Three phase partitioning as a rapid and efficient method for purification of plant-esterase from wheat flour

Abstract Three-phase partitioning (TPP) was used to purify plant-esterase from wheat flour. Effect of various process parameters has been evaluated and plant-esterase was purified to 11.35-fold by optimized single step TPP system (50%, (w/v) (NH4)2SO4 saturation, 1:1 (v/v) ratio of crude extract: t-butanol at pH 4).The enzyme was found to be exclusively partitioned in the aqueous phase. Using TPP system, plant-esterase quickly purified to homogeneity with very high purity and activity. On the basis of single factor research, purification process was optimized by using response surface method, established a new type of high efficient purification plant-esterase method. To the best of our knowledge, this is the first report for purification and characterization of plant-esterase by using three phase partitioning (TPP). The results indicated that, TPP is a simple, quick, economical and very attractive process for purification of plant-esterase compared to conventional chromatographic protocols.


INTRODUCTION
Organophosphorus compounds (OPs) have become key components in current agricultural pesticides and herbicides. While these compounds have achieved enormous commercial success, they have also become major environmental pollutants. Due to their wide spectrum of insecticidal activity, organophosphorus pesticides account for 38% of the total pesticides applied in crop protection all over the world 1, 2 . In addition to agricultural applications, OPs have also found more sinister uses as nerve agents. In the event of a chemical weapon attack, it would be imperative to have a rapid on-site OP monitoring system in place.
As Acetyl cholinesterase(AchE) is a biorecognition element highly sensitive to the inhibition of organophosphates, carbamate pesticides, and nerve gases, AchE-based biosensors for OPs have been extensively investigated in past decades 3-7 .Nevertheless, both the high cost and lesser accessibility (extracted from animal blood or tissue) of AChE greatly limit its wide application. Recently, as an alternative of AChE, plant-esterase (EC 3.1.1.X) has received much attention because plant esterase and AChE share a similar sensitivity and inhibition mechanism with OP pesticides. Moreover, plant-esterase can be extracted from a number of plants such as wheat, soybean, rice, and sorghum at low cost and with easy obtainment 8-10 , thus holding great potential in OPs detection.
In our previous study, we developed a much faster and simpler optical detection protocol based on spectrophotometric changes of the H 2 TPPS 1 -plant-esterase complex in both liquid and solid state 11 . Recently, we developed a new cost-effective, simple, sensitive, reliable PLaE--CS/ AuNPs-GNs composite-based biosensor holds great potential in OPs detection for food and environmental safety 12 . However, the commercial-scale production of OPs sensors based on plant-esterase would be hampered by the requirement for large quantities of highly purifi ed enzyme with appropriate properties.
Various researchers have developed several methods for the purifi cation of plant-esterase 10, 13, 14 . However, almost all these methods involve a large number of steps. In our previous study, purifi cation of plant-esterase from fl our in an aqueous two-phase system (ATPS) was developed 8 . It is known that the higher the number of steps, the higher the loss of product yield. Furthermore, the scale-up of these methods is diffi cult and expensive. It may be noted that 50-90% of the production cost is due to the purifi cation steps 15 . Hence, effi cient and economical large-scale bioseparation methods are needed, to provide high purity, high yield plant-esterase that is biologically activity. One purifi cation method that meets all these criteria is Three-phase partitioning (TPP).
TPP is a simple and effi cient method for the separation and enrichment of protein compounds such as enzymes from complex mixtures. It consists in the sequential addition of a suffi cient amount of salt (typically ammonium sulphate) and an organic solvent (mainly t-butanol) in the crude extract and after agitation and decantation, the mixture separates into three distinct phases: a t-butanol rich layer and an aqueous layer which are formed above and under the precipitated protein layer 16 . Pigments, lipids and inhibitors contained in crude extract are generally concentrated in the top phase, whereas polar compounds such as saccharides are enriched in the lower aqueous phase 17 . The third phase is formed by a protein-enriched intermediate layer and generally, an increase of purification fold in this intermediate layer can be attained 18 . During the TPP process, activation of enzyme is also possible. This may be due to the phenomenon that the increased fl exibility of enzyme can lead to higher catalytic activity in TPP process 19, 20 . A remarkable increase in the catalytic activity and yield of fi cin 19 and invertase 21 obtained from TPP has been reported. Sulfate ion and t-butanol are known to be excellent protein structure markers or kosmotropes 18 . Kosmotropes, inversely to chaotropic agents, cause water molecules to favorably interact, which also stabilizes intramolecular interactions in the proteins 22 .

Chemicals
Wheat fl our was purchased from the local market (Chongqing, China). Bovine serum albumin (BSA) used for calibration of protein estimation was purchased from Sigma (St. Louis, MO, USA). 1-Naphthyl acetate (1-NA) used for calibration of Plant-esterase activity was purchased from YuanJu Biotechnology (Shanghai, China). Sodium dodecyl sulfate (SDS), Coomassie Blue R-250 and N,N,N',N'-tetramethyl ethylene diamine (TEMED) were purchased from Bio-Rad Laboratories (Hercules, CA,USA). Fast Blue B salt were purchased from YuanJu Biotechnology (Shanghai, China). Other analytical grade chemicals were purchased from Beibei Chemical Reagent Factory (Chongqing, China). Water used for preparation of aqueous solutions was from a Millipore Direct-Q Water system (resistivity, 18.2 MΩ cm).

Preparation of crude extract
A 1:5 (w/v) solution of fl our in distilled water was made. The mixture was incubated at 35 o C for 30 min in a water bath, then centrifuged at 8000 rpm for 10 min at 4 o C. The clear supernatant was collected and was kept frozen at -20 o C for future use.

Purifi cation of plant-esterase by three-phase partitioning (TPP)
TPP experiments were carried out as described by Dennison 18 with slight modifi cations. Briefl y, the crude plant-esterase enzyme extract (5 ml containing 67.47 units and 33.83 mg protein) was saturated at room temperature with 20% ammonium sulfate, followed by addition of a half volume of t-butanol. The mixture was gently vortexed and then allowed to stand at 35 o C for 40 min in a water bath. Afterwards, the mixture was centrifuged at 4500 rpm for 10 min at 4 o C to facilitate the separation of the three phases. The upper t-butanol phase was removed by a Pasteur pipette. The lower aqueous phase and the interfacial phase were separated carefully and analyzed for enzyme activity and protein content. The interfacial precipitate was dissolved in 0.04 M, pH 8.0 sodium phosphate buffer.
The temperature of 4 o C, 15 o C, 25 o C, 35 o C, 45 o C, 55 o C were chosen to study its effects on partitioning of plant--esterase. The rest parameters were kept constant such as pH 7, ammonium sulfate 50% and crude extract to t-butanol ration of 1:1.
After that, effects with different pH values of medium study were tested. Crude extract was saturated with 50% ammonium sulfate and pH was adjusted to 3, 4, 5, 6, 7, 8 and 9, then 1:1 t-butanol was added and the incubation temperature was 35 o C.
After optimization of the parameters, three repetitions were conducted to confi rm the overall results using 50% ammonium sulfate, 1:1 ratio crude extract to t-butanol and a pH of 4.0 and the incubation temperature was 35 o C. The interfacial phase always containing the higher plant-esterase activity was collected, dissolved in 0.04 M, pH 8.0 sodium phosphate buffer, dialyzed overnight against the same buffer and concentrated by ultrafi ltration using Amicon Ultra device witha 10 kDa MWCO. The concentrated enzyme was stored at -20 o C until use for further characterization studies in order to determine the general biochemical properties.

Enzyme activity assay
Plant-esterase activity was determined by the colorimetric method of VanAsperen 23 . The general buffer was 0.04 M sodium phosphate, pH 6.5. 1-Naphthylacetate (1-NA) (16 mM) was used as substrate. The mixture consisting of phosphatebuffer (1.95 ml), enzyme solution (0.5 ml) and 1-NA (50 μl) was incubated at 35 o C for 15 min in a water bath. The hydrolysis of 1-NA was terminated by adding 0.5 ml of Fast Blue B salt-SDS solution. The absorbance at 595 nm was measured by spectrophotometer (PerkinElmer Instruments, USA) after 10 min.

Protein determination
The total amount of soluble proteins present in the sample was measured by the modifi ed Bradford dye-binding assay 24 . Interference from phase-forming components was eliminated by using identical phase systems without sample as a blank in the spectrophotometric assay.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SDS-PAGE of the protein samples with 10% gel was performed according to the method of Laemmli 25 with slight modifi cation. For protein staining, the gel was stained with Coomassie Brilliant Blue R-250 for 1 h and then destained with 50% (v/v) methanol and 12% (v/v) acetic acid for 2-3 h.

Optimization of PlaE purifi cation process by response surface method
TPP process was optimized using response surface methodology. For this purpose, four factors, namely ammonium sulphate saturation(w/v%), temperature, pH and crude extract/t-butanol(v/v) were selected, Purifi cation fold as a response value. 29

Eff ect of ammonium sulfate saturation on Plant-esterase
Salting out can be used to separate proteins based on their solubility in the presence of high salt concentration. Ammonium sulfate is the traditional kosmotropic salt used for protein salting out because of its high solubi-50% (NH 4 ) 2 SO 4 concentration, it was selected as optimum value or next set of experiments. This salt concentration was suffi cient enough to concentrate the plant-esterase, a fi nding in agreement to its known property to precipitate at 50-60% (NH 4 ) 2 SO 4 saturation 31, 32 .

Eff ect of crude extract to t-butanol ratio on plant-esterase partitioning
After the selection of (NH 4 ) 2 SO 4 saturation, the effect of crude extract to t-butanol ratio for plant-esterase partitioning in the TPP system was investigated. t-Butanol has been selected here due to its several advantages over other solvents and from the reference of earlier published reports on TPP 18, 19, 33, 34 . Due to its size and branched structure, t-butanol does not easily permeate inside the folded protein molecules and hence does not cause denaturation. It also shows signifi cant kosmotropic and crowding effects that enhances the partitioning of enzyme 26 . Different crude extract: t-butanol ratio effect for the partitioning of plant-esterase at the constant saturation of (NH 4 ) 2 SO 4 (50%, w/v), at 35 o C is shown in Fig. 2. It can be seen from Fig. 2 that the highest purity was obtained at the ratio of 1:1 and purity fold decreased with subsequent increase in crude to t-butanol ratio. At 1:0.5 and 1:0.75 ratios, there is less purifi cation fold which may be due to smaller amount of t-butanol does not adequately synergize with (NH 4 ) 2 SO 4 18, 26 . Similar results have been reported by Ozer et al. who used the TPP for the purifi cation of invertase from tomato 17 . If it is higher than 1:1, it is likely to cause protein denaturation as reported in many earlier studies 19, 35 . Otherwise, many previous studies and in agreement to our fi ndings, a ratio of 1:1, crude extract to t-butanol was reported to give maximum partitioning 17, 26, 28 Thus, further experiments were carried out at 1:1 ratio of feed to t-butanol.
Additionally, NH 4 + and SO 4 2-are at the ends of their respective Hofmeister series and have been shown to stabilize protein structure. Ammonium sulfate concentration plays a major role in TPP as it is responsible for protein-protein interaction and precipitation. It causes protein precipitation by salting out mechanism. Protein solubility is infl uenced by the ionic strength of the solution which subsequently depends on salt concentration 27 . On addition of suffi cient salt proteins precipitation occur, this is known as salting out effect. At higher salt concentration, water molecules are attracted by salt ions result in stronger protein-protein interactions and the protein molecules coagulate through hydrophobic interactions 28 . Generally, in TPP process, the researchers start minimum salt concentration of 20% (w/v) to optimize the partitioning conditions 18 .
Herein, the experiment was carried out by keeping the experimental parameters as follows: ammonium sulfate loading 20% (w/v), crude extract: t-butanol ratio 1.0:0.5, temperature 35 o C and pH 7. Ammonium sulfate concentration was varied from 20% to 70% w/v of the crude extract concentration. At lower concentration (20 and 40%) ammonium sulfate may not able to alter the hydrophobic surface of plant-esterase. Similar trend has been observed in the extraction of peroxidase 29 . Fig. 1. reveals that with an increase in (NH 4 ) 2 SO 4 concentration, purity of plant-esterase is signifi cantly increased up to 6.42-fold. Further increase in (NH 4 ) 2 SO 4 concentration reduces fold purity. Higher salt concentrations leads to the reduction of purifi cation fold, which may be due to the irreversible denaturation of protein as reported in earlier studies 18, 30 . As maximum purity was achieved at  Eff ect of temperature on plant-esterase partitioning Temperature is an important parameter that affects the enzyme confi guration and overall stability. It is well known that the reaction rate of enzymes increases with increasing temperature, but after a certain temperature activity, it decreases due to denaturation 17, 36, 37 . As TPP operates through multiple effects, which includes conformational tightening and changes in protein hydration, it was considered worthwhile to explore the effect of varying process temperature. Most concentration processes are usually carried out at low temperatures. The use of low temperatures in solvent or salt precipitation dissipates the heat generated, ensuring minimal protein denaturation. The requirement of low temperatures in TPP has not been clearly reported in the literature, however Dogan and Tari have observed the improved yield and purifi cation of exo-polygalacturonase using TPP at 25 o C rather than 37 o C 38 . We investigated the effect of temperature from 4 to 55 o C while keeping all other parameters at a constant value such as, 50% (NH 4 ) 2 SO 4 saturation concentration, pH 7, broth to t-butanol ratio 1:1 (v/v). Fig. 3 illustrated the effect of temperature on partitioning behavior of plant-esterase in TPP. Purity of enzyme was increased with an increase in the temperature. At 35 o C temperature, purity was found 9.07-fold. Decrease in purifi cation fold at higher temperature (above 35 o C) may be due to thermal deactivation of enzyme. Probable reason behind this may be , as already explained, at 4-

Eff ect of pH on plant-esterase partitioning
One of the other important parameter effecting to the enzyme activity is pH. Protein concentration by salting out depends on the sulphate concentration and pH-dependent net charge of the proteins. Electrostatic forces and binding of sulphate anions to cationic protein molecules, which promote macromolecular contraction and conformational shrinkage, are the main causes of the strong sulphate pH dependency in salting out. Proteins tend to precipitate most readily at their pI (isoelectric point). Below the pI, proteins are positively charged and can be quantitatively precipitated out by TPP.
On the other hand, negatively charged proteins are more soluble and not easily precipitated. Reports of invertase 17 , laccase 34 precipitating selectively at its pI and leaving most of the contaminant proteins in the aqueous phase, confi rms the relevance of pH as an important parameter. Fig. 4. shows the effect of pH on the partition behaviour of plant-esterase during TPP. The purifi cation fold of plant-esterase increases when the pH is increased from 3 to 4. With further increase in pH, the purifi cation fold falls down again. The purifi cation fold of 11.35 at pH 4 could be optimum value for TPP purifi cation due to the better conformational stability of plant-esterase towards t-butanol at this pH.

Establishment of regression equation and signifi cance checking for response surface design
Experimental design scheme and results such as Table 1. The results showed that the purifi cation ratio of PlaE was between 4.95515 and 11.5491. Using Design--Expert 8 software to analyze the data in Table 1

Response surface analysis and optimization
To intuitively illustrate the main factors that affect the response value and the interaction between factors, the software draws a three-dimensional response surface map and a two-dimensional contour map based on the experimental data, as shown in Fig. 5 and Fig. 6. The response surface diagram and contour map refl ect the infl uence of other factors on the response value when two factors remain at 0 level. For example, the response surface diagram Fig. 5 AB and the contour map Fig. 6 AB refl ect the interaction between the reaction of pH (C) and the temperature (D) in 0 water, and the reaction between the saturation of ammonium sulfate (A) and the ratio of material to liquid (B). Fig. 5AB data showed that the extraction rate increased signifi cantly with the increase of ammonium sulfate saturation (A); With the increase of material liquid ratio (B), the extraction rate increased signifi cantly. The curve slope of the former is obviously greater than that of the latter, indicating that the effect of ammonium sulfate saturation (A) on the purifi cation multiple of PLaE is greater than that of the liquid ratio (B) on the PLaE purifi cation multiplier, of which ammonium sulphate saturation (A) is at 0.8 level, the corresponding ammonium sulfate saturation is 58%, the ratio of material to liquid (B) is 0.5, and the corresponding ratio of liquid to liquid is 1:1.125, its PLaE purifi cation ratio reached the maximum, reaching 10.7390. Similarly, the best conditions of the other 5 response surfaces can be obtained. The results are shown in Table 3.
The contour map can directly determine the infl uence of the interaction of factors on the response value. The circular indicates that the interaction is not signifi cant, and the ellipse indicates signifi cant interaction. From Fig. 7, we can see that the interaction between A and B and the interaction between A and C have a signifi cant impact on response value.  Verifi cation of the best process By solving the equation by Design-Expert 8.0 software, the optimum purifi cation conditions are as follows: the saturation level of ammonium sulfate is 1, the corresponding saturation is 60%, the ratio of material to liquid is 0.8, the corresponding ratio of material and liquid is 1:1.2, the level of pH is -0.75, the corresponding pH is 3.25, the corresponding temperature is 0, the corresponding value is 35, and the corresponding PLaE purifi cation times The maximum value of the number theory is 11.9323. The experiment was repeated 3 times according to the optimum conditions, and the actual value was 12.0326. The results showed that the model was accurate and reliable, and could be used for the purifi cation process of plant esterase.

SDS-PAGE ANALYSIS
As shown in the SDS-PAGE gel analysis (Fig. 7A), TPP system can signifi cantly improve the purity of the as-prepared plant-esterase (lane 1 vs lane 2). The molecular weight of the purifi ed plant esterase was measured to be about 68 kDa, which is in good agreement with the reported value 8, 12 . Fig. 7B shows the purifi ed plant--esterase in white powder after freeze-drying.

CONCLUSIONS
To the best of our knowledge, this is the fi rst report for purifi cation and characterization of plant-esterase by using three phase partitioning (TPP). In comparison to chromatographic methods, TPP is very cheap, simple and effi cient method. Effect of various process parameters has been evaluated and plant-esterase was purifi ed to 11.35-fold by optimized single step TPP system (50%, (w/v) (NH 4 ) 2 SO 4 saturation, 1.0:1.0 (v/v) ratio of crude extract: t-butanol at pH 4).The enzyme was found to be exclusively partitioned in the aqueous phase. Using TPP system, plant-esterase quickly purifi ed to homogeneity with very high purity and activity, showing an effective activity compared to the classical purifi cation protocols. It can be concluded that, as fast elegant non-chromatographic separation technique, TPP could be attractive for the purifi cation of plant-esterase.

Acknowledgment
The present study was supported by The Sichuan Provincial Education Offi ce Project(17ZA0267),Talent introduction project of Sichuan University of Science and Engineering(2018RCL24) and Key Laboratory of fi ne chemicals and surfactant in Colleges and universities of Sichuan Province(2018JXY05), Sichuan Science and Technology Department Project (2019YJ0463), Innovation and entrepreneurship training program for College Students(S201910622037&cx2019050).
Confl ict of Interest: The authors declare that they have no confl ict of interest.