Extraction and purification of beta-amylase from stems of Abrus precatorius by three phase partitioning

Food Chemistry 183 (2015) 144–153

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Extraction and purification of beta-amylase from stems of Abrus precatorius by three phase partitioning Sorel Tchewonpi Sagu a, Emmanuel Jong Nso a, Thomas Homann b, César Kapseu a, Harshadrai M. Rawel b,⇑ a b

Department of Process Engineering, National School of Agro-Industrial Sciences (ENSAI), University of Ngaoundere, P.O. Box 455, Adamaoua, Cameroon Instrumental Analysis in Nutritional Science, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany

a r t i c l e

i n f o

Article history: Received 15 September 2014 Received in revised form 24 February 2015 Accepted 10 March 2015 Available online 20 March 2015 Keywords: Purification Beta-amylase Abrus precatorius Three phase partitioning Doehlert design

a b s t r a c t The stems of Abrus precatorius were used to extract a beta-amylase enriched fraction. A three phase partitioning method and a Doehlert design with 3 variables (ratio of crude extract/t-butanol, the ammonium sulphate saturation and pH) were used. The data was fitted in a second-order polynomial model and the parameters were optimized to enrich beta-amylase. Experimental responses for the modulation were recovery of activity and the purification factor. The optimal conditions were: a ratio of crude extract/t-butanol of 0.87 (v/v), saturation in ammonium sulphate of 49.46% (w/v) and a pH of 5.2. An activity recovery of 156.2% and a purification factor of 10.17 were found. The enriched enzyme was identified as a beta-amylase and its molecular weight was 60.1 kDa. Km and Vmax values were 79.37 mg/ml and 5.13 U/ml, respectively and the highest activity was registered at a temperature of 70 °C and a pH between 6 and 6.5. A significant stabilization of the beta-amylase was observed up to 65 °C. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Amylases are important industrial enzymes used in starch liquefaction to produce glucose, fructose and maltose and also in brewing, baking, textile, paper, detergent and sugar industries (Noman, Hoque, Sen, & Karim, 2006). They represent about 25–33% of the world enzyme market (Nguyen, Rezessy-Szabó, Claeyssens, Stals, & Hoschke, 2002). Amylases are represented in plants, animal tissues and microorganisms. Although amylase are widely used in the food and chemical industries, the production of these enzymes on a large scale is only restricted to certain microorganisms underlining the necessity to find alternative sources for their production (Amid & Abd Manap, 2014). In this context, cereals have also been exploited for the isolation of potential enzymes (Muralikrishna & Nirmala, 2005). Even if the plant tissues are cumbersome to handle, vegetable samples such as stems and leaves have additionally served as substrate for extraction of some enzymes like papain and bromelain (Esti, Benucci, Lombardelli, Liburdi, & Garzillo, 2013). Amylases from higher plants other than cereals were less well characterized although amylases have been purified from leaf and seeds of some species (Witt & Sauter, 1996). A review of the literature on plant amylases

⇑ Corresponding author. Tel.: +49 (03 32 00) 88 5525; fax: +49 (03 32 00) 88 5582. E-mail address: [email protected] (H.M. Rawel). http://dx.doi.org/10.1016/j.foodchem.2015.03.028 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

suggests that enzymes from higher plants still lack the necessary research attention. Under these premises, the stems of Abrus precatorius have been identified as potentially rich in amylolytic enzymes. A. precatorius (known commonly as jequirity or rosary pea) is a creeper plant of the family of Fabaceae, originating from Indonesia and mainly found in subtropical area (Tripathi & Maiti, 2003). Crude extracts of A. precatorius are used since decades in Northern Cameroon by the local populations to sweeten gruels made from millet. These crude extracts of A. precatorius are also used in process of a traditional beer production called bili bili using sorghum. In this case, extracts are used to facilitate the mashing procedure and to extract the fermentable sugars. These observations suggested us that the crude extracts of stems of A. precatorius could contain amylases and could be therefore use as a potential new source for amylase production. Three-phase partitioning (TPP) is a simple and efficient method for the separation and enrichment of protein compounds such as enzymes from complex mixtures. It consists in the sequential addition of a sufficient 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 (Dennison & Lovrien, 1997). 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

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145

Nomenclature AAD ANOVA APS AR ELSD HPLC k i, j Km N MMP p PF R2 RSM SDS–PAE TEMED

average absolute deviation analysis of variance ammonium persulfate activity recovery evaporative light-scattering detection high-performance liquid chromatography number of variables independent variables Michaelis constant number of experiments Matrix metalloproteinase probability level purification factor coefficient of determination response surface methodology sodium dodecyl sulfate polyacrylamide gel electrophoresis N,N,N0 ,N0 -tetramethylethylenediamine

lower aqueous phase (Özer, Akardere, Çelem, & Önal, 2010). The third phase is formed by a protein-enriched intermediate layer and generally, an increase of enzymatic activity in this intermediate layer can be attained (Dennison & Lovrien, 1997). TPP has also been used in one step for concentration and purification of several proteins and enzymes e.g. invertase from tomato (Özer et al., 2010), glucoamylase from Aspergillus niger and pullulanase from Bacillus acidopullulyticus (Mondal, Sharma, & Gupta, 2003), proteins secreted from Corynebacterium pseudotuberculosis (Paule et al., 2004), aryl alcohol oxidase from Pleurotus ostreatus (Kumar & Rapheal, 2011), catalase from sweet potato tubers (Duman & Kaya, 2013). From these studies, it was observed in each case that an increase over 100% of enzymatic activity in the medial layer containing the precipitated enzyme could be achieved. In this short survey no mention on the use of this technique for precipitation of amylases has been reported. It is in this context that the present study was undertaken to extract and purify/enrich amylases from a crude extract of stems of A. precatorius using a TPP process. The response surface methodology (RSM) with Doehlert design was used in order to model and optimize three parameters of the enrichment process. The highperformance liquid chromatography (HPLC) was used for identification of the amylase activity and the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) was used to determine its relative molecular weight. The effects of temperature, pH and selected chemicals were evaluated and the kinetic constants were defined. 2. Materials and methods 2.1. Materials The stems of A. precatorius were harvested in Ngaoundere (Adamawa, region in Northern Cameroon). Stems were sorted, cleaned, cut and dried at 45 °C during 3 days in a ventilated electric dryer CKA-2000 type. They were then ground, sieved to obtain a powder with a particle size less than 1 mm, sealed in plastic packages and then stored at 20 °C. D-Maltose used for calibration of amylase activity was obtained from Serva (Heidelberg, Germany) and Bovine serum albumin (BSA) used for calibration of protein estimation was procured from Merck KG&A (Darmstadt, Germany). Potato starch used was from Sigma Aldrich

TPP U v Vmax w xi X Y Y i;cal Y i;exp

three-phase partitioning enzymatic units volume, l maximum velocity weight, g coded variables given par the Doehlert table real variables response variable the calculated response the experimental response

Greek symbols bij coefficient of the interactions terms bii coefficient of the quadratic terms bi coefficient of the linear terms b0 constant term

(Steinheim, Germany). All the other chemicals and HPLC solvents were reagent-grade or gradient-grade, respectively.

2.2. Extraction and purification of beta amylase 2.2.1. Extraction For the completion of extraction, 5 g of powdered A. precatorius stems were weighed into centrifuge tubes of 45 ml, extraction solvent (35 ml) were added and the mixture was stirred for 2 h at 4 °C. The whole mixture was subsequently centrifuged at 4000g for 10 min at 4 °C. Supernatant was cloth filtered to remove any precipitated particles. Filtrate was kept at 20 °C for 2 h, then thawed in a refrigerator at 4 °C and centrifuged at 6000g for 10 min. Supernatant constituting the crude extract of amylase was collected and aliquots were stored at 20 °C for evaluation of the amylase activity and protein concentration. A preliminary study was performed with three solvents, namely 50 mM phosphate buffered saline (PBS buffer) pH 7.4, 200 mM phosphate buffer pH 7.4 and distilled water. Distilled water gave the best results and was therefore used as the final extraction solvent in this work.

2.2.2. Purification of beta-amylase by three-phase partitioning (TPP) Amounts of ammonium sulphate given by the experimental design were weighed and introduced into the 15 ml tubes. 5 ml of crude extract were added and the whole was mixed until dissolution of salt. The pH of system was adjusted by addition of either HCl or NaOH depending on the initial and final pH and after the addition of t-butanol (according to the proportion given by the experimental design) followed by homogenisation; the tubes were kept for 1 h at 4 °C for separation of different phases. In order to allow a good separation of phases, tubes were centrifuged at 4000g, 4 °C for 5 min. The upper phase consisting of the organic solvent was carefully removed using a micropipette, followed by the lower aqueous phase. The precipitate thus recovered was dissolved in 2 ml of distilled water and stored at 20 °C for 2 h. Tubes were thawed at 4 °C, centrifuged at 10,000g, 4 °C for 10 min and supernatant constituting the enriched amylase was collected and stored at 20 °C. Various analyses were performed to assess the purified amylase activity as well as the efficiency of the purification process.

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2.3. Doehlert design for optimisation of TPP parameters

The specific activity is defined as the enzymatic unit per mg protein in the enzyme preparation (U/mg protein).

TPP process was optimized using Doehlert design and response surface methodology. For this purpose, three factors, namely ratio of crude extract/t-butanol (v/v), ammonium sulphate saturation (% w/v) and pH were selected. Ranges of variation of these factors were respectively: ratio of crude extract/t-butanol (X1): 1:0.5 (2) to 1:2 (0.5); ammonium sulphate saturation (X2): 20–60% (w/v) and pH (X3): 4–9. Doehlert design allows description of a region around the optimal response and contains k2 + k + 1 experiments, with k describing the number of factors. For three variables, a set of 13 experiments were generated and 4 replications at central point were performed. Coded values given by Doehlert design and corresponding real values for each experiment are presented in Table 1. Each of the 17 experiments was performed in duplicate. Mathematical models describing the effect of each factor, in term of their linear, quadratic and interaction effects were described by a second-order polynomial equation as presented in Eq. (1).

Y ¼ b0 þ

X

bi X i þ

X

bii X 2i þ

X

bij X i X j

ð1Þ

where, Y is the experimental response, Xi and Xj are the levels of variables, b0 is a constant term, bi and bj are the coefficients of the linear terms, bii are the coefficients of the quadratic terms, bij are the coefficients of the interactions terms and i or j represent the independent variables. The response surfaces were represented with model equations. For analysis and interpretation of the results, multiple regression analysis based on the least square method was performed using STATGRAPHICS Centurion software (Version XVI). Significance of the effects was checked by analysis of the variance (ANOVA) and using p-value significance levels (p < 0.05; p < 0.01 and p < 0.001). The two experimental responses subjected to optimization were the activity recovery and the purification factor. The activity recovery (AR) was determined as the ratio of total activity of purified amylase to that in crude extract and expressed as percentage:

AR ð%Þ ¼ ðtotal activity of the purified enzyme= total activity of crude extractÞ  100

ð2Þ

The purification factor (PF) was determined as the ratio of the specific activity of the purified beta-amylase to the specific activity of the crude extract:

PF ¼ ðspecific activity of the purified enzyme= specific activity of crude extractÞ  100

ð3Þ

2.4. Dialysis A fraction of the purified beta-amylase obtained at the optimal conditions was transferred into the molecular porous membrane tubing of 6–8000 molecular weight cut-off and immersed in distilled water for 24 h of dialysis at 4 °C. Distilled water was continuously stirred using a magnetic stirrer throughout the process and was changed regularly. At the end of process, the solution of beta-amylase was collected and stored at 20 °C for the lyophilisation process. An aliquot of the dialysed sample was also stored for further analyses. 2.5. Lyophilisation Samples of beta-amylase were lyophilized to obtain powders in order to facilitate packaging and storage. To this end, dialysed beta-amylase was collected in suitable flasks, stored at 20 °C for 2 h and then introduced in freeze drying systems Alpha 1–4 (Martin Christ, Germany) for 20 h. Powders obtained were stored at 20 °C for further analyses. 2.6. Analyses 2.6.1. Protein contains Protein concentration was measured by the method of (Lowry, Rosebrough, Farr, & Randall, 1951) using bovine serum albumin as standard. 2.6.2. Amylase activity by DNS method Amylase activity was measured by the method of (Bernfeld, 1955) with some modification. Briefly, 200 ll of appropriately diluted amylase containing sample were added to 200 ll of substrate (1% w/v starch solution) and then incubated at 50 °C for 5 min. The reaction was stopped by adding 200 ll of 3,5-dinitrosalicylic acid (DNS solution) and the mixture was heated at 100 °C for 5 min for colour development. After cooling at room temperature, 900 ll of distilled water were added and the absorbance was read at 540 nm against a blank prepared under the same conditions using a spectrophotometer (Pharmacia Biotech, Novaspec II, Massachusetts, USA). An enzymatic extract used as a negative control was previously denatured at 100 °C for 15 min and then analysed under the same conditions. The corresponding

Table 1 Doehlert design: coded variables, real variables and experimental responses of purification of beta-amylase from Abrus precatorius by TTP using ammonium sulfate and t-butanol. Exp. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Coded variables

Real variables

Experimental responses

x1

x2

x3

X1 (v/v)

X2 (% w/v)

X3

Activity recovery

Purification factor

0.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.00 0.50 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.87 0.87 0.87 0.87 0.29 0.29 0.29 0.58 0.29 0.58 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.82 0.82 0.82 0.82 0.82 0.82 0.00 0.00 0.00 0.00

1.25 2.00 0.50 1.63 0.88 1.63 0.88 1.63 0.88 1.63 1.25 0.88 1.25 1.25 1.25 1.25 1.25

40.0 40.0 40.0 60.0 20.0 20.0 60.0 46.7 33.3 33.3 53.3 46.7 26.7 40.0 40.0 40.0 40.0

6.5 6.5 6.5 6.5 6.5 6.5 6.5 9.0 4.0 4.0 4.0 9.0 9.0 6.5 6.5 6.5 6.5

156.9 ± 4.3 149.4 ± 4.4 178.8 ± 1.4 163.6 ± 5.5 32.2 ± 0.9 21.9 ± 3.1 142.2 ± 3.3 132.8 ± 4.8 124.4 ± 3.1 78.0 ± 1.6 156.2 ± 1.2 142.7 ± 4.6 59.9 ± 3.6 157.8 ± 6.2 158.6 ± 4.5 158.9 ± 2.6 161.7 ± 4.5

8.61 ± 0.04 6.43 ± 0.03 8.38 ± 0.03 6.61 ± 0.03 1.51 ± 0.02 0.83 ± 0.00 7.78 ± 0.04 7.04 ± 0.03 10.62 ± 0.04 8.27 ± 0.05 10.22 ± 0.04 5.90 ± 0.03 3.52 ± 0.01 8.38 ± 0.06 7.93 ± 0.03 7.70 ± 0.04 8.24 ± 0.07

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concentration of reducing sugars was determined with a standard curve using maltose (main product of amylase activity). The enzymatic activity was expressed as the International Unit per ml (U/ml) corresponding to one mole of maltose released per minute and per ml at 50 °C. This method was used for analysis of the enzymatic activity of the crude extract and purified samples during the whole optimization process. 2.6.3. Amylase activity by colorimetric method A colorimetric method was also used as a second method to confirm the beta-amylase activity. This method is based on the breakdown of starch by amylase. 200 ll of substrate (40 mg of soluble starch according to Zulkowsky GR, 0.85 g NaCl, 0.86 g benzoic acid, 2.7 g disodium phosphate in 100 ml distilled water, pH 7) were introduced in Eppendorf tubes and pre-incubated at 50 °C for 1–2 min. 40 ll of amylase sample were introduced and incubated for 7.5 min; 200 ll of colour reagent (0.8 g of KI, 63.75 mg of KIO3, 1 ml HCl 37% and 24 ml distilled water) were added and the volume was filled up to 2 ml with distilled water. The blank was prepared under the same conditions, but without amylase solution. After mixing, tubes were kept 15 min at room temperature and absorbance was read at 590 nm (the zero was set with distilled water). Amylase activity was expressed in U/dl and calculated as in Eq. (4):

Activity ðU=dlÞ ¼ ½ðabsorbance ðblankÞ  absorbance ðassyÞÞ= absorbance ðblankÞ  1:00

ð4Þ

2.6.4. Determination of protease activity by Azocasein method To detect the presence of protease activity in the crude extract sample and in the amylase enriched fraction, a modified method using azocasein presented by (Garcia de Fernando & Fox, 1991) was used. 200 ll of azocasein solution 2% (w/v) and 300 ll of a buffer solution of sodium bicarbonate 0.5% (w/v), pH 8.0 were added to 100 ll of samples. The mixture was incubated at room temperature for 20 h, and then reaction was stopped by adding 500 ll of 20% (w/v) trichloroacetic acid. The mixture was well mixed, incubated at room temperature for 5 min and then centrifuged 5 min at 10,000g. 500 ll of supernatant were taken and added to 1000 ll of 1 M NaOH, and absorbance was read at 440 nm. One unit of activity was defined as the amount of activity that gives a change of one unit of absorbance at 440 nm/min/ml of enzyme preparation. 2.7. Characterisation

147

2.7.2. HPLC analysis The detection and characterisation of beta-amylase activity was performed by analysing the products of the enzymatic reaction with a Shimadzu HPLC system (Shimadzu Europa GmbH, Duisburg, Germany) equipped with an evaporative light-scattering detection (details are provided in the supplementary data, Fig. S2). 2.7.3. Effect of temperature and pH on activity and stability The optimum pH of purified beta-amylase was studied over a pH range of 2.0–10.0 at 50 °C using 1% (w/v) potato starch solution as substrate. The effect of pH on enzyme stability was evaluated by measuring the residual activity after a pre-incubation time of 4 h at 4 °C and at various pH (2.0–10.0) using 200 mM phosphate buffer. Residual activity was expressed in percent and was determined under standard assay conditions. To investigate the effect of temperature, purified beta-amylase activity was assessed at different temperatures (20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 and 100 °C) with distilled water using 1% (w/v) potato starch solution as substrate. For thermal stability, residual activity of enzyme was determined after a pre-incubation time of 1 h at different temperatures (20–100 °C). Enzymes solutions were tested at the optimal conditions and residual beta-amylase activities were expressed in percent. 2.7.4. Kinetic constants The Michaelis constant (Km) and maximum velocity (Vmax) were determined for purified beta-amylase by applying the Lineweaver– Burk plot. Different concentrations of starch from 1 to 20 mg/ml were used and activity was assessed at 50 °C and at pH 6.0. Apparent values of Km and Vmax were calculated by the equation of linear regression of Lineweaver–Burk plot. 2.7.5. Effect of different compounds on activity of purified betaamylase The effects of various metal ions and chemicals on the partly purified beta-amylase activity were investigated using KCl, NaCl, MgCl2, CuSO4, CaCl2, FeCl3, ZnSO4, AgNO3, citric acid, Tannic acid, salicylic acid, Boric acid, lactic acid and acetic acid (at concentrations of 1, 5 and 10 mM). Enzyme preparations were pre-incubated with metals ions and chemicals for 30 min at 4 °C and the residue beta-amylase activity was determined under the standard assay conditions. The activity of beta-amylase assayed without metals ions and chemicals was considered as 100%. 2.8. Statistical analysis

2.7.1. Electrophoresis 2.7.1.1. SDS–PAGE. SDS–Page (T = 14–18%) according to Laemmli (1970) was adapted for determination of the molecular weight of the proteins. Samples were diluted with a ratio of 1:1 in sample buffer and were heated at 90 °C for 10 min. Sample buffer contained 0.05 M Tris–HCl buffer pH 6.8 containing 4 g sodium dodecyl sulphate, 12 g glycerol, 5 g 2-mercaptoethanol and 0.01 g Coomassie Brilliant blue R 250. Low molecular weight calibration kit for SDS Electrophoresis (GE Healthcare Europe GmbH, Freiburg, Germany) was applied. After separation, the gel was stained overnight at room temperature with Coomassie blue solution (in 10% acetic acid) and then de-stained with 10% acetic acid for 2–3 h. The band intensity and the relative molecular mass of amylase were estimated using scanning utilities and gel image analysis software (Image Lab Software, Bio Rad laboratories, Germany).

where Yi,exp is the experimental response, Yi,cal is the response calculated using the model equation, and N is the total number of experiments. The response model with a lowest AAD and a highest R2 (close to 1.0) was validated as a proper model equation for expression of the response.

2.7.1.2. MMP Zymogen gel assay. The basic protocol for this SDS– PAGE procedure is essentially the same as described previously (details are given in the supplementary data, Fig. S1).

2.8.2. Experimental measurements The results reported here are the averages of at least three measurements. Data are reported as mean value ± standard deviation.

2.8.1. Validation of model equations To validate the different model equations, average absolute deviation (AAD) and coefficient of determination (R2) values were determined. AAD was calculated as described by (Sagu, Nso, Karmakar, & De, 2014) by Eq. (5):

AAD ¼

N X ðjY i;exp  Y i;cal j=Y i;exp Þ=N

ð5Þ

i¼1

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3. Results and discussions A preliminary study was done to determine the most suitable solvent for extraction. For this purpose, three different buffers namely: 50 mM phosphate buffered saline pH 7.4 (PBS buffer), 200 mM phosphate buffer pH 7.4 and distilled water were used. DNS method was used to determine the amylase activity. The results showed a better activity of the crude extract of beta-amylase with distilled water (20.42 ± 1.61 U/ml) compared to phosphate buffer (15.26 ± 0.98 U/ml) and PBS buffer (16.85 ± 1.41 U/ ml). Based on these results, distilled water was selected as the appropriate extraction solvent. In the same way, the first step of purification process by TPP was to decide upon the most influential factors. Ratio of crude extract/t-butanol (X1), ammonium sulphate saturation (X2) and pH (X3) as factors, and activity recovery and purification factor as experimental responses were selected according to the recommended reports (Dennison & Lovrien, 1997). The ranges of variation of each factor were selected according to the results of a preliminary study (data not shown). Experimental and theoretical responses of the 17 experiments are presented in Table 1. It is observed that a variation of purification factor ranging from 0.8 ± 0.00 (experiment 6) to 10.6 ± 0.04 (experiment 9). This result indicates an increase in concentration of amylase during the three phases partitioning up to 10 times. It is also observed that a value of the activity recovery varied from 21.9 ± 3.1% (experiment 6) to 178.8 ± 1.4% (experiment 3). It is clear that excepting experiments 5, 6, 10 and 13, values of the relative activity of beta-amylase obtained during TPP were all greater than 100% (Table 1). This observation corroborates with the results obtained by Özer et al., 2010. In this work, invertase was purified from tomato using TPP method and values of the activity recovery of 190% were recorded. This increasing of the activity of beta-amylase may be a result of a greater structural flexibility of the enzyme molecule (Dennison & Lovrien, 1997). The removal of the surrounding matrix, wherein the proteins are formerly embedded in the plant material allows a better interaction of the enzyme with the substrate. In fact, during the TPP process, simultaneous use of ammonium sulphate and t-butanol also allows the enzyme to be released of many concomitant compound effects from pigments, lipids, enzyme inhibitors and polar compounds including polysaccharides. Ammonium sulphate coupled with an organic solvent is often protective of biological molecules and in this environment, enzymatic activity is thus increased (Dennison & Lovrien, 1997). 3.1. Effects of ratio of crude extract/t-butanol, ammonium sulphate saturation and pH on the experimental responses 3.1.1. Effects on the activity recovery Table 2 presents the regression coefficients of mathematical models for the activity recovery. It is observed that the ratio of crude extract/t-butanol (p < 0.05), the ammonium sulphate saturation X2 (p < 0.001), its second-order factor X 22 (p < 0.001) and the secondorder factor of pH (X 23 ) (p < 0.01) had a significant effect on the ratio of activity. In Fig. 1a, it can be observed that the effect of ammonium sulphate saturation was positive, activity recovery varying from 43.6% (for a value of ammonium sulphate at 20% w/v) to reach a value of 170.7% when the salt amounted to 47% w/v. In the Fig. 1b, it can be noted that when the ratio of crude extract/t-butanol was 0.5 and the ammonium sulphate saturation 40% w/v, the activity recovery increased with the pH up to 166% at pH 5.7. Thereafter, this value decreased, reaching 112% at pH 9. The regression model representing the effect of t crude extract/ t-butanol, ammonium sulphate saturation and pH on activity recovery of beta-amylase, in terms of their real level, is given as:

Table 2 Coefficients of regression, R2 and AAD values for the mathematic models of ratio of activity and the purification factor. Term

Activity recovery (%)

Purification factor

b0 b1 b2 b3 b11 b12 b13 b22 b23 b33 R2 AAD

354.842 128.18* 17.620*** 59.511 9.458 1.057 6.914 0.175*** 0.265 4.545** 0.9635 0.03

1.255 2.891* 0.926*** 2.762*** 1.524 0.015 0.938* 0.009*** 0.001 0.054 0.9607 0.05

b represents the coefficients of equations different of models with b0 the constant term; b1, b2 and b3 the linear effects; b11, b22 and b33 the quadratic effects; b12, b13 and b23 the interaction. With 1, 2 and 3 respectively the ratio of enzyme extract to tbutanol, the ammonium sulfate saturation and pH. * Significant at p 6 0.05. ** Significant at p 6 0.01. *** Significant at p 6 0.001.

AR ð%Þ ¼ 392:84  59:62X 1 þ 17:28X 2 þ 60:05X 3 þ 15:05X 21 þ 1:06X 1 X 2 þ 6:91X 1 X 3  0:18X 22  0:19X 2 X 3  4:79X 23

ð6Þ

The value of coefficient of determination R2 for the above equation is 0.9541. This value indicates that the regression model is able to explain 95.41% of variability of the data. The value of AAD (0.03) that measures the relative average deviation between predicted and experimental response show that the model describes the values of activity recovery of beta-amylase adequately. The rest of lack of fit can be due to the unexpected effect of the solvent. Tonova & Lazarova, 2008 reported that for systems containing salt, the interfacial tension between the aqueous phase and the organic solvent increases with the salt concentration, making the system more unstable. The positive signs of linear coefficients of ammonium sulphate saturation and pH observed on the mathematical model indicate that they had a positive effect on the activity recovery of beta amylase. Since the positive linear effect of pH was not significant and that the quadratic factor of pH had a significant negative effect on the activity recovery (Table 2), we can generally conclude that pH had a cumulative negative effect on the activity recovery. 3.1.2. Effects on the purification factor The purification factor of beta amylase is a major parameter providing information on the efficiency of the TPP enrichment process. Fig. 1d and e shows the effect of independent variables on the purification factor. It is observed that the purification factor increased with the ammonium sulphate saturation whereas it decreased with pH. For example, when the ratio of crude extract/ t-butanol was 0.5, the purification factor was increased from 1.44 at 20% w/v to 8.92 at an ammonium saturation of 45% w/v (Fig. 1c). At same time, purification factor was reduced from 12.42 at pH 4–4.76 at pH 9 (Fig. 1d). As in Table 2, the ratio of crude extract/t-butanol (X1) (p < 0.05), the ammonium sulphate saturation (X2) (p < 0.001), the pH (X3) (p < 0.001) and the quadratic term of ammonium sulphate saturation (X 22 ) (p < 0.001) contributed with a significant effect on the purification factor of beta-amylase. This result shows that each parameter acted independently and while acknowledging the interaction between the ratio of crude extract/t-butanol and pH (p < 0.05), there were no real synergistic effects between the ratio of crude extract/t-butanol and ammonium sulphate saturation, or between the ammonium sulphate saturation and the pH. The

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149

Fig. 1. Response surfaces of activity recovery as function of (a) ratio of enzyme extract to tert-butanol and ammonium sulfate saturation, (b) ratio of enzyme extract to tertbutanol and pH; and of the purification factor as function of (c) ratio of enzyme extract to tert-butanol and ammonium sulfate saturation, (d) ratio of enzyme extract to tertbutanol and pH; keeping the constant variable at central point ((a and c): pH 6.5; (b and d): 40% ammonium sulfate saturation).

regression model representing the effect of the three factors on the purification factor in terms of their coded level is given as:

PF ¼ þ 1:26  2:89X 1 þ 0:93X 2 þ 2:76X 3 þ 1:52X 21 þ 0:02X 1 X 2 þ 0:94X 1 X 3  0:009X 22  0:001X 2 X 3  0:05X 23

ð7Þ

ammonium sulphate leads to the precipitation of enzymes by reducing their solubility and by increasing their surface hydrophobicity. Thus, efficiency of enzyme precipitation by TPP depends not only on the concentration of salt, but also on the charge of the protein which is strongly dependent of the pH of solution (Pike & Dennison, 1989).

2

The value of the coefficient of determination R (0.9607) for this equation and the value of AAD (0.05) indicate a good fitting of the model with the experimental data. These results show a perfect agreement between the experimental values and those generated by mathematical models, which indicate that the model is adequate and can be used for optimization. The minus sign of the two factors X1 and X3 indicates that that ratio of crude extract/ t-butanol and pH have negative influence. In fact, it is well known that enzymes contain side chains of amino acid polar sites that can be positively or negatively charged. This polarity makes them very sensitive to changes in pH. Also, saturation of solution by

3.2. Optimisation of the TPP of beta amylase The numerical optimization approach was adopted to determine the best experimental conditions for purification of the beta-amylase from stems of A. precatorius by TPP. This optimization of process factors (ratio of crude extract/t-butanol, ammonium sulphate saturation and pH) have been carried out using STATGRAPHICS Centurion software (Version XVI). The optimum processing conditions were investigated for the two experimental responses (activity recovery and purification factor). The activity

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recovery and the purification factor (i.e. the specific activity) are likely related, the first one represents the activity by volume unit (ml) and the second by total protein amount (mg). The conditions taken in consideration for optimization were to maximize the values of activity recovery and purification. The ranges of three independent parameters were: the ratio of crude extract/t-butanol (0.5–2), ammonium sulphate saturation (20–60% w/v) and pH (4–9). These ranges were chosen according to the results of a preliminary study (data not shown). Optimum conditions generated by the numerical optimization with a maximum overall desirability of 0.922 were: a ratio of crude extract/t-butanol of 0.87, an ammonium sulphate saturation of 49.46% w/v and a pH of 5.2. The corresponding values of experimental responses were, activity recovery: 156.2% and purification factor: 10.17. In order to confirm the validity of values obtained from the numerical optimization, three confirmation experiments with final optimal experimental conditions were carried out. The average values of 158.27 ± 3.81% of activity recovery and 10.09 ± 0.11 of purification factor were obtained. In terms of the results of the confirmation experiments and the corresponding values predicted, these data confirmed the optimal values generated by numerical optimization and it can be concluded that the regression models obtained were reasonably accurate for predicting the activity recovery and the purification factor of beta-amylase extracted from stems of A. precatorius during the purification by TPP.

(a)

(b)

Effect of pH Stability at pH

100

The purified beta-amylase derived from TPP optimal conditions was subjected to dialysis for 24 h at 4 °C against distilled water, in order to remove the ammonium sulphate used for the precipitation. Samples obtained were frozen at 20 °C and then lyophilized for 24 h. A summary of purification steps of beta-amylase from stems of A. precatorius (supplementary data Table S1). A reduction both of total activity and specific activity of the betaamylase after dialysis and lyophilisation steps was observed. For example, the total activity of crude extract and purified beta-amylase (TPP-interfacial precipitate) were respectively (20.42 ± 1.07) and (32.32 ± 1.22) U/ml. A value of (14.97 ± 0.66) and (11.38 ± 0.85) U/ml were registered after dialysis and lyophilisation respectively (supplementary data Table S1). Similarly, the specific activity of the crude extract and TPP-interfacial precipitate were 3.79 and 38.24 U/mg. These values decreased to 24.59 and 19.99 U/mg respectively after dialysis and lyophilisation. It is known that after dialysis, enzyme is freed of residues of salt used for precipitation, which enables an improvement in its activity. The results obtained are contradictory and it appears that a degradation of the beta-amylase during dialysis and lyophilisation occurs. These results conducted us to investigate and find the reasons of the decrease of activity. The crude extracts from plants generally contain several compounds including protease activities, hence the difficulty to isolate and purify a particular molecule from such mixtures. For this purpose, protease activity was checked in crude extract as well as in the purified beta-amylase. 3.4. Protease activity In order to understand the reduction of beta-amylase activity after dialysis and lyophilisation, protease activity was tested. The method of azocasein was used and the values obtained were: crude extract: 0.503U and purified beta-amylase: 0.237U. The results confirmed the presence of protease activities. It is observed that this activity was higher in the crude extract, indicating that a portion was removed during the process of TPP. To confirm this result, MMP Zymogene gel electrophoresis (12% polyacrylamide) impregnated with casein was carried out (supplementary data, Fig. S1). It

Relative activity (%)

80

3.3. Dialysis and lyophilisation

60

40

20

0 1

2

3

4

5

6

7

8

9

10

11

pH Fig. 2. Temperature (a) and pH (b) profiles of purified beta-amylase from Abrus precatorius.

is clear from this figure that crude extract and purified beta-amylase contained a protease activity, characterized by a clear band. The position of these clear bands on the top of gels means that the protease had a high relative molecular weight. The presence of protease activity in the purified beta-amylase fraction did not significantly affect subsequent measurement of enzyme activity because of the lower concentrated nature of protease activity. The apparent molecular weight of the protease is very high, since the protein does not wander far in the gel. It remains at the beginning of the separation gel and therefore with the standards applied it was not possible to determine the molecular weight. In Fig. 3 the band at the beginning of the separation gel represents the protease. Finally, purified beta-amylase as from optimized TPP without further dialysis and lyophilisation was used for further studies of characterization because of its high activity. 3.5. Characteristics of purified beta-amylase 3.5.1. High performance liquid chromatography analysis of hydrolysis products obtained by purified enzyme The chromatograms obtained from this analysis are available as supplementary data (Fig. S2). Interestingly, it was observed, that only a single peak resulted as a product from the enzyme activity

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Samples from experiments of Doehlert design M

P

CE

1

2

3

4

5

6

7

8

9

10

11

12

13

M

94 67 43 31

20.1 14.4

Fig. 3. Electrophoresis of samples of Abrus precatorius. The sample of lane 1 was prepared by mixing 71 mg of powder of Abrus precatorius with 1 ml of sample buffer for 1 h. The mixture was heated at 90 °C for 10 min and then centrifuged 5 min at 10,000g. The samples of lane 2–15 were prepared by mixing samples to be analysed with a sample buffer in the ratio 1:1. The whole was heated at 90 °C for 10 min and then cooled at room temperature before being loaded in the wells of gel. Volumes and dilutions were adjusted to maintain constant protein loading in each lane. Lanes: M: marker. P: powder of Abrus precatorius, CE: crude extract of Abrus precatorius, 1–13: samples of purified beta-amylase of Doehlert design, experiments number 1–13 respectively.

of crude extract and purified amylase. This peak has been identified as maltose. According to the reaction mechanism and the product observed, this activity may be allocated to beta-amylase activity. In fact, beta-amylases are exo-amylases, which catalyse the successive removal of maltose from the non-reducing ends of the glucose polymers with inversion of the anomeric configuration (Derde, Gomand, Courtin, & Delcour, 2012). This result conducted us to conclude that enzyme extracted and purified from the stems of A. precatorius is a beta-amylase. 3.5.2. Molecular weight estimation of purified beta-amylase by electrophoresis SDS–PAGE allowed the determination of relative molecular masses of purified beta-amylase by comparison of the migrated bands with standard molecular masses. According to Cudney & McPherson (1993), the molecular mass of beta-amylase is ranging between 42 and 110 kDa. In our study, the molecular mass of purified beta-amylase was tentatively assumed to be 60 kDa from the migration using 14% separating gel (Fig. 3 and supplementary data, Fig. S4). We did try to identify this 60 kDa band using in-gel tryptic digestion and combining it with mass spectrometric methods (MALDI_TOF_MS), but the sequence is not available in the protein data bank and alignment or comparison with other beta-amylases from other biological sources did not give any satisfying results. Further experiments are directed towards these questions. Our assumption to this allocation to 60 kDa also comes from these experiments (supplementary data, Fig. S4). Beta-amylase extracted from plant leaves had molecular masse of 55 kDa (Monroe & Preiss, 1990). Recently while analysing germinating millet seeds, the molecular weight was estimated to be 58 kDa based on its mobility on SDS–PAGE and gel filtration, which showed that it is composed of a single unit (Yamasaki, 2003). Also, it is observed that for all the samples of purified beta-amylase (lane 1–13); proteins migrated similarly with a clear band characteristic of the beta-amylase purified except the lane 5 and 6 (Fig. 3). These two lanes correspond to the experiments 5 and 6 of Doehlert design of TPP process. The determination of protein concentration by the Lowry method showed very low protein concentration. 3.5.3. Kinetic study of purified beta-amylase Kinetic constants Km and Vmax of purified beta-amylase were determined from initial rate measurements for hydrolysis of starch

from potato at different concentration up to saturation. Using Michaelis–Menten plot and the values of apparent Km and Vmax were found as 79.37 mg/ml and 5.13 U/ml, respectively (supplementary data, Fig. S3). Yamaguchi et al., 1996 extracted beta-amylase from Bacillus cereus and found a Km of 0.90 and 1.02 mM using maltotriose and maltoheptose respectively as substrate. Batlle, Carbonell, & Sendra, 2000 reported a Km of barley beta-amylase of 1.49 g/L using potato amylopectin as substrate. Also, Hirata et al. (2004a) found values of Km of B. cereus beta-amylase ranging between 0.73 and 1.9 mg/ml using potato amylopectin as substrate. These do differ from the values reported here. 3.5.4. Effects of pH and temperature Fig. 2 shows the effect of temperature and pH on the activity of purified beta-amylase. It is observed that a high activity in temperature range of 45–75 °C with an optimum activity registered at 70 °C (Fig. 2a). The enzyme is relatively thermostable, maintaining more than 60% of its activity after pre-incubation of purified beta-amylase for 1 h without substrate from 20 to 65 °C. After 65 °C, the relative activity dropped quickly to rich 19% of residual activity at 70 °C. This activity has been less than 5% when the purified beta-amylase was pre-incubated at 80 °C for 1 h. In a work of (Kwan, So, Chan, & Cheng, 1993), beta-amylase from Bacillus circulans was stable at 45 °C for 30 min but lost half of its activity after 30 min at 50 °C. It is well known that increasing the temperature up to the optimum speeds up the reaction significantly but at higher temperatures the effect on protein denaturation becomes dominant and the conversion rate decrease again (Heinz, Buckow, & Knorr, 2005). Similarly, the effect of pH on amylase activity was studied by incubating the enzyme in 200 mM phosphate buffer solutions at 50 °C and a pH range from 2.0 to 10.0. A good activity was observed between pH 4.5 and pH 7.5 with a maximum activity between pH 6 and 6.5 (Fig. 2b). The profile of activity obtained for the pH stability was quite similar to the profile obtained for the effect of pH (Fig. 2a). The purified beta-amylase presented higher stability and a maximal residual activity around pH 6–6.5 but a considerable loss of activity was observed at extreme acidic or basic pH. Thermal stability and pH stability of an enzyme are two important parameters that determine his potential use for an industrial process. These results are in perfect agreement with the previous works, with a high activity of beta-amylase found in the temperature range of 30–80 °C and in the pH range of 3.5–8.0, this

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Table 3 Effect of some chemicals on the activity of amylase purified from Abrus precatorius. Chemicals

CaCl3 NaCl KCl MgCl2 ZnSO4 CuSO4 FeCl3 AgNO3 Urea Boric acid Lactic acid Acetic acid Citric acid Tannic acid Salicylic acid

Residual activity (%) 1 mM

5 mM

10 mM

92.3 ± 4.6 87.7 ± 0.3 95.2 ± 3.5 106.1 ± 3.6 108.7 ± 4.0 1.1 ± 0.1 0.5 ± 0.1 5.7 ± 1.4 88.8 ± 0.6 83.5 ± 7.5 49.4 ± 6.5 88.9 ± 8.1 24.2 ± 4.4 19.7 ± 3.3 36.4 ± 0.3

117.4 ± 0.0 106.3 ± 6.4 105.7 ± 7.9 102.4 ± 2.7 96.4 ± 6.3 1.4 ± 0.3 0.8 ± 0.7 3.5 ± 0.4 90.5 ± 5.5 82.7 ± 8.1 15.8 ± 3.4 46.2 ± 3.8 5.6 ± 0.6 5.9 ± 0.6 2.4 ± 1.9

119.7 ± 4.3 101.6 ± 7.2 97.7 ± 8.3 97.8 ± 1.9 95.2 ± 5.2 2.1 ± 0.3 1.2 ± 0.8 2.4 ± 2.8 89.7 ± 7.9 82.6 ± 1.9 9.7 ± 1.5 36.0 ± 5.4 2.1 ± 0.2 3.2 ± 3.2 0.3 ± 0.3

depending on the origin and the different conditions used for activity assay (Hirata, Adachi, Utsumi, & Mikami, 2004b; Lizotte, Henson, & Duke, 1990). 3.5.5. Effects of some chemicals The effects of various metal ions and chemicals at concentrations of 1, 5 and 10 mM on the activity of the purified beta-amylase were studied at pH 6.5 and at 50 °C by the incubation of enzyme with the respective compounds. The results are presented in Table 3. MgCl2 (106.1 ± 3.6%) and ZnSO4 (108.7 ± 4.0%) each at 1 mM had an increasing effect on the activity whereas at 10 mM, this effect was no longer observed (Table 3). It was shown that CaCl3 at 1 mM slightly reduced the activity. At concentrations of 5 and 10 mM, its influence caused it to increase to 117.4 ± 0.0% and 119.7 ± 4.3%, respectively. In the literature, it is reported a relative increase in amylase activity at various concentrations of MgCl2, ZnSO4 and CaCl3 (Petersen, Ueda, Hall, & Gray, 1977). Monovalent salts KCl and NaCl moderately activated beta-amylase activity at a concentration of 5 mM. Urea and boric acid had a moderate inhibitory effect on the beta-amylase activity (between 80% and 90%) independent of their concentration. Organic compounds (lactic acid, acetic acid, citric acid, tannic acid and salicylic acid) caused a high inhibitory effect. FeCl3 and AgNO3 at concentrations 1, 5 and 10 mM also inhibited more than 90% the activity of betaamylase (Table 3). 4. Conclusion A beta-amylase was extracted from A. precatorius stems and purified by three phase partitioning. A total of 17 experiments were made and the recovery of activity and the purification factor have been taken as experimental response to check the efficiency of purification process. Application of RSM with Doehlert design allowed optimizing the main variables such as the ratio of crude extract/t-butanol, the ammonium sulphate saturation and pH. Dialysis and lyophilisation were carried out and a protease activity was detected. HPLC analysis permitted to identify clearly the activity of purified enzyme to be related to beta-amylase and its relative molecular weight was as expected. Some characteristics of the purified beta-amylase such as optimum temperature, pH, and kinetic constants were determined. The intention was to produce a protein enriched fraction, where both the protein content and high activity of amylase can be achieved for use in local fermentation processing of food products in Cameroon. Therefore, a complete homogeneity of the sample was not a ‘‘must’’ requirement. A further purification may result in a more appropriate sample, but the effort will not be

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