Liang Zhou, Suzanne M. Budge, Abdel E. Ghaly*, Marianne S. Brooks and Deepika Dave
Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada
Received date: November 08, 2012; Accepted date: December 17, 2012; Published date: December 19, 2012
Citation: Zhou L, Budge SM, Ghaly AE, Brooks MS, Dave D (2012) Extraction and Purification of Chymotrypsin from the Intestine of Red Perch (Sebastes marinus) Using Reverse Micelles: Optimization of the Forward Extraction Step. J Food Process Technol 3:157. doi:10.4172/2157-7110.1000157
Copyright: © 2012 Zhou L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Visit for more related articles at Journal of Food Processing & Technology
Fish waste; Chymotrypsin; Extraction; Purification; Ultrafiltration; Fractionation; Microemulsions; Enzyme activity; Protein concentration; Recovery yield
Canada has 25% (244,000 km) of the world’s coastline and 16% of the world’s fresh water (755,000 km2). About 80% of total fish landings come from the Atlantic fishery while the Pacific fishery accounts for up to 16% . In 2009, Canada’s total seafood and fish landings were 1,880,896 t/y tons with the total value of 3.3 billion dollars .
A large portion of the fish (80%) landed in Canada is processed. The most common fish processing operation includes three steps: (a) removing the viscera (b) removing the head, tail, fins and skin and (c) removing the frame and producing fillets. Fish waste is generated from the unwanted parts of the fish which can generally be divided into two streams: (a) solid waste including heads, tails, fins, frames, offal (guts, kidney and liver) and skin and (b) wastewater from cleaning the fish and equipment [3,4]. Fish processing solid waste can account for 27% from the removal of heads and guts from the fish, 25-30% from tuna canning production, 66% from the production of fillets and up to 80% of material from the production of surimi [3-5].
Currently, fish waste is an approved substance for disposal at sea and the Canadian fish industry is dumping most fish waste into the sea because there is no economical way of utilizing the waste off shore and it is costly to transport large amount of fish waste to meal plants or land-based waste disposal systems [6,7]. It is estimated that about 1,034,492 tons of fish waste can be produced annually in Canada of which 88,000-130,000 metric tons are disposed in the ocean . The decomposition of large volumes of wastes lower the level of dissolved oxygen in the water and generates toxic by-products [3,9].
However, fish processing wastes contain high value proteins and fats which can potentially be reused to produce valuable by-products such as organic fertilizers, fishmeal and fish silage . Fish wastes are also known to contain highly valued fatty acids including omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), enzymes (pepsin, trypsin, chymotrypsin), collagen and biodiesel [10-12]. These valuable compounds can be used in the medical, food and transportation industries .
Chymotrypsin has wide application in food, leather processing, chemical and clinical industries. Industrially, chymotrypsin is produced from the pancreas of cattle or swine and is commonly made in either a tablet form for oral consumption or as a liquid for injection. The price of chymotrypsin is related to the purity of the product and the cost of the raw materials. Using fish waste, rather than fresh cattle or swine pancreases, could dramatically lower the cost of chymotrypsin production .
Currently, ammonium sulphate is used to precipitate crude chymotrypsin from raw material which is further purified with chromatography. Reverse micelles are thermodynamically stable molecules that can extract large biomolecules like proteins through electrostatic interaction that attracts soluble proteins into the interior of the reverse micelles [15,16]. They can form amphiphilic structures in polar organic media incorporating a surfactant such as AOT which can be used to extract large amounts of proteins in the aqueous phase without denaturation.
The reverse micelles extraction process is divided into two steps: forward extraction and back extraction. During the forward extraction step, the aqueous and organic phases are separately prepared and homogenized. After the transfer of protein from the aqueous phase to the organic phase, the two phases are separated by centrifugation [15-17]. Selection of surfactants in the forward extraction step plays a significant role in protein stabilization. Sodium di-2-ethylhexyl sulfosuccinate (AOT) is the most common surfactant used in chymotrypsin purification [15-17]. In the backward extraction step, protein is transfered from the reverse micelles to the aqueous solutions. The backward extraction step is usually very slow and salt is added into the aqueous phase to assist the process . Increasing chloride ion concentration will decrease chymotrypsin yield by competing with chymotrypsin in the extraction process and the effect is particularly significant at low ionic strength [15,18]. Jolivalt et al.  reported that pH influences ionic molecular interactions in solution and therefore, influences the efficiency of extraction by reverse micelles.
The main focus of this study was to evaluate the effectiveness of the reverse micelles method for purifying chymotrypsin from fish processing waste and to optimize the forward extraction step. The specific objectives were: (a) to study the effects of AOT concentration (1, 5, 10, 15, 20, 35 and 50 mM) and pH (6.0, 6.5, 7.0, 7.5, 8.0 and 8.5) during the forward extraction step on enzyme activity (AE), protein concentration (Cp), specific activity (SA), purification fold (PF) and recovery yield (RY) and (b) compare the effectiveness of reverse micelles to that of the ammonium sulphate precipitation method.
Tris HCl, CaCl2, NaCl, ammonium sulphate, benzoyl-tyrosine ethyl ester (BTEE), methyl alcohol and n-butyl alcohol isobutyl alcohol were obtained from Sigma-Aldrich, Oakville, Ontario, Canada. AOT, isooctane (2,2,4-trimethylpentane), chymotrypsin, isobutyl alcohol and BSA (bovine serum albumin) were obtained from Fisher Scientific, Ottawa, Ontario, Canada.
The fish, red perch (Sebastes marinus), used in the experiment were obtained from Clearwater Seafoods Ltd., Halifax, Nova Scotia, Canada. The intestines were separated from fish and washed with cold water and isotonic saline solution to remove the blood in the tissue according to the procedure described by Chong et al.  and Boeris et al. . They were chopped into small pieces (1 cm3), weighed, marked and stored at -20ºC for later use.
Crude enzyme extraction
The extraction procedure (Figure 1) described by Heu et al.  and Castillo-Yáñez et al.  was followed. The prepared frozen samples were thawed at 4°C overnight. A 50 g (wet basis) sample of fish gut was mixed with 150 mL isotonic saline solution and homogenized using a laboratory homogenizer (Polytron PT1035, Brinkmann Instruments, Toronto, Ontario, Canada) for 5 min then incubated for 8 hr at 4°C to activate the chymotrypsinogen in the samples. After incubation, the sample was centrifuged at 20,000 g at 4°C for 30 min (MP4R, International Equipment Company, Needham, Massachusetts), then filtered and defatted with 50 mL CCl4. The supernatant was considered a crude enzyme extract. The volume at each step was measured and the activity and concentration of crude enzyme were determined, three replicates were carried out.
Chymotrypsin purification by ammonium sulphate (AS)
Chymotrypsin was purified from the crude enzyme using the ammonium sulphate precipitation method (Figure 1) following the procedure described by Kunitz . Ammonium sulphate was slowly added to the crude extract to reach 35% saturation with continuous stirring. The mixture was stirred for a further 30 minutes at 4°C and then centrifuged at 20,000 g for 15 min (MP4R, International Equipment Company, Needham, Massachusetts). The supernatant was collected and the saturation was then adjusted to 70% by addition of ammonium sulphate. After 30 min, the suspension was centrifuged at 20,000 g for 15 min. The pellets collected from the 35% and 70% saturation steps contained precipitated enzymes. Dialysis was performed on the pellets using Tris-HCl buffer. The enzyme concentration, activity and yield were then determined.
Chymotrypsin purification by reverse micelles (RM)
The reverse micelles procedure used to purify chymotrypsin is shown in Figure 1 and the parameters studied and their levels are shown in Table 1. The AOT reversemicelles were prepared by dissolving AOT in reagent-grade isooctane and adjusting the AOT concentration to 1, 5, 10, 15, 20 or 35 mM. Crude protein sample solution was prepared by mixing the same volume of crude extract with buffer (0.1 M Tris-HCl) and adjusting the pH to 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5. Equal volumes (5 mL) of the aqueous protein solution and AOT solution were mixed with magnetic stirrer (Classic C24, New Brunswick Scientific Company, Edison, New Jersey, USA) for 30 min to reach equilibrium. The mixture was then centrifuged for 15 min at 4000 rpm (MP4R, International Equipment Company, Needham, Massachusetts) in order to separate the two phases as described by Hentsch et al. . In the backward extraction step, the organic phase collected from the first step was mixed with 5 mL new aqueous phase and 15% v/v isobutyl alcohol with magnetic stirring for one hour to reach equilibrium. The mixture was then centrifuged for 15 min at 4000 rpm. The enzyme activity, protein concentration, specific activity and purification fold of the purified chymotrypsin in the water phase was measured.
|AOT||1 mM, 5 mM,10 mM,15 mM, 20 mM, 35mM|
|pH||6.0, 6.5, 7.0, 7.5, 8.0, 8.5|
|Salt Concentration||1.5 M|
Table 1: Experimental parameters.
Determination of protein concentration (Cp)
The Bradford method was used for the determination of protein concentration according to the procedures described by Yang et al.  and Castillo-Yáñez et al. . Two standard curves were developed using a series of concentrations of bovine serum albumin (BSA): the standard curve (assay range 10-150 μg/mL) and the micro standard curve (assay range 1-10 μg/mL). The following solutions were prepared: (a) 0.1 g of BSA was dissolved in 10 mL of Tris-HCl buffer at room temperature (b) the stock BSA solution was diluted to span the 100- 1,500 μg/mL range and (c) BSA solution in the range of 100-900 μg/mL was diluted ten times more for the micro standard curve. The two standard curves are combined together as shown in Figure 2.
10 μL of each standard was mixed with 5 mL of Bradford reagent. Each sample was allowed to incubate at room temperature for 10 minutes and the absorbance of each standard was measured at 595 nm against a blank that was composed of 10 μL of buffer and 5 mL of Bradford reagent. 0.1 mL diluted sample (concentration between 5 to 100 μg/L) was mixed with 5 mL Bradford reagent, incubated for 5 min and then the absorbance was measured at 595 nm . The result was compared with the standard curve to determine the sample protein concentration three replicates were carried out.
Determination of enzyme activity (AE)
The activity of chymotrypsin was defined as the change of absorbance measured at 256 nm in one minute caused by the addition of 1 mL of chymotrypsin protein solution . The substrate used in the experiment was benzoyl- tyrosine ethyl ester (BTEE). The release of p-nitroaniline was followed by recording the increase in the absorbance every min for 5 min at 256 nm [13,26]. 1.5 mL Tris-HCl buffer (0.08 M tris, pH 7.8, 0.1 M CaCl2), 1.4 mL of 0.00107 M BTEE and 0.1 mL test enzyme solution were placed into cuvettes. The enzyme activity was calculated as follows.
AE : Enzyme activity (Units/mL Enzyme)
ΔU256: The change of the absorbance at the wave length 256 nm per minute.
3 : Volume of reaction mixture (mL)
Df : Dilution factor
0.964 : Millimolar extinction coefficient of BTEE at 256 nm
0.10 : Volume of test enzyme solution used in assay (mL)
Determination of total activity (TA)
The total activity is defined as the change in the absorbance value per min for the total chymotrypsin extracted from the entire sample of red perch intestine. The total activity was calculated as follows.
Determination of specific activity (SA)
Specific activity is defined as the ability of 1 mg enzyme to hydrolysis BTEE in one min at a pH of 7.5 and a temperature of 25°C. 0.1 mL enzyme solution was added into cuvettes and the change in absorbance was measured at 256 nm every half minute for 5 minutes. The specific activity was calculated using the following equation.
SA: Specific activity (Units/mg Protein)
Determination of purification fold (PF)
Purification fold is used to evaluate the increase in purity of the enzyme after the purification step. It was calculated using the following equation:
Determination of recovery yield (RY)
Recovery yield is defined as the ratio of total refined enzyme activity and total crude enzyme activity. Recovery represents the chymotrypsin activity remaining in the purification process. When combined with specific activity, it can show the effectiveness of a purification method.
Crude protein extraction
Crude protein was extracted from the intestine (50 g) of red perch and the total volume (TV) was measured after homogenization, centrifugation and dilution. The enzyme activity (AE), total activity (TA), specific activity (SA), protein concentration (Cp), purification fold (PF) and recovery yield (RY) were determined as shown in Table 2. After centrifugation, the TV, TA and Cp decreased from 183 to 144 mL (21.32%), from 146.40 to 135.36 U (7.54%) and from 4486.2 to 1975.8 μg/mL (55.96%), while the AE and SA increased from 0.80 to 0.94 Unit/mL (17.50%) and from 0.18 to 0.48 Unit/mg (169.1%), respectively. The purification fold and the recovery yield were 2.69 and 92.7%, respectively.
|After Dilution and pH adjustment||288||0.47||135.36||987.90||0.48||-||100.00|
Table 2: Crude protein extraction parameters.
Ammonium sulphate extraction
The crude extract was purified using the ammonium sulphate precipitation method. The AE, TA, Cp, SA, PF and RY were determined (Table 3). The AE, TA and Cp values of the purified enzyme were lower than those of the crude enzyme. The AE, TA and Cp decreased from 0.47 to 0.22 Unit/mL (53.3%), from 2.37 to 1.10 Unit/mL (53.3%) and from 987.90 to 41.64 μg/mL (95.8%), respectively. On the other hand, the SA and PF of purified enzyme were much higher than those of crude enzyme. The SA increased from 0.48 to 5.31 Unit/mg (1012%) and the PF increased from 2.69 to 11.1 (313.64%). The final yield was 46.72%.
|AE (Unit/mL)||0.22 ± 0.01|
|TA (Unit)||1.10 ± 0.05|
|Cp (µg/mL)||41.64 ± 1.16|
|SA (Unit/mg)||5.31 ± 0.19|
|PF (-)||11.10 ± 0.39|
|RY (%)||46.72 ± 2.23|
Table 3: Extraction parameters after ammonium sulphate precipitation.
Reverse micelles extraction
During the optimization of the forward extraction, two parameters were studied: AOT at six levels (1, 5, 10, 15, 20 and 35 mM) and pH at six levels (6.0, 6.5, 7.0, 7.5, 8.0 and 8.5). The volumes of the water phase (Vw) and organic phase (Vo) and the TV were measured and the VR was calculated by dividing Vo by Vw. The AE, TA, SA, Cp, PF and RY were determined. The analysis of variance performed on the data (Table 4), indicated that the effects of pH and AOT were significant at 0.0001 level. A significant interaction between the pH and AOT was also observed at 0.0001 level.
Table 4: Analysis of variance for the various parameters.
The TV increased slightly with increased pH and decreased with increased AOT concentration (Figure 3). When the pH was increased from 6.0 to 8.5, the TV slightly increased by 0.98-1.15%, depending on the AOT concentrations. On the other hand, when the AOT concentration was increased from 1 to 35 mM the TV decreased by 8.28-11.44%, depending on the pH.
The VR decreased slightly with increased pH whereas AOT concentration did not have any effect on the VR (Figure 4). When the pH was increased from 6.0 to 8.5, the VR slightly decreased (1.0-1.1%), depending on the AOT concentration.
The AE increased with initial increases in the pH and AOT concentration and then decreased with further increase in pH and AOT concentration (Figure 5). When the pH was increased from 6.0 to 7.0, the AE initially increased (by 20-80%), and then decreased (by 28.6-50.0%) when the pH was further increased from 7.0 to 8.5. When the AOT concentration was increased from 1 to 20 mM (2000%), the AE initially increased (by 110.0-192.3%) and then decreased (by 33.3- 63.3%) when the AOT concentration was further increased from 20 to 35 μM (75%).
The Cp increased with initial increases in the pH and AOT concentration and then decreased with further increases in the pH and AOT concentration (Figure 6). When the pH was increased from 6.0 to 7.0, the Cp increased (by 7.72- 36.67%) and then decreased (by 2.63- 36.3%). When the AOT concentration was increased from 1 to 20 mM (2000%) the Cp increased (by 51.6-105.8%), and then decreased (by 23.6-49.0%) when the AOT concentration was further increased from 20 to 35 μM (75%).
The SA initially increased with initial increases in the pH and AOT concentration and then decreased with further increases in the pH and AOT concentration (Figure 7). When the pH was increased from 6.0 to 7.0 the SA increased (by 12.4-37.8%), and then decreased (by 16.9- 29.6%) when the pH was further increased from 7.0 to 8.5. When the AOT concentration was increased from 1 to 20 mM (2000%) the SA increased (by 29.5-46.2%), and then decreased (by 3.4-20.3%) when the AOT concentration was further increased from 20 to 25 mM (75%).
The PF increased with initial increases in the pH and AOT concentration and then decreased with further increases in the pH and AOT concentration (Figure 8). When the pH was increased from 6.0 to 7.0 the PF increased (by 12.1-37.8%), and then decreased (by 16.9-29.7%) when the pH was further increased from 7.0-8.5. When the AOT concentration was increased from 1 to 20 mM (2000%) the PF increased (by 29.5-46.3%) and then decreased (by 3.3-20.3%) when the AOT concentration was further increased from 20 to 25 mM (75%).
The RY increased with initial increases in the pH and AOT concentration and then decreased with further increases in the pH and AOT concentration (Figure 9). When the pH was increased from 6.0 to 7.0 RY increased (by 9.5-30.3%), and then decreased (by 19.3- 33.3%) when the pH further increased from 7.0-8.5. When the AOT concentration was increased from 1 to 20 mM the RY increased (by 35.8-50.3%) and then decreased (by 16.3 to 43.1%) when the AOT concentration was further increased from 20 to 36 mM (75%).
After homogenization and centrifugation, the enzyme activity (AE), total activity (TA), protein concentration (Cp) and recovery yield (RY) decreased which indicated that a portion of chymotrypsin was lost during the crude extraction process. However, the increases in specific activity and purification fold indicated that the remaining portion of chymotrypsin was concentrated during the crude extraction process.
During the forward extraction step, a minimum amount of surfactant (AOT) was required to form the reverse micelles structure in the organic phase. However, high AOT concentrations can cause difficulties in separating protein molecules from the reverse micelles during the backward extraction step . The pH on the other hand affects the net charge of protein molecules which means that the electrostatic interaction force between chymotrypsin and the surfactant head groups is pH dependent. Changes in pH can, therefore, affect the extraction efficiency [16,27,28].
Generally, increasing the AOT concentration from 1 to 35 mM resulted in a slight decrease (7.36-10.16%) in the TV while increasing the pH (from 6.0 to 8.5) resulted in a slight increase (0.7-3.8%) in the TV. When the AOT concentration increased, a third unclear phase (emulsion) appeared between the organic and aqueous phases. The volume and stability of the third phase increased with increasing AOT concentration. Wang and Cao  reported a decrease in TV during the extraction of kallikrein using the reverse micelles method. They also observed the formation of a stable oil-water mixture between the organic phase and aqueous phase after stirring for half an hour at low temperature. Hentsch et al.  reported a third phase (stable whiteemulsion) when the AOT concentration was higher than 50 mM. Chen et al.  observed a stable third phase which could not be separated by ultra-centrifugation (10,000 rpm) at a pH lower than 7. Hu and Gulari  added tributyl phosphate (TBP) to inhibit the formation of the third phase.
Increasing the pH from 6.0 to 7.0 resulted in initial increases in the AE, Cp, SA, PF and RY which was then followed by decreases with further increases in the pH (from 7.0 to 8.5). The primary driving force for extraction of chymotrypsin from the aqueous phase to the reverse micelles water pool is the attractive electrostatic interaction between chymotrypsin molecules and the inner layer charge of the reverse micelles. Hu and Gulari  reported that when the pH of the aqueous phase was lower than the isoelectric point (pI) of chymotrypsin, the positive charge on the chymotrypsin surface interacted with the negative charge on the head structure of the AOT molecule causing the chymotrypsin to transfer from the aqueous phase to the organic phase. Theoretically, the lower the pH of the aqueous phase (compared to the pI of chymotrypsin), the stronger the interaction would be and the easier the transfer of chymotrypsin from the aqueous phase to the organic phase. Since the pI of chymotrypsin is 8.5, increasing the pH from 7.0 to 8.5 weakened the electrostatic interaction between chymotrypsin and AOT which then resulted in dramatic decreases in AE, Cp, SA, PF and RY.
Increasing the AOT concentration from 1 to 20 mM also resulted in initial increases in AE, Cp, SA, PF and RY followed by decreases in those parameters with further increases in the AOT concentration (from 20 to 35 mM). AOT forms a “microcapsule” structure in the organic phase which acts as a colloidal extractor for chymotrypsin molecules. Thus, the AOT concentration significantly affects the extraction ability of the reverse micelles system. When the AOT concentration was increased from 1 to 20 mM, the “microcapsule” structure increased, resulting in increases in AE, Cp, SA, PF and RY but when the AOT concentration was further increased to 35 mM, decreases were observed in those parameters likely due to the difficulties in separating chymotrypsin from the reverse micelles structure as reported by Grandi et al. , Lang et al.  and Hentsch et al. .
Hebbar et al.  reported that the SA increased from 28 to 49 Unit/mg when the pH was increased from 6.0 to 8.0 and then decreased when the pH was further increased from 8.0 to 10.0 while extracting bromelain from pineapple waste. They also found that when the cetyl tri methyl ammonium bromide (CTAB) concentration was increased from 50 to 150 mM, the SA increased from 22.49 to 40.32 Unit/mg and then decreased to 21.71 Unit/mg when the CTAB concentration was further increased to 200 mM. Wang and Cao  extracted kallikrein from crude kallikrein with CTAB reverse micelles and obtained a maximum SA of 200 Unit/mg at pH 12. Heu et al.  studied the effect of AS, Benzamidine-Sepharose 6B, Sephadex G-75, first DEAESephacel, second DEAE-Sephacel and Sephacry extraction methods on purification of chymotrypsin from anchovy viscera using BTEE as subtract and obtained SA values of 6.62, 6.14, 21.3, 41.7 and 98.27 Unit/mg, respectively. Castillo-Yáñez et al.  reported that the SA of Monterey sardine chymotrypsin obtained using BTEE as subtract in AS fraction, followed by purification with gel filtration and ionic exchange chromatography were 10, 18 and 43 Unit/mg, respectively. It is clear from their studies that the SA value will depend on the enzyme substrate and extraction methods used. The SA obtained in this study ranged from 7.5 to 14.19 Unit/mg depending on the pH and AOT concentration used in the process. The purpose of this study was to find a cheaper efficient and large scale method which can replace the traditional crude purification step in industry. However, if higher SA is required the RM method should be combined with a chromatography purification step.
The PF obtained in this study was in the range of 15.81-29.63. This PF trend is similar to those reported in the literature. Hebbar  found that the PF increased to a maximum of 5.3 when the pH increased from 6.0 to 8.0 and then decreased when the pH was further increased from 8.0 to 10.0 during the extraction of bromelain from pineapple waste. They also found that when the CTAB concentration was increased from 50 to 150 mM, the PF increased from 2.9 to 5.2 and then decreased to 2.8 when the CTAB concentration was further increased to 200 mM. Wang and Cao  extracted kallikrein from crude kallikrein with CTAB reverse micelles and found the maximum PF to be 7.15. The PF obtained in this study is higher than those reported by other researchers because the SA of the crude enzyme obtained here was much lower than those in other reports. The SA of crude extract obtained from rabbit fish, Monterey sardine and anchovy viscera were 1.3, 6 and 0.82 Unit/mg [20,21] while the SA of crude extract obtained in this study was only 0.47 Unit/mg, including impurities in the crude extract.
In this study, 88.9% RY was achieved under the forward extraction condition. Chen et al.  used AOT reverse micelles to extract matrine and reported a recovery yield of 70.3%. Wang and Cao  reported a recovery yield over 80% when extracting kallikrein using CTAB reverse micelles. Hebbar et al.  extracted bromelain from pineapple waste at an optimal pH of 8.0 and obtained an RY of 106%. RY > 100% could occur because of the recovery of the impurities with chymotrypsin. The crude extraction used in this study contained other proteins so the Cp of the purified solution did not solely represent the chymotrypsin concentration but it can be used to determine the extraction ability of reverse micelles.
The highest AE, Cp, SA, PF and RY obtained with the reverse micelles method were reached at a pH of 7 and AOT concentration of 20 mM (Table 5). The optimum pH (7) of the forward extraction determined in the study is within the range of 6.8-7.2 reported by other researchers [16,34] while the optimum AOT concentration (20 mM) was higher than those reported by Lang and Jada  and Hentsch et al.  of 15 mM.
|pH||AOT concentration (mM)|
Table 5: Optimum pH and AOT concentration for forward extraction.
The reverse micelles (RM) procedure was compared to the ammonium sulphate (AS) method (Table 6). The values of extraction parameters obtained with the RM method were much higher than those obtained with the AS method. The AE, TA, SA, PF and RY obtained with the RM method were higher than those obtained with the AS method by approx 2-3 folds. However, the Cp obtained with RM was lower (0.76) than that obtained with AS. This is likely because chymotrypsin purification with the RM method is more specific and transfers chymotrypsin from the crude extract into the RM capsule structure while the AS method precipitated other proteins in addition to chymotrypsin.
|AE (Unit/mL)||0.22 ± 0.01||0.48 ± 0.01|
|TA (Unit)||1.10 ± 0.05||2.39 ± 0.05|
|Cp (μg/mL)||41.64 ± 5.48||31.87 ± 1.31|
|SA (Unit/mg)||5.31 ± 0.19||14.98 ± 0.49|
|PF||11.08 ± 0.39||31.27 ± 1.02|
|RY (%)||46.72 ± 2.23||100.85 ± 2.23|
Table 6:, Effect of methods on chymotrypsin extraction parameters.
The effects of the forward extraction pH (6.0, 6.5, 7.0, 7.5, 7.0 and 8.5) and AOT concentration (1, 5, 10, 15, 20 and 35 mM) on the purification of chymotrypsin from red perch were studied. The results showed that TV decreased with increases in AOT concentration and increased with increases in pH. pH and AOT concentration had no effect on VR. Higher amount of surfactant formed a stable oil-water mixture structure at low temperature. The highest TV was achieved at pH 8.5 with AOT concentration of 1 mM. AE, Cp, SA, and RY all reached a max at pH 7.0 and AOT concentration of 20 mM. This maximum was due to the increased electrostatic interaction between the net charge on the surface of the protein molecule and the reverse micelles inner charge layer caused by increasing pH and the increased the stability of the reverse micelles structure caused by increased AOT concentration. The decrease in these parameters with pH > 7.0 and AOT concentration > 20 mM were due to the decreased net charge on protein molecules which weakened the electrostatic interaction between chymotrypsin and the reverse micelles and the high AOT concentration which increased the difficulties in the backward extraction step. The AE, TA, SA, PF and RY obtained with RM method were higher than those obtained with the AS method by 2.18, 2.17, 2.82, 2.82, and 2.16 folds, respectively. However, the Cp obtained with RM was lower (0.76) than that obtained with SA. This is because chymotrypsin purification with the RM method is more specific and transfers chymotrypsin from the crude extract into the RM capsule structure while the AS method precipitated other proteins in addition to chymotrypsin.
The project was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada through a strategic grant.