1-Butyl 3-methyl imidazolium

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1 27 CHAPTER 3: MATERIALS AND METHODS 3.1. Materials Carica Papaya latex was collected from local garden (Thanjavur, Tamilnadu, India) in the month of December at mornings and authenticated by Dr.N.Ravichandran, CARISM, SASTRA University, Thanjavur. Polyethylene Glycol (6000,4000,8000), ammonium sulfate, Sodium chlorid e, Copper sulfate, Nickel sulfate, Zinc sulfate, cobalt chloride, cadmium carbonate were purchased from Himedia Laboratories, Mumbai, India. 1-Butyl 3-methyl imidazolium chloride, 1-Butyl 3-methyl imidazolium bromide, 1-methyl 3-octyl imidazolium chloride, Tetra butyl phosphonium bromide, Tetra butyl ammonium bromide was procured from SIGMA Aldrich, Bangalore, India. Reactive dyes CIBACRON Blue F3GA, PROCION Red HE3G and PROCION Yellow HE3G were provided at free of cost from Lakshmi Enterprises Dyes & chemicals, Salem, Tamilnadu, India. Standard papain and Bovine Serum Albumin were procured from SIGMA Aldrich, Mumbai, India. Casein, Sodium hydroxide, Trichloro Acetic (TCA) acid, Di potassium hydrogen phosphate, Potassium dihydrogen phosphate, Copper sulfate pentahydrate, sodium potassium tartarate, Follin-Ciocalteu reagent were purchased from Himedia Laboratories, Mumbai, India. All chemicals used in the extraction processes were of analytical grade.

2 28 Papaya Crude latex Centrifugation Clarified enzyme solution Integrating Ligands Removal of bottom Phase Papain partitioning using conventional ATP system: (PEG6000+ Ammonium sulphate) Forward extraction: parameter studies Addition of NaCl Papain Partitioning using ATPi System (PEG6000-Ligand+ (NH 4 ) 2 SO 4 ) ATPi System preparation Using IL, Metal and Dye ligands RSM-CCD Study Selection of Significant parameters. Backward extraction Removal of Top phase Bottom phase Selection of optimized ATPi System ATPi- Phosphonium IL ATPi Imidazolium IL Purity studies by GFC Final Confirmation studies Bottom phase of backward extraction Papain Partitioning using ATPi_ IL System Forward extraction Backward extraction SDS Page/FTIR analysis Thermodynamic Studies Scale up studies Figure 3. 1: Scheme of ATPi extraction of papain

3 Preparation of papaya latex solution Crude papaya latex was collected from the unripe fruit of Carica Papaya planted in the local farms of Thanjavur. Thin longitudinal incisions for about 3 mm depth were made on the skin of Carica Papaya with a stainless steel knife. The exuding milky latex was allowed to flow down the fruit and drop into the collecting device attached to the trunk. Following collection, the milky latex was transferred to a storage vial containing phosphate buffer solution (PBS) maintained at ph 7.2 and the mixture was stirred until it attains homogeneity [81]. The homogeneous latex solution was then centrifuged at 4000 rpm (High speed cooling centrifuge, REMI Instruments Ltd, Mumbai, India) to remove insoluble. The process was repeated at 6000 rpm in order to get a clarified papaya latex solution as supernatant phase, the phases were removed carefully and the clarified solution was stored at 4 C until used for the study. A 10 ml of the clarified papaya solution maintained at 4 C was used in the following aqueous two phase extraction studies Papain partitioning in Aqueous two phase system An aqueous two phase (ATP) system composed of Polyethylene glycol (PEG) and ammonium sulfate salt was used for the partitioning of papain. Phase diagram and tie line length (TLL) for the ATP system (composed of PEG and ammonium sulfate) were reported in the previous work [82, 83]. To the clarified enzyme latex solution the stock solutions of polymer and salt were mixed together and stirred well, so as to get the final concentration of 15%w/v PEG (4000, 6000 & 8000) and 12%w/v ammonium sulfate. The final volume of the system

4 30 was made to 10 ml by the addition of deionized water. The ph of the system was adjusted to 7.2 using K 2 HPO 4 / KH 2 PO 4 solution and the above ATP mixture was stirred well for about 30 minutes. Then the mixture was transferred into graduated centrifuge tubes and centrifuged at 6000 rpm for 10 minutes to attain phase separation. The phase volumes were measured and the top and bottom phases were separated to determine the partition coefficient. Initially, PEGs (4000, 6000 and 8000) and ammonium sulfate salt with different concentrations were tried for aqueous two phase system formation and their ranges are tabulated in Table 3.1. The effect of ph on papain extraction was also studied for 15% w/v PEG 6000 and 12% w/v ammonium sulfate aqueous two phase system. Table 3. 1: Phase forming components and their ranges in ATP system formation Component Range PEG Molecular mass, MW 4000, 6000, 8000 PEG concentration, %w/v 10, 12, 15, 18 Salt concentration, %w/v 8, 10, 12, 15

5 Papain partitioning studies in integrated Aqueous Two Phase (ATPi) system A ligand assisted aqueous two phase system is termed here as an integrated aqueous two phase extraction (ATPi) system and the schematic diagram is shown in Figure 3.1. ATPi system was formed by integrating papain affinity groups (ligand) with the phase forming compound (either polymer or salt) [84]. The metal salts, dyes and ionic liquids were used as ligands for this investigation. For the ATPi system formation, stock solutions of PEGs (4000, 6000 and 8000), ligands (Ionic liquids, Tri-azine dyes and Metal salts ) and ammonium sulfate solution were prepared separately. Initially, the ligand and PEG were mixed in the deionized water to form PEG-ligand complex solution. Papain latex solution and ammonium sulfate were added later to the PEG complex and stirred gently. The ph of the system was adjusted with the phosphate buffer solution prepared in deionized water. The above ATPi mixture was allowed to reach saturation with gentle mixing at room temperature for 45 minutes and then centrifuged at 6000 rpm for 10 minutes. The resulting two phases were separated carefully by micropipettes without disturbing the interface and used for further studies. The above procedure was repeated for each of nine ligands and the ATPi systems were prepared Design of Experiment A four-factor central composite design was developed and evaluated using regression analysis for which Response Surface Methodology (RSM) (Design -Expert Version 9 software, State-Ease Inc., Minneapolis MN, USA) was employed to study and optimize the factors which are responsible for the maximum extraction of papain from its clarified latex solution [85]. RSM comprised of central composite design and regression

6 32 analysis are used to assess the various influencing factors even under complex relations. Central Composite Design (CCD) in RSM is a statistical approach to analyze the interactions of the process parameters and develops process model for the experimental design and used to optimize the process conditions for the maximum response of the system with less number of design points thereby reducing the overall cost. An experimental data of 30 runs were applied to optimize the ATPi system formation where the central point was repeated six times to estimate the experimental error variance for the extraction of target protein. The four factors taken for the CCD approach are PEG molecular mass (A), PEG concentration (B), ligand concent ration (C) and ph (D), and are considered as independent variable (X). The interrelationship between the process parameters and its responses as dependent variables (Y) were also analyzed through the central composite design approach. The levels and ranges chosen for the factors in ATPi_IL, ATPi_Dye and ATPi_Metal systems are shown in table 3.2, 3.3 and 3.4. The maximum response of the factors obtained from the RSM analysis tends to increase the protein affinity towards the ligand bound PEG rich phase.

7 33 Table 3. 2: Design of Experiment for ATPi_IL system Code Factors Range studied Actual coded levels Low (-1) Medium (0) High (+1) A PEG (MW) B PEG, %w/v) C Ionic Liquid (µg/ml) D ph Table 3. 3: Design of Experiment for ATPi_Dye system Code Factors Range studied Low (-1) Actual coded levels Medium (0) High (+1) A PEG (MW) B PEG (% w/v) C Dye (µg/ml) D ph

8 34 Table 3. 4: Design of Experiment for ATPi_Metal system Code Factors Range studied Low (-1) Actual coded levels Medium (0) High (+1) A PEG (MW) B PEG ( % w/v) C Metal (mg/ml) D ph Regression analysis A quadratic model was employed for data analysis as shown in Equation (3.1). i = + o i i i ii ii 2 i ij ij i j (3.1) Where Y i is the predicted response, β 0, β i, β ii and β ij are the regression coefficients for the intercept, linear, quadratic and interaction coefficients respectively, X i and X j are the coded independent variables. Analysis of Variance (ANOVA) was performed using Design-Expert (Version 9), and probability values (P -values) were used to identify statistical significance of each statistical parameters like F-test and model terms[86]. The interaction between regression terms were, in addition, visualized using three dimensional surface plots. Through numerical optimization process, the levels of each factor were optimized for maximum

9 35 response. Validity of the model was checked by testing various optimized parameter combinations with maximum response and the same was applied in ATPi, using both dyes for the papain extraction Analytical methodology Total protein estimation The total protein estimation was determined for the enzyme fraction by the Lowry s method with BSA as standard[87]. In Lowry s protein estimation 0.5 ml of the sample solution was mixed with 4.5 ml of Lowry s reagent. The reactive mixtures were well stirred and incubated for 10 minutes at room temperature. After incubation, 0.2ml of Folin-Ciocalteu reagent was added and mixed well. Absorbance was measured at 680 nm after 30 minutes incubation at room temperature. The papain concentration was determined from the BSA calibration chart Protease assay The papain activity of samples were determined by casein digestion assay [88]. In this method 10µl of enzyme solution was mixed with 5 ml of 1% casein and the reaction mixture was incubated for 5 minutes at 37 C. After incubation the reaction was stopped by the addition of 5 ml of TCA and again, incubated for 30 minutes at 30 C.Then, the sample was filtered and absorbance was measured at 280nm.The enzyme concentration was determined from tyrosine standard chart. One unit is the amount of papain enzyme that will liberate one µg of tyrosine after one minute of digestion at 37 C from a standard casein substrate solution at ph 7.0.

10 Determination of partition coefficient The partition coefficient of papain (K P ) and ligand (K L ) were calculated as the ratio between the concentration in the top phase to the concentration in the bottom phase as shown in equation (3.2). K P = C C t b (3.2) Where C t represents the concentration of papain/ligand in top phase and C b represents the concentration of papain/ligand in bottom phase [89] Purity factor (PF) The purity factor (PF) of ATP and ATPi systems were determined as the ratio of specific enzyme activity of papain in the top phase to the specific enzyme activity in the feed sample Percentage Yield crude. Percentage Yield was calculated as the ratio of protein in sample to the protein in Enzyme activity One unit of enzyme activity is defined as the amount of papain giving an increase in absorbance at 280 nm under the assay condition.

11 Specific enzyme activity Specific enzyme activity was measured as the ratio between enzyme activity and the total protein concentration Optimization of ATPi extraction system The RSM methodology shows the optimized values of all system parameters and exhibits the significant model through regression analysis. The nine ligand systems studied were arranged in descending order, in terms of partition coefficient, and the top two systems were chosen for further forward and backward extraction studies Forward extraction The concentrations of PEG and ligand used for the forward extraction were considered from the optimized factors of the RSM analysis. At defined concentration, the integrated PEG_ligand stock solution and ammonium sulfate stock solution were mixed in a graduated centrifuge tube and then the clarified papain solution was added to the mixture placed on the shaker to conduct forward extraction. The ph of the system was adjusted to 7.0 with the phosphate buffer solution and a final volume of 10 ml from the mixture was taken and the two phase separation was carried out by performing centrifugation at 6000 rpm for 10 minutes, then the resulting top phase was carefully separated to perform backward extraction Backward extraction In the backward extraction process, aqueous two phase system was formed by blending 20% of NaCl salt with the top phase separated from the forward extraction. The centrifugation of the mixture at 8000 rpm for 10 minutes resulted in two phase formation and the salt rich bottom phase containing the target papain enzyme was separated by

12 38 micropipette and the Gel filtration chromatography analysis was carried out to perform purification studies on papain enzyme Phase diagram/ Tie line length determination The binodal curve for the optimized ATPi system was determined using the cloud point method [90]. Stock solution of concentrated PEG 6000 was prepared and then ammonium sulfate stock solution was added drop wise into the solution. Initial addition of ammonium sulfate salt solution produces monophasic system, but the repetitive addition of salt makes the mixture turbid. The point at which turbidity occurs was noted for binodal curve, followed by the drop wise addition of deionized (ultrapure) water until the mixture becomes clear. The procedure was repeated several times with various concentrations of PEG and ammonium sulfate. All the analytical methods were performed in triplicates and found the mean with standard deviation. The system compositions were calculated within an accuracy of ±10-5 g by using an analytical balance. For the determination of tie line lengths (TLL), a mixture from the two phase region was selected, vigorously agitated and allowed to reach equilibrium by separating the two phases for 12 hours at temperature 298 K. After the separation, concentration of phase forming compounds in top and bottom phases were determined. Conductivity meter was used for the salt concentration determination, whereas polymer concentration was determined by refractometer. Finally, each individual tie line was determined by the application of lever rule to express the effect of aqueous two phase system composition

13 39 on partitioned material. The tie-line length was calculated from the Equation (3.2) given below. S S TLL = P P top 2 2 bottom top bottom (3.2) Where [P] and [S] are the polymer and salt concentrations determined in each phase[90] Purification of papain by using Gel filtration chromatography (GFC) A Sepahdex G-100 column (AKTA Prime Plus, GE, Sweden) wa s used for the GFC purification with the column size of 5ml. The column was previously equilibrated with ph 7.2 buffer solution and the mobile phase of 20mM sodium phosphate was allowed with the flow rate 2.5ml/min. The samples isolated from the backward extraction was injected to remove the ions present in the salt solution. A sample volume of 0.5 ml was introduced into the column through an injection port. By changing the load to injection mode, the sample was injected. The sample elution occurred at the same flow rate were collected separately with fraction collector. The elution was monitored by UVvis spectrophotometer at 280nm. When bound molecules elute from the column, chromatographic peaks were observed. The final fraction of 0.5 ml was collected and the chromatographic peaks and its respective volumes were recorded Scale up studies on ENZextractor The papain extraction by ATPi system was conducted in a three litre ENZextractor (Customized bench top extractor, shown in Figure 3.2) with a working

14 40 volume of 1.5 L. The ENZextractor was equipped with perforated disc and they were placed in central rotating shaft fixed to a head plate motor. In the extractor, six discs were placed at various distances at constant spacing. Discs were drilled with 6 holes with a diameter of 9 mm and they were operated at different rotating speed. The internal diameter of the vessel and discs were 24 mm and 18 mm respectively. ATPi extraction parameters such as temperature, ph and phase volume ratio were continuously monitored with respective probes. ATPi extraction solution (15% w/v PEG 6000 loaded with IL3 ligand and 12% w/v ammonium sulphate) was filled in the ENZextractor. The solution was added with papain latex (150mg) and the final ph 7.2 of the system was adjusted by the addition of phosphate salt. The extraction was carried out in batch mode operation for 90 minutes at an impeller speed of 90 rpm. The resulting sample was allowed for setting (50 rpm) at room temperature (24 3) C for 45 minutes which results in the phase separation. The samples were analyzed for total protein, partition coefficient and papain activity.

15 41 Figure 3. 2: Customized bench top enzyme extractor, ENZextractor Final Fraction conformation Sodium Dodecyl Sulphate ( SDS) page SDS page was used to evaluate the purity of papain in the top phases, using 12% bis-acrylamide gel. The samples were first dissolved in SDS buffer (62.5 mm Tris HCl, ph 6.8, SDS 2% w/v, bromophenol blue 0.01% w/v and 10% glycerol) and then subjected to SDS page at a constant 90 mv current using running buffer followed by 0.1% of Coomassie Brilliant Blue staining along with the standard papain[18].

16 Fourier Transform Infra Red (FTIR) Spectroscopy analysis The FTIR was performed in order to conform the final fraction. The FTIR spectroscopy analysis was carried out using a ATR technique for the purified final fraction of papain to identify the functional groups present in the sample[91]. The Infra Red light was passed through the ATR crystal in such a way that it reflects atleast one of the internal surface in contact with the sample. The samples were scanned in the wave number ranging from 4000 cm -1 to 400 cm -1.