Pharmacognosy Magazine

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 14  |  Issue : 57  |  Page : 319--326

An aspartic-metalloprotease from an endemic plant tuber (Burnatia enneandra micheli): Purification and biochemical characterization


Ngangoum Eric Serge1, Mezajoug Kenfack Laurette Blandine2, Sanjit Kumar3, Tchiegang Clerge2, Mookambeswaran Vijayalakshmi3,  
1 Department of Food Sciences and Nutrition, Bioprocess Laboratory, Institute University of Technology, University of Ngaoundere, Ngaoundere, Cameroon; Centre for Bio-Separation Technology, VIT University, Vellore, Tamil Nadu, India
2 Department of Food Sciences and Nutrition, Bioprocess Laboratory, Institute University of Technology, University of Ngaoundere, Ngaoundere, Cameroon
3 Centre for Bio-Separation Technology, VIT University, Vellore, Tamil Nadu, India

Correspondence Address:
Tchiegang Clerge
Universite de Ngaoundéré, Laboratoire des Bio-Procédés, Unité de Recherche en Biochimie-Technologie Alimentaires et Nutrition, BP 455 Ngaoundéré
Cameroon

Abstract

Background: The objective of this work was to isolate, optimize, and characterize protease from Burnatia enneandra which is an endemic plant found abundantly in the Far-Nord Region of Cameroon. The optimum condition to extract maximum quantity of protease from B. enneandra with respect to pH, the ratio (m/v), and agitation frequency was defined as 5.1%, 4%, and 100 rpm, respectively. Materials and Methods: The enzyme was purified using ammonium sulphate precipitation, double gel filtration chromatography sephadex G200 followed by sephadex G75 and the purified protease was further characterized. With an apparent molecular weight of 23 kDa on SDS-PAGE, the purified protease showed maximum activity at 5.1 and 40°C respectively for pH and temperature. Its activity was enhanced by metal ions such as Ca2+ and Ni2+, while Fe2+and Zn2+ showed significant inhibition. Results: B. enneandra protease activity was not affected by proteases inhibitors such as phenylmethylsulfonyl fluoride, aprotinin, and iodoacetamide but was strongly inhibited by Pepstatin A and ethylenediaminetetraacetic acid which allowed to classify this new protease as aspartic-metalloproteases. Using casein as substrate, protease from B. enneandra had a maximum rate of reaction (Vmax) and Michaelis-Menten constant (Km) of 64.935 (U/mL) and 373.941 (μg/mL), respectively. Abbreviations used: CCD: Central composite design; Km: Michaelis–Menten constant; Vmax: Maximum Velocity; PBD: Plackett–Burman design; PMSF: Phenylmethylsulfonyl floride; AAD: Absolute Average Deviation; AF: Accuracy Factor; BSA : Bovine serum albumin; BF: Bias factor; EDTA: Ethylene diamine tetraacetic acid; RSM: Response surface methodology; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.



How to cite this article:
Serge NE, Laurette Blandine MK, Kumar S, Clerge T, Vijayalakshmi M. An aspartic-metalloprotease from an endemic plant tuber (Burnatia enneandra micheli): Purification and biochemical characterization.Phcog Mag 2018;14:319-326


How to cite this URL:
Serge NE, Laurette Blandine MK, Kumar S, Clerge T, Vijayalakshmi M. An aspartic-metalloprotease from an endemic plant tuber (Burnatia enneandra micheli): Purification and biochemical characterization. Phcog Mag [serial online] 2018 [cited 2021 Nov 27 ];14:319-326
Available from: http://www.phcog.com/text.asp?2018/14/57/319/240774


Full Text



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SUMMARY

Purification of an Aspartic-metalloprotease from an Endemic Plant Burnatia enneandra micheliBiochemical characterization of metalloprotease Effect of different substrates on metalloprotease activityEffect of metal ions on metalloprotease activity

 Introduction



Proteases are a group of enzymes which are distributed in plants, animals, and microorganisms where they play a significant role in the physiological process.[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] Proteases isolated from various organisms are commercially used in various industries such as in food, pharmaceutical, leather, waste treatment, detergent, and textile industries.[4],[14],[15] Most of the commercial proteases are either isolated from microorganisms or animals.[6],[14],[16],[17] There are very few proteases which are isolated, characterized from plants.[2] The purification and characterization of new promising proteases from plant sources have also incurred greater attention by various food and biotechnology-based industries due to their properties being significant active in a broad range of pH and temperature.[3] Hence, plant-based proteases have been extracted from various species as well as from all parts of the plant.[2],12,[18],[19],[20] However, due to higher demand for protease globally, it is necessary to continue searching for newly available sources with low prices. In Cameroon, some plants are used conventionally according to their potential. Burnatia enneandra which is one of them is an endemic plant belonging to Alismataceae family largely found in Far-Nord Region of Cameroon. Its tubers are used in food system of the local population, especially in local beverages production. In 2014, Mezajoug-Kenfack et al. reported that B. enneandra tubers could be an important source of proteases for industries after purification and characterization Mezajoug-Kenfack, Ngangoum, Tchiégang, and Linder.[21]

The main objective of this work was to determine the optimal condition to extract protease from B. enneandra tubers and also to characterize the purified enzyme using various biochemical methods and to determine its applicability in industrial application. Therefore, the Plackett–Burman design was investigated to select the main parameters which can significantly influence protease extraction from B. enneandra tubers.[22] These significant parameters were there after optimized using response surface methodology (RSM) through center composite experimental design.

 Materials and Methods



The tubers of B. enneandra used in this work were collected from a farm at Yagoua Far-Nord Region of Cameroon (10°20′ 34″ N, 15°14′ 26″ E). The tubers were treated according to the method described by Mezajoug-Kenfack, Ngangoum, Tchiégang, and Linder. They were peeled, cleaned, cut in the small disk, and dried into the convective dryer (CKA 2000-AUF) at 40°C for 48 h. Dry tubers were pounded in mortar immediately after drying to avoid humidity, then ground in a micro hammer mill Culatti and sifted. Powder samples with particles size lower than 400 μm were retained for proteases extraction.

Extraction and purification

Plackett-burman design

Protease extraction from plants can be influenced by many factors. The Plackett–Burman design was used to study the effect of the ratio m/v, pH, temperature, extraction time, stirring speed, and NaCl concentration on the protease extraction from the powder of B. enneandra tubers, taking protease activity as a response. All the factors were studied at two levels: high (level + 1) and low (level-1) [Table 1]. Statgraphics Centurion XVI was used as software to generate and analyze the experimental matrix of Plackett–Burman [Table 2]. The relation between code and real values is expressed by the following equation:{Table 1}{Table 2}

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Where ai is the coded value of the parameter Ai, A0 is the value of Ai at the center point, and ΔA is the step change of an independent parameter.

Central composite design

The significant parameters selected through Placket–Burman design were optimized using RSM method with central composite design (CCD). Seventeen experiments were carried out to study each selected parameters in five levels: two alpha levels (−1.35313, +1.35313), low (−1), medium (0), and high (+1) levels. The measured responses were subjected to analysis of variance (ANOVA) using Statgraphics Centurion XVI as software. Based on the experimental data, an empirical second order polynomial equation was established to evaluate the relationships between the independent variables and the process dependent on Giovanni.[22] A second order polynomial equation is express as follows:

[INLINE:3]

where Y is the predicted protease activity defined as response, α0: Is the intercept term, αi: Is the linear coefficients; αii: Is the quadratic term coefficients, and αij: Is the cross product coefficients, xi and xj are independent variables.

The acceptation of the given model was evaluated by calculating the determination coefficient (R2), the absolute average deviation (AAD), the accuracy factor (AF), and the Bias factor (Bf) which the formula was given as follows:

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Where N is the number of trials in the experimental design, Ȳ is the response average, Yical and Yiexp are calculated and experiment responses, respectively.

Protease activity assay

The protease activity of the purified protease as well as crude extract was measured according to the modified method described by Yezli et al.[23] Briefly, 1 mL of enzyme solution was mixed with 5 mL of 0.65% w/v casein prepared in 50 mM phosphate buffer solution (pH 6.0). The test tube containing the enzyme and substrate was incubated into the shaker for 30 min at 37°C. After an incubation period, the reaction was stopped by adding 0.7 mL of 10% trichloroacetic acid and allowed to stand at room temperature for 20 min. The blank tube was made by substituting the enzyme solution with MilliQ water and used as a control. Both test and blank were centrifuged at 8000 rpm for 25 min, and the supernatant was collected. A volume of 200 μL of the supernatant was mixed with sodium bicarbonate solution 0.5M, and Folin–Ciocalteu reagent (0.67N) and the absorbance was measured at 660 nm.[24]

One unit of enzyme activity was defined as an amount of enzymes that release 1.0 μmole of L-tyrosine per min, in the assay conditions. The proteolytic activity was calculated using the following equation:

[INLINE:8]

Where, T: Mass (μg) of tyrosine released during hydrolysis; Vt: Total volume of the assay, Vu: Volume of enzyme solution used, t: Time of hydrolysis, Vc: Volume used in color development.

Protein determination

Protein content was estimated in the crude extract, and after each purification step, using the method described by Bradford.[25] The bovine serum albumin (BSA) was used as a standard.

Purification of protease

The crude extract obtained in the optimal defined conditions from B. enneandra tubers was used as the starting material for protease purification. The crude extract was subjected to ammonium sulfate precipitation at different concentrations of saturation (20%, 40%, 60%, and 80%) at 4°C. The precipitate was collected by centrifugation at 8000 rpm for 25 min at 4°C, and dissolved in 50 mM sodium phosphate buffer (pH 7.2) and dialyzed to remove the salt against the same buffer at 4°C for 24 h with continuous stirring. The dialyzed suspension was concentrated and subjected to gel filtration chromatography column Sephadex G-200 (1.5 cm × 140 cm) previously equilibrated with sodium phosphate buffer (50 mM, pH 6) containing 0.1 M NaCl. The protease was eluted with the same buffer and fractions of 1 mL were collected at the flow rate of 250 μL/min. The active fractions containing some impurities were pooled concentrated and loaded in Sephadex G-75 (1.6 cm × 67 cm) column previously equilibrated with sodium phosphate buffer (50 mM, pH 6). The protease was eluted with the same buffer-containing 0.1 M NaCl and fractions of 1 mL were collected at the flow rate of 0.50 mL/min. Protein concentration, proteolytic activity was determined in each of the fractions and the enzyme purity was verified by electrophoresis sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Molecular-weight determination

The molecular weight of the purified protease from B. enneandra was evaluated by SDS-PAGE[26] in 12% resolving gel as described by Laemmli. Electrophoresis was carried out using PowerPac, Basic™ from BIO-RAD as apparatus and the protein bands were stained with 0.1% Coomassie Brilliant Blue R-250 solution, then destained with destaining solution which was the mixture of milliQ water (50%), methanol (40%), and acetic acid (10%). The molecular mass of purified protease was determined by using a low-molecular-weight protein maker calibration.

Effect of pH on protease activity and stability

The effect of pH on protease activity was evaluated by incubating the protease at 37°C for 30 min at different pH ranging from 2 to 12. The appropriate pH was obtained with various buffer systems: glycine-HCl (pH 2–3), sodium acetate (pH 4–5), sodium phosphate (pH 6–8), Tris-HCl (pH 9–10), and glycine-NaOH (pH 11–12). For the evaluation of pH stability, the protease was preincubated at different pH for 1 h at 40°C, and the residual activity was then determined as described above.

Effect of temperature on protease activity and thermal stability

The influence of the temperature on protease activity was determined at different temperatures ranging from 10°C to 80°C with 10°C interval. One milliliter of protease solution and 5 mL of casein 0.65% w/v prepared in phosphate buffer (50 mM, pH 7.5) were added, and the mixture was incubated for 30 min at different temperatures. The enzyme activity was determined after the incubating period as described above. The thermal stability of purified protease was evaluated by preincubating the enzyme for 1 h at different temperatures vary from 10°C to 80°C, respectively, and the residual activity was determined.

Effect of protease inhibitors on protease activity

To determine the nature of the purified protease from B. enneandra, the enzyme was incubated with different proteases inhibitors. The inhibitors in which the effect was studied were phenylmethylsulfonyl fluoride (PMSF), aprotinin, Pepstatin A, iodoacetamide, and ethylenediaminetetraacetic acid (EDTA). The purified protease was preincubated in 5 mM and 10 mM of the inhibitors solutions and then incubated with casein solution for 30 min at 40°C and the remaining protease activity was determined. A control tube of the protease activity was done without inhibitors, and its activity was taken as 100%.

Effect of metal ions on protease activity

The effect of different metal ions (MgCl2, ZnCl2, NiCl2, SnCl2, FeCl2, CaCl2, KCl, and NaCl) on protease activity was evaluated. The mixture of protease and substrate were incubated with metal ions solution, and the remaining protease activity was determined according to the method described above. A control tube of the protease activity was done in the absence of metal ions, and its activity was taken as 100%.

Effect of different substrates on protease activity

The effect of the natural substrate which was protein from defatted flours of Ricinodendron heudelotii (Euphorbiaceae) as well as different synthetics substrates such as hemoglobin, casein, gelatin, and BSA on purified protease activity was evaluated. The enzyme activity was determined after incubating each substrate with protease solution at 40°C for 30 min.

Determination of kinetic parameters

The Michaelis–Menten constant (Km) and the maximum velocity (Vmax) of the purified protease were calculated by fitting the data of protease activity obtained from the increasing casein concentrations to a linear regression after Lineweaver–Burk transformation.

 Results and Discussion



Screening of the variables affecting protease activity

The Plackett–Burman design (PBD) was investigated to screen and select the main significant parameter which influences protease extraction from B. enneandra. The experimental matrix along with response is presented in [Table 2].

Base on the experimental data and the related independent variables, a first order polynomial equation representing protease activity (Y1) as the response was established.

Y1 =29.950 − 1.790 × X1 +12.160 × X2 +0.555 × X3− 0.401 × X4 +0.339 × X5− 2.807 × X6

The ANOVA of the PBD experiments was done to evaluate the significance of the parameters. They were judged statistically significant or not if their P ≤ 0.05 or ≥0.05, respectively [Table 3]. The effect of pH, the ratio (w/v), and stirring speed was found to be significant, while the effect of time, temperature, and NaCl concentration was not. In fact, their P value that are 0.6315, 0.4397, and 0.5754 were higher than 0.05, which demonstrating that the three variables produced not significant effects on proteases extraction from B. enneandra. The pH, ratio (w/v), and stirring speed were, therefore, chosen for further optimization of protease extraction.{Table 3}

Optimization of the selected variables

A total of 17 experiments were carried out using CCD to study interactions between the pH, ratio (w/v), and stirring speed and also to determine their optimal values. The experimental design which contains the different combinations is presented in [Table 4], along with the experimental (Y2Exp) and the calculated (Y2Cal) responses.{Table 4}

Base on the experimental data, a second order polynomial equation was established to predict protease extraction from the powder of B. enneandra tubers.

Y2 =145.163 + 73.474 × X1− 16.697 × X2 +10.6609 × X3− 10.317 × X12 − 8.6805 × X1× X2 +7.523 × X1× X3 +19.2667 × X22+15.0463 × X2× X3 +12.3135 × X32

To determine the accuracy of the polynomial model, the coefficient of determination Joglekar and May, Ross, the AAD Bas and Boyaci,[27] the bias factor, and the AF Dalgaard and Jørgensen were calculated and the results are presented in [Table 5].{Table 5}

All calculated parameters values were in range with the norms found in the literature Bas and Boyaci, Dalgaard, and Jørgensen.[27],[28] To confirm this accuracy, protease was extracted in the optimal defined condition. The protease activity obtained was 311.954 mU/mL which was close to the predicted value 308.145 mU/mL. These results suggest that the established model was acceptable and can be used to predict protease extraction from B. enneandra, this with the respect of the independent variables range.

The ANOVA at the level of 5%, of the center composite design (optimization) experiments is given in [Table 6]. Base on this ANOVA, the means parameter to consider for protease extraction from B. enneandra were ratio (m/v), pH, interaction between pH, stirring speed, and the quadratic effect of pH, this because of their P < 0.05 which was the analysis level. To better understand, the effect of pH and ratio (w/v), the three-dimensional curve was plotted [Figure 1] using Sigma plot software version 11.0. From this [Figure 1] we can see that protease activity increase when the pH value increase. However, up to about pH 6 any further increasing of pH decrease the protease activity. This can be due to the fact that, in solution protein behavior depends on pH condition of the environment. The optimal condition for protease extraction from B. enneandra was found to be: 4%, 5.1 and 100 rpm, for the ratio (m/v), pH, and stirring speed, respectively. The implementation of this condition gave 308.145 mU/mL as protease activity which was closed to the predicted value 311.954 mU/mL calculated with the regression polynomial second order model.{Table 6}{Figure 1}

Purification and molecular weight of protease

Protease was extracted from B. enneandra tubers and subjected to three steps purification all carried out at 4°C. The purification procedure is summarized in [Table 7]. Ammonium sulfate fractionation was the first step in purification and the protease of interest was obtained maximally at 60% of the saturation of salt with 10.69 U/mg as specific activity and 3.12 folds purification compare to crude extract [Table 7]. The fraction obtained at 60% saturation was dialyzed, concentrated, and loaded on Sephadex G200 column and two Peaks I and II [Figure 2] were eluted and analyzed for protein concentration as well as protease activity. The second peak of elution [Figure 2] showed activity but was not pure according to gel electrophoresis analysis [Figure 2]. Fractions constituting this peak were pooled, concentrated, loaded on Sephadex G75 column, and eluted with phosphate buffer (50 mM, pH 6). During this last step of purification, one single peak was obtained from Sephadex G75 column [Figure 3] with 37.09 folds purification and the specific activity was enhanced to 127.23 U/mg.{Table 7}{Figure 2}{Figure 3}

The homogeneity of the eluted protease through the second gel permeation chromatography was confirmed by polyacrylamide gel electrophoresis with the presence of one single protein band. The standard protein markers used were phosphorylase b (97.4 kDa), BSA (66 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa).

The purified protease from B. enneandra showed single band and the molecular weight was evaluated at 23 kDa [Figure 4].{Figure 4}

Effect of pH on protease activity and stability

The effect of pH on protease activity was evaluated at different pH range 3–12 using casein as substrate [Figure 5]. Protease activity increased with increasing pH value and the maximum activity was found at pH 5. Above this value, any increasing of pH lead to decrease protease activity. The stability of protease from B. enneandra was also done after preincubating the enzyme for 1 h at the same range of pH. The purified protease was stable at pH range 3–10 and retained more than 60% of its original activity after preincubating the protease for 1 h. Similar results were reported by Balqis and Rosma who purified protease from Artocapus. integer leaves.[29]{Figure 5}

Effect of temperature on protease activity and thermal stability

The temperature profile of the purified protease was studied at pH 5 by incubating the enzyme at different temperatures ranging from 10°C to 80°C. From [Figure 6], it is obvious that protease from B. enneandra showed its highest activity at 40°C, and then, the activity rapidly decreased with increasing temperature. This result is in accordance with some reported from plants proteases which exhibited their maximum activity in the temperature range 40°C–50°C Mohamed and Habbani; Devi, and Hema-Latha. However, the results found here are in disagreement with those of Sanatan et al. Sanatan, Lomate, Giri, and Hivrale, who reported protease with optimal temperature at 60°C from Periplaneta americana.[30]{Figure 6}

Protease from B. enneandra was stable up to 60°C after 1 h incubation and more than 50% of its original activity remained at this temperature [Figure 6].

Effect of protease inhibitors on protease activity

To determine the class in which belongs the protease from B. enneandra, the effect of many proteases inhibitors on protease activity was investigated. Protease inhibitors such as serine protease inhibitor (Aprotinin, PMSF) and cysteine protease inhibitor (iodoacetamide) displayed no significant effect on protease activity. This result involved that B. enneandra protease is not belong to the class of serine or cysteine protease. At the concentration of 1 mM, peptatin A which is the inhibitor of aspartic proteases shows no great inhibition (1.2%) on protease activity. However, at 10 mM concentration 42.05% inhibition was observed with the same inhibitor proved that the protease is an aspartate protease Kumar, Sharma, Saharan and Singh, Raposo and Domingos. B. enneandra protease activity was also inhibited up to 48.59% and 90.18% following incubation with EDTA at 1 mM and 10 mM concentration, respectively [Figure 7]. These results consistent with previous findings demonstrating that B. enneandra protease also belonging to metalloprotease class are strongly inhibited by EDTA.[31] Protease extract from B. enneandra tubers was inhibited both by aspartic and metalloprotease inhibitor which permitted to classify this protease as an aspartic-metalloprotease. This protease can be used in dairy industry, meat tenderization as well as protein hydrolysates production.{Figure 7}

Effect of metal ions on protease activity

Effect of different metal ions either monovalent (K+, Na+) or divalent (Ca2+, Mg2+, Fe2+, Zn2+, Ni2+, Li2+, Co2+, and Sn2+) on protease activity was determined using casein as substrate. Metal ions such as Li2+, Na+, K+ Sn2+, and Co2+ have no effect on protease activity, while Mg2+ showed slightly increasing enzyme activity. Ca2+ and Ni2+ ions significantly enhanced the protease activity when Zn2+ and Fe2+ ions greatly shown inhibition [Figure 8]. These results are in line with those reported by some authors Mahajan, Nayak, and Lele.[32] However, Anandharaj et al. Anandharaj, Sivasankari, Siddharthan, Rani and Sivakumar found that Ni2+ ion inhibited metalloprotease from Bacillus alkalitelluris TWI3 Isolated from Tannery Waste.{Figure 8}

Effect of different substrates on protease activity

The protease activity of purified extract from B. enneandra tubers was assayed using both natural (protein from defatted flours of R. heudelotii) and synthetic substrates (casein, BSA, gelatin and hemoglobin). The enzyme was incubated with different substrates and its optimal condition and activities were determined compared to casein, which was taken as 100%. The protease activity decreased from synthetic substrates BSA (83.91%), hemoglobin (64.10%) and gelatin (58.63%) to natural substrate protein from R. heudelotii defatted flours (45.56%) [Figure 9]. Purified protease from B. enneandra is more active on BSA and gelatin than that extracts from the leave of A. integer, but in revenge this protease was more active on hemoglobin. In fact, Balqis and Rosma Balqis and Rosma reported 138, 81 and 24% as a relative activity of protease from the leave of A. integer on hemoglobin, BSA and gelatin, respectively, compared to casein.{Figure 9}

Determination of kinetic parameters

The effect of increasing casein concentration was investigated and the protease activity was calculated as described above. The maximum rate of reaction Vmax and Km were evaluated graphically from regression Lineweaver-Burk plot [Figure 10]. The results revealed that purified protease from B. enneandra tubers had Vmax and Km of 64.935 (U/mL) and 373.941 (μg/mL), respectively.{Figure 10}

 Conclusion



The optimal conditions to extract protease from B. enneandra tubers were found to be 5.1, 40°C and 100 rpm for pH, temperature and agitation frequency, respectively. Crude enzyme extract was purified successively using ammonium sulfate precipitation, gel filtration chromatography Sephadex G200, and gel filtration chromatography Sephadex G75. Characterization of the purified protease showed that its activity was inhibited both by pepstatin A and EDTA proved that it is an aspartic-metalloprotease. The purified protease has optimal pH in acid zone that is 5.1 and its activity is enhanced by Ca2+ ion which suggested that this protease may be useful in the industries for milk clotting. However, to conclude this outcome, this enzyme needs more detailed study on milk clotting properties.

Acknowledgements

SK thanks the SERB for financial assistance under the Fast Track Scheme (SB/FT/LS-190/2012). MA Vijayalakshmi acknowledges the financial assistance from the Department of Science and Technology (DST), Ministry of Science and Technology.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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