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 chloride,

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.

 28

Papaya Crude latex

Centrifugation

Clarified enzyme solution

Integrating Ligands

Papain partitioning using conventional Papain Partitioning using ATPi System

ATP system: (PEG6000+ Ammonium (PEG6000-Ligand+ (NH4)2SO4)
sulphate)  ATPi System preparation
Forward extraction: parameter studies
Using IL, Metal and Dye ligands
 RSM-CCD Study
 Selection of Significant
Removal of Addition of NaCl
parameters.
bottom Phase

Backward extraction

Removal of Selection of optimized ATPi

Top phase System
Bottom phase  ATPi- Phosphonium IL
 ATPi –Imidazolium IL

Purity studies Bottom phase of

by GFC backward extraction
Papain Partitioning using
ATPi_ IL System
 Forward extraction
 Backward extraction
Final Confirmation
studies

Scale up studies
Thermodynamic
SDS Page/FTIR
Studies
analysis

Figure 3. 1: Scheme of ATPi extraction of papain

 29

3.2. 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.

3.3. 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
 30

was made to 10 ml by the addition of deionized water. The pH of the system was adjusted

to 7.2 using K2HPO4/ KH2PO4 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

 31

3.4. 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.

3.4.1. 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
 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 concentration (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.
 33

Table 3. 2: Design of Experiment for ATPi_IL system

Code Factors Range Actual coded levels

studied
Low Medium High
(-1) (0) (+1)

A PEG (MW) 4000-8000 4000 6000 8000

B PEG, %w/v) 10-20 10 15 20

C Ionic Liquid (µg/ml) 4-10 4 7 10

D pH 4-10 4 7 10

Table 3. 3: Design of Experiment for ATPi_Dye system

Code Factors Range Actual coded levels

studied
Low Medium High
(-1) (0) (+1)

A PEG (MW) 4000-8000 4000 6000 8000

B PEG (% w/v) 10-20 10 15 20

C Dye (µg/ml) 12-18 12 15 18

D pH 4-10 4 7 10
 34

Table 3. 4: Design of Experiment for ATPi_Metal system

Code Factors Range Actual coded levels

studied
Low Medium High
(-1) (0) (+1)

A PEG (MW) 4000-8000 4000 6000 8000

B PEG ( % w/v) 10-20 10 15 20

C Metal (mg/ml) 15-48 15 32 48

D pH 4-10 4 7 10

3.4.2. Regression analysis

A quadratic model was employed for data analysis as shown in Equation (3.1).

i =  o +  i i   ii i   ij i  j
2
(3.1)
i ii ij

Where Yi is the predicted response, β0, βi, βii and βij are the regression coefficients

for the intercept, linear, quadratic and interaction coefficients respectively, Xi and Xj 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
 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.

3.5. Analytical methodology

3.5.1. 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.

3.5.2. 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.

 36

3.5.3. Determination of partition coefficient

The partition coefficient of papain (KP) and ligand (KL) 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).

Ct
KP = (3.2)
Cb

Where Ct represents the concentration of papain/ligand in top phase and Cb

represents the concentration of papain/ligand in bottom phase [89].

3.5.4. 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.

3.5.5. Percentage Yield

Percentage Yield was calculated as the ratio of protein in sample to the protein in

crude.

3.5.6. 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.

 37

3.5.7. Specific enzyme activity

Specific enzyme activity was measured as the ratio between enzyme activity and

the total protein concentration.

3.6. 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.

3.6.1. 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.

3.6.2. 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
 38

micropipette and the Gel filtration chromatography analysis was carried out to perform

purification studies on papain enzyme.

3.7. 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
 39

on partitioned material. The tie-line length was calculated from the Equation (3.2) given

below.

TLL = P
top  P bottom   S top  S bottom 
2 2
 (3.2)

Where [P] and [S] are the polymer and salt concentrations determined in each

phase[90].

3.8. Purification of papain by using Gel filtration chromatography (GFC)

A Sepahdex G-100 column (AKTA Prime Plus, GE, Sweden) was 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 UV-

vis 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.

3.9. 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
 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.
 41

Figure 3. 2: Customized bench top enzyme extractor, ENZextractor

3.10. Final Fraction conformation

3.10.1. 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].
 42

3.10.2. 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.