Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex

Abstract

Context: A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties.

Objective: Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity.

Materials and methods: Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed.

Results: Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4–12). Bidhi amylase activity increased to 260% by addition of 10⁻³ mM Fe²⁺ or 10⁻² mM Cu²⁺, and was strongly inhibited by Mg²⁺ and EDTA. In the presence of Ca²⁺ and Mg²⁺, Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C.

Discussion and conclusion: Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was β-fructose, α(1–4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

Keywords::

• Saccharose
• glucose
• metal ion
• detergents
• HPLC

Introduction

Amylases are among the most important enzymes used in modern biotechnology, particularly in the processes involving starch hydrolysis. The extensive application of amylases in food, starch liquefaction and saccharification, detergent, textile, paper, brewing and distilling industries has paved the way for their large-scale commercial production (Gupta et al., 2003). Although amylases originate from different sources, plants, animals and microorganisms, most of the available commercially produced ones are of microbial origin (Pandey et al., 2000). Because of the industrial importance of amylases, there is an increasing worldwide interest in screening of new microorganisms producing amylases suitable for new industrial applications (Burhan et al., 2003; Gupta et al., 2003). Today, a large number of microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis of starch in the starch processing industry (Pandey et al., 2000). Moreover, a large number of plants remain to be investigated for the screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex from Kahli and Bidhi varieties that differ by the color of their fruits. Bidhi means white, the color of the mature fruit is green, and Kahli means black, the color of the mature fruit is dark purple. Saint Pedro figs produce two crops every year. In spring, the first crop (breba) forms on wood from the previous year and matures in May to June. The second crop (main crop) sets on new season wood in March to May. This crop usually matures between July and August. The edible fig is a number of the genus Ficus that belongs to the family Moraceae and to the order Urticales (Hutchinson, 1959). Moraceae encompasses trees and shrubs that characteristically have a milky juice containing remarkable molecules with several biological properties. Lazreg Aref et al. (2011) reported that F. carica latex contains polyphenols, sesquiterpens, coumarins and flavonoids. The latex of at least five of the genera plants contains high proteolytic activity (Robbins & Lamson, 1934; Tauber, 1949), but the amylolytic content of this species has never been studied.

The aim of this study is to determine the characterization of amylase from the latex of Tunisian indigenous Saint Pedro immature (breba) crop figs. These varieties were screened, characterized and compared.

Material and methods

Extraction of crude latex

Latices of Kahli and Bidhi were obtained by collection at the end of April and the beginning of May 2010 from severed fruit stalks of unripe fruit growing in the variety plot at the high school of horticulture of Department of Agriculture, and Arboriculture, Chott Meriam Sousse, Tunisia. All trees were labeled and reliability of identification was assured by Pr. Massoud Mars, Professor of Arboriculture. The droplets of latex were collected directly into polyethylene centrifuge tubes, frozen immediately in dry-ice and maintained in frozen state at −20°C until the analyses were performed. The gum, approximately 30% by weight in F. carica latex, was removed from the aqueous solution by centrifugation in a refrigerated centrifuge (Bakemann Avanti TM30) at 15 000 rpm for 60 min at 0°C. The clear, straw-colored aqueous solution that was designated as crude extract, which contained between 10 and 17% protein, was frozen and stored at −25°C until required for analysis. Approximately 90% of this protein has proteolytic activity (Sgarbieri et al., 1964).

Amylase assay

Enzyme assay was performed by measuring the amount of reducing sugar released according to the dinitrosalicylic acid (DNSA) method (Bernfeld, 1955). The reaction mixture consists of 0.5 mL of 1% (w/v) soluble potato starch (Sigma) in 0.1 M phosphate buffer (pH 7) for Kahli extract and 0.1 M acetate buffer (pH 6.5) for Bidhi extract and 0.5 mL of the appropriately diluted enzyme. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing end groups per minute. d-Glucose was used to construct a standard curve. All the experiments were performed independently in triplicate, and the results are presented as the mean of three.

Effect of pH on amylase activity and stability

The effect of pH on amylase activity was studied in the pH range 3–12 at 45°C. For measurement of pH stability, the crude enzyme was incubated at 4°C for 60 min in different buffers and residual activity was determined under the enzyme assay conditions. The following buffer systems were used: 100 mM glycine–HCl buffer (pH 2–4); 100 mM acetate buffer (pH 4–6); 100 mM Tris–HCl buffer (pH 7–8); and 100 mM glycine–NaOH buffer (pH 9–12).

Effect of temperature on amylase activity and stability

The effect of temperature on amylase activity was studied from 20 to 70°C. Thermal inactivation of the crude enzyme was also examined by incubating the enzyme preparation at various times (15–120 min) at 80°C. Aliquots were withdrawn at desired time intervals and the remaining activity was measured under enzyme assay conditions. The non-heated latex amylase was considered as control (100%).

Effect of metal ions and inhibitors on amylase activity

A stock solution of each salt was prepared at concentration ranges of 10⁻⁶ to 1 mM. The effect of some salts/cations (CaCl₂, CuCl₂, MgCl₂, FeSO₄, ZnSO₄•H₂O) on enzyme activity was also determined. All the used metals were in the chloride and sulfate forms. The substrate/salts mixture was also incubated before it was used for enzyme assay. The enzyme activity was assayed under standard assay conditions, and activity in the absence of any additives was taken as 100%. The influence of enzyme inhibitors on amylase activity was also studied using 1, 5 and 10 mM of phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA). The crude enzyme was pre-incubated with inhibitors at 45°C for 15 min after which the residual activity was calculated using 1% potato starch as a substrate in standard assay conditions. The activity of the enzyme assayed in the absence of inhibitor was taken as 100%.

Evaluation of enzyme for use in detergent formulation

The detergent bands used were Ariel^® (Proctor and Gamble, Suisse), Omino Bianco^® (Bolton, Belgium), Dixan^® (Henkel, Spain), OMO^® (Unilever, France) and Nadif^® (Henkel-Alki, Tunisia). They were diluted in double distilled water to a final concentration of 7 mg/mL to stimulate washing conditions and heated at 100% for 15 min to inactivate the enzyme that could be part of their formulation. The detergents were added to the reaction mixture and the reaction was carried out under standard assay conditions. To determine the stability of Bidhi and Kahli amylase in the presence of the different detergents, 10 µL of crude enzyme was added in detergent solution and incubated at 80°C for 60 min. Aliquots of 0.5 mL were taken at different time intervals and the residual activity was determined at 45°C and compared with the control sample incubated at 80°C without any detergent (Phadatare et al., 1993; Banerjee et al., 1999).

Chromatographic analysis of the starch hydrolysis products

TLC analysis

The starch hydrolysis products were subjected to thin-layer chromatography (TLC) with silica gel 60 (Merck, Germany) in the solvent system composed of butanol/ethanol/water (5:3:2, v/v/v). The spots were visualized by spraying TLC plates with H₂SO₄/methanol (5:95, v/v) followed by heating at 120°C for 10 min (Fontana et al., 1988).

HPLC analysis

A reverse-phase high-performance liquid chromatography (HPLC) technique was developed to identify and quantify the starch hydrolysis compounds. Concentrations were calculated based on peak areas compared to those of external standards. The HPLC chromatograph was a Schimadzu apparatus equipped with an LC-10ATvp pump and SPD-10Avp detector. The column was 4.6 × 250 mm (Shim-pack, VP-ODS) and the temperature was maintained at 40°C. The flow rate was 0.5 mL/min. The mobile phase used was 0.1% acetic acid in water (A) versus 80% acetic acid in acetonitrile (ACN) (B) for a total running time of 40 min, and the gradient was changed as follows: Solvent B started at 20% and increased immediately to 50% in 30 min. After that, elution was conducted in the isocratic mode with 50% solvent B within 5 min. Finally, solvent B decreased to 20% until the end of running time.

Results and discussion

Effect of temperature on amylase activity and stability

The effect of temperature on amylase activity was measured at pH 6.5 and 7 for Bidhi and Kahli extracts, respectively, over a temperature range of 20–70°C. As shown in Figure 1A, the same effect of temperature was observed in both extracts. The amylase activity increased with the temperature to reach an optimum at 45°C and thereafter, decreased slowly at higher temperature. These values closely resembled those of α-amylase (Barly amy III) from barley Hordium vulgare L. (Poaceae) (Al-bar, 2009) and those of finger millet Eleusine coracana (Poaceae) α-amylase α-1(b), α-3 (Nirmala & Muralikrishna, 2003). An optimum temperature of 55°C was reported for α-amylase from cockroach Periplaneta americana Linnaeus (Blattdae) (Dué et al., 2008) and Safflower, wheat and pearl millet (Nirmala & Muralikrishna, 2003; Muralikrishna & Nirmala 2005). The effect of temperature on the stability of both extract varieties was also checked (Figure 1B). Our results showed that the Bidhi amylase was stable up to 45 min of incubation at 80°C. In contrast under the same conditions, the Kahli amylase retained only 70% of its initial activity. Figure 1B also showed that the amylase of Kahli and Bidhi displayed a half life (t_1/2) of 60 and 85 min, respectively, at 80°C. However, the amylase activity was rapidly lost and no activity was detected after 120 min of incubation. Al-bar (2009) reported that amylase from barley H. vulgare was stable up to 40°C, and the enzyme lost 50% of its activity after 30 min of incubation at 50°C. Thermic incubation study of P. americana α-amylase showed that the enzyme retained 7.6% of its original activity after being incubated at 55°C for 60 min and 85% of its activity at 37°C and the enzyme was stable for 20 min until 55°C; above this temperature, the enzyme was rapidly inactivated (Dué et al., 2008).

Figure 1.  (A) Effect of temperature on Bidhi (-□-) and Kahli (-▪-) amylase activity. (B) Thermal inactivation of Bidhi and Kahli amylase at 80°C. The enzyme activity was tested at various temperatures using soluble potato starch as substrate at pH 6.5 and 7 for both extracts, respectively. Residual activity was determined from 0 to 120 min. Values are means of three independent experiments. The initial activity before pre-incubation was taken as 100%. Standard deviations were <5%.

Effect of pH on amylase activity and stability

The effect of pH on amylase activity was studied using soluble potato starch as a substrate at various pH values at 45°C. The pH activity profile of both Kahli and Bidhi amylase is shown in Figure 2A. Interestingly, both enzymes were highly active between pH 3 and 12 with an optimum around pH 6.5 and 7 for Bidhi and Kahli amylases, respectively. An optimum pH of 6 was reported for amylases from malted wheat α-1, malted barley α-1 and malted finger millet α-2 (Kruger & Lineback, 1987; Muralikrishna & Nirmala, 2005). The optimum pH for Kahli amylase can be considered similar to α-amylases from archaeon Haloferax mediterranei (Halobacteriaceae) (pH 7–8) (Pérez-Pomares et al., 2003). Like the amylolytic enzymes characterized from different organisms, the amylases from Kahli and Bidhi were also stable in a wide range of pH as shown in Figure 2B; both amylases were quite stable in acidic and basic ranges of 4–12 and over 65% of their original activities were retained, but Kahli amylase seemed to be more stable at basic pH ranging from 9 to 12. It was reported that Rhizobia amylases were also very stable, retaining more than 70% of their original activities in the pH range of 4.5–9.5 (De Oliveira et al., 2010).

Figure 2.  Effect of pH on Kahli (-▪-) and Bidhi (-□-) amylase activity (A) and stability (B). The enzyme activity was tested at various pHs using soluble potato starch as substrate at 45°C. The pH stability of both amylases was determined by incubating each enzyme in different buffers for 1 h at 4°C and the residual activity was measured at pH 6.5 and 7 for Kahli and Bidhi amylase, respectively, at 45°C. Buffer solutions used for pH activity and stability are mentioned in Materials and Methods section. The initial activity before pre-incubation was taken as 100%. Standard deviations were < 5%.

Effect of various metal ions and inhibitors

The effect of various metal ions on Bidhi and Kahli amylases was also investigated and their activities were measured at pH 6.5 and 7, respectively, at 45°C in the presence of increasing concentrations of Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺ and Fe²⁺. As shown in Figure 3A and 3B, the effect of metal ions revealed that Mg²⁺ caused an interesting activating effect on Kahli amylase at the concentration of 10⁻⁶ mM, but it strongly inhibits the activity of Bidhi amylase. Similar result was obtained by Dué et al. (2008), who reported that Mg²⁺ activated P. americana α-amylase. It can also be noticed that Bidhi and Kahli displayed a 2.5-fold increase in their amylase activity values when adding 10⁻⁶ and 10⁻⁵ mM Ca²⁺ to the medium of reaction (Figure 3A and 3B). These findings are in accordance with earlier reports showing plant and animal α-amylases are metalloenzymes that contain a Ca²⁺-binding domain that is important for the stabilization of the tertiary structure (Vallee et al., 1959; Berbezy et al., 1996). Cereal α-amylases are known to be metalloenzymes containing at least one Ca²⁺ per molecule (Janeck & Belaz, 1992) and its number may go up to 10 (Vihinen & Mäntsälä, 1989). It was suggested that Ca²⁺ ion appears to stabilize the active site cleft by inducing an ionic bridge between the domains. All plant α-amylases appear to contain loosely bound Ca²⁺ compared to microbial enzymes and its removal results in both irreversible as well as reversible inactivation resulting in the loss of thermal stability (Greenwood & MacGregor, 1965; Thoma et al., 1971).

Figure 3.  Effect of increasing Zn²⁺ (□), Cu²⁺ (☆), Fe²⁺ (▵), Mg²⁺ (▾) and Ca²⁺ (○) ion concentrations on Kahli (A), and Bidhi (B) amylase activities was determined after 5 min incubation in potato starch (1%) as substrate at pH 6.5 and 7, respectively, and 45°C. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).

The obtained results showed that Zn²⁺ had no effect on Kahli and Bidhi amylases; although the ions Fe²⁺ and Cu²⁺ had no effect on Kahli, they exhibited an interesting activation effect on Bidhi amylase at 10⁻⁴ and 10⁻³ mM, respectively. Cu²⁺ had a slight inhibitory effect on enzyme activity from central Amazonian Rhizobia strains at concentrations of 1 and 5 mM (De Oliveira et al., 2010).

The effect of some inhibitors was also studied (Figure 4A and 4B). This figure shows that when Kahli amylase was incubated in minimal concentrations of EDTA, PMSF and β-mercaptoethanol, the enzyme activity was retained at 85, 100 and 100% of the original activity, respectively. As shown in Figure 4A and 4B, in contrast to the Kahli enzyme, the Bidhi amylase was strongly inhibited by the EDTA and only 15% of its activity was retained. Comparable with these results, amylase activity from the Rhizobia strains was reduced by up to 30% in the presence of EDTA (De Oliveira et al., 2010).

Figure 4.  Effect of inhibitors on Kahli (A) and Bidhi (B) amylase activities was determined using potato starch (1%) as substrate at pH 6.5 and 7, respectively, and 45°C. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).

Compatibility and stability of enzyme with commercial detergents

Studies on the effect of detergents on amylase activity (Figure 5A, 5B, 5C and 5D) showed that Bidhi and Kahli enzyme activities increased in the presence of OMO^® and Omino Bianco^® and were almost the same as those of the control in the presence of Ariel^® with little differences in activity levels (Figure 5A and 5B). On the other hand, Bidhi amylase was severely inhibited in the presence of Nadif^® and Dixan^® but Kahli amylase saved over 50% of its activity. The compatibilities of Bidhi and Kahli amylases with certain commercial detergents were shown to be good as the enzymes retained 98 and 89%, 98 and 87% and 80 and 70% of their activities after 30 min of incubation at 80°C in the presence of the detergent brands, Omino Bianco^®, OMO^® and Ariel^®, respectively (Figure 5C and 5D).

Figure 5.  Compatibility of amylase activities from Bidhi (A), (C) and Kahli (B), (D) with commercial detergents (-▪- Omino Bianco^®, -○-OMO^®, -★- Ariel^®, -▿- Nadif^®, -♦- Dixan^®). The activities are expressed as a percentage of the activity levels in the absence of detergents (100%).

Product profile of the enzyme

TLC technique was used for analyzing the hydrolytic products of the enzyme action on soluble starch (Figure 6A and 6B). Hydrolyzed products in various time courses (0–60 min of incubation) of Kahli amylase digestion are shown in Figure 6B, exhibiting the degradation of substrate into products mainly saccharose (G₂) and maltotetraose (G₄). Glucose (G₁) and other malto-oligomers (maltotriose G₃ and maltopentaose G₅) were absent during the course of the substrate hydrolysis. On the other hand, after 15 min of reaction with Bidhi amylase, G₂ was observed as a predominant product (Figure 6A) and when further hydrolysis was performed, G₁, G₃, G₄ and G₅ appeared; G₁ was produced after 30 and 60 min of incubation. The study of the influence of time on amylases activities was confirmed by HPLC (Figure 7). This showed that the increase in reaction time leads to the decrease in saccharose concentration for Kahli amylase (Table 1), and the increase of glucose and decrease of saccharose for Bidhi amylase (Table 2); fructose appeared after every 30 min of incubation, and then after 30 min a part of glucose was transformed into fructose. Dué et al. (2008) reported that the action of purified amylase from cockroach (P. americana) on soluble starch revealed the presence of maltose and maltodextrin but no glucose in the reaction medium after 2 h of reaction. These results indicated the endoamylolytic character of Bidhi enzyme that can be classified as β-fructose and α(1-4) glucose. One of the most significant technological advances in starch technology is the conversion of starch to glucose, and subsequent conversion of glucose into fructose by glucose isomerase to yield high fructose syrups (Greenwood & MacGregor, 1965).

Table 1.  Hydrolyzed products in various time courses (0–60 min) of digestion by Kahli and amylase.

Time (min)	0	15	30	45	60
Saccharose, g/l	—	8.088	6.652	6.169	4.700
Glucose, g/l	—	—	—	—	—
Fructose, g/l	—	—	—	—	—

Table 2.  Hydrolyzed products in various time courses (0–60 min) of digestion by Bidhi amylase.

Time (min)	0	15	30	45	60
Saccharose, g/l	4.010	11.288	7.211	9.654	4.969
Glucose, g/l	—	—	4.042	—	—
Fructose, g/l	5.502	—	8.776	—	2.372

Figure 6.  TLC analyses of reaction products of Bidhi (A) and Kahli (B) amylase from the left to right; standard oligosaccharides (lane 1), saccharose (lane 2), reaction products for 0 min (lane 3), 5 min (lane 4), 15 min (lane 5), 30 min (lane 6), 45 min (lane 7) and 60 min (lane 8). Lane 1 indicates the standard oligosaccharides (G₁, glucose; G₂, maltose; G₃, maltotriose; G₄, maltotetraose; G₅, maltopentose).

Figure 7.  High-performance liquid chromatography of starch hydrolysate produced during serial time of incubation (0, 15, 30, 45 and 60 min) by Bidhi (2, 3, 4, 5, 6) and Kahli (7, 8, 9, 10, 11) amylase. Standard (1): saccharose, glucose and fructose.

Conclusion

The present study adds further information about the enzymatic activities of F. carica latex, which contains interesting amylolytic activities. The studied amylases from these varieties of F. carica are highly thermostable in the presence of starch and insensitive to inhibition by glucose. These properties may provide a very efficient way of producing glucose from starch and justify this study on the amylolytic activity of F. carica. Bidhi enzyme could be a potential candidate to be used on its own for complete hydrolysis of high-concentration raw starch to glucose and fructose. Considering its high activity and stability in a wide range of pH (4.5–12), at high temperatures (80°C) and Ca²⁺-independent hydrolyzing mechanism, Bidhi amylase is a good choice for application in starch, food and detergent industries but also in medical and therapeutical field.

Acknowledgments

The authors are grateful to Pr. Ben Ouada Hafed (Directeur de l’Institut Supérieur des Sciences Appliquées et de Technologie de Mahdia) and Pr. Abdelwaheb Fekih (Laboratoire de Chimie, 03/UR/1202, Faculté de Médecine Dentaire, 5000 Monastir, Tunisia) for providing facilities for this work.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.