Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)

Introduction

α Amylase (α-1,4-gluco-oligosaccharide, EC3.2.1.1) is a hydrolyzing enzyme that randomly cleaves an internal α-1,4-glycosidic linkage in starch and glycogen to produce malto-oligosaccharide and glucose [1]. This enzyme family is widely applied in many purposes including health promotions and industries, such as paper, sugar, textile, ethanol, etc., and it currently accounts for approximately 30% of the world’s industrial enzyme production [2]. Commonly available ones on the market are those from microorganisms, but there are limitations due to their specificities, activities and stabilities. Thus, novel amylases, especially from nature, is important. Those with high activity and stability have been sought out for many years. Recently, the enzymes involved in the fruit ripening stage is outstanding. The predominant carbohydrate in the raw stage of fruit is starch, which is replaced to a large extent by sugars, and α amylases are responsible for this process. Plant and fruit amylases are attractive because of their high activities in converting starch to sugars within a short period [3]. This always occurs at high temperatures over 40°C.

Mango (Mangifera indica Linn.) is a famous tropical fruit, commonly consumed as a dessert. Mango fruit mainly contains water and carbohydrates, with little protein and fat content. In the unripe stage, when carbohydrates is abundant, the carbohydrates are then replaced with sugars such as sucrose, glucose and fructose [4]. For starch content, amylose and amylopectin are the major ones in the fruit. Amylose is a glucose polymer linked by α (1–4) glycosidic bonds in linear structure. Amylopectin is a glucose polymer, as well, but linked by 94%–96% α (1–4) bonds in the linear portion and 4%6% α (1–6) bonds in the branch structure [5]. Ok-Rong mango (M. indica Linn. cv. Ok-Rong) is a native Thai mango commonly grown in all areas of Thailand (local name; Mamuang Ok-Rong). It is a large green tree that grows up to 20 m tall. This mango is edible, both in the green and ripe stage. The ripening one is defined by a soft texture, a yellow peel and unique sweet taste. For Ok-Rong mango, the plastid α amylase gene from Ok-Rong mango has been clarified to be a 594-amino acid residue sequence [6]. Three dimensional structure and catalytic site were computationally predicted. In this study, we intended to further study the α amylase iso-enzymes. They were purified by affinity chromatography using β-cyclodextrin, and characterized for its biochemical properties.

Materials and methods

Results

Purification of α amylases

From the β-cyclodextrin affinity chromatography profile, a single peak of protein with α amylase activity was obtained (Figure 1). The peak was in fractions 21–24. The α amylase from Ok-Rong mango was purified 105-fold with a 4.67% recovery yield and 59.27 U mg⁻¹ of specific activity (Table 1). After being checked by SDS-PAGE and the zymographic method, the α amylase solution exhibited two protein bands with amylase activity toward starch (Figure 2).

Figure 1:

Affinity chromatography elution profile of Ok-Rong mango α amylases from the β-cyclodextrin Sepharose 6B column. The elution of β-cyclodextrin is indicated by an arrow labeled β-CD. The α amylase activity fractions are indicated in a circle.

Table 1:

Purification of the α amylases from Ok-Rong mango.

Procedure	Protein (mg)	Activity (U)	Specific activity (U mg protein−1)	Yield (%)	Purification (fold)
Crude extract	1020.75	570.00	0.56	100	1
70%Amomonium sulfate precipitation	76.35	123.33	1.62	21.63	2.89
β-cyclodextrin Sepharose 6B	0.45	26.67	59.27	4.67	105.83

Figure 2:

SDS PAGE and zymogram analysis of purified amylases from Ok-Rong mango. (A) Crude protein stained with Coomassie blue; (B) purified amylase proteins stained with Coomassie blue; (C) purified amylase protein stained with silver stain; (D) amylase activity detection by zymogram staining. These results represent the two isoforms of α amylase, (E).

Molecular weight and pI determination

Two isoforms of α amylase were successfully separated on a 2D-PAGE profile. After being analyzed with ImageMaster 2D Platinum Software, the upper band weighed 64 kDa and had a pI of 5.0. The lower band was found to be a 60 kDa protein with a pI of 4.6 (Figure 3).

Figure 3:

Isoelectric point analysis of the two α amylase isoforms.

Enzyme characterization

Effect of pH and temperature on α amylase activity

The effect of pH was examined at 37°C using pH values ranging from 2 to 11. The maximum activity of the two isoforms was observed at pH 4 (Figure 4A). To probe the pH stability, the enzyme solution was pre-incubated with different buffers for 1 h. The residual relative activity was higher than 50% at pH 5–9 after pre-incubation and had a decrease of 80% by pH 11 (Figure 4B). To study the effect of temperature, the α amylase activities of two isoforms were determined at different temperatures ranging from 30°C to 90°C. The α amylase activity remained higher than 80% at 40°C–70°C. The optimal temperature was 70°C (Figure 5A). For temperature stability, α amylases activities were studied by pre-incubating the enzyme at different temperatures for 1 h. The α amylase activity remained higher than 80% over the range of 30°C–40°C (Figure 5B).

Figure 4:

Optimal pH (A) and pH stability (B) of the two isoforms of Ok-Rong mango α amylase (solid line, isoform I and dotted line, isoform II). The optimal pH was detected using various buffers. The relative activity was observed as the percentage of the optimum activity. To probe stability, the enzyme was incubated at 25°C for 1 h, and its remaining activity was assayed. Each point represents the mean±SD of three independent experiments.

Figure 5:

Optimal temperature (A) and temperature stability (B) of two isoforms of Ok-Rong mango α amylase (solid line, isoform I and dotted line, isoform II). The relative activity was observed as the percentage of optimum activity. To probe stability, the enzyme was incubated at different temperatures for 1 h, and the remaining activity was assayed. Each point represents the mean±SD of three independent experiments.

Effect of metal ions and reagents on α amylase activity

Zinc ion did not affect α amylase activity, whereas Hg²⁺ completely inhibited the enzyme activity (Table 3). β-Mercaptoethanol had some effect on the activity. The enzyme solution retained more than 80% of its activity. Both isoforms of α amylase were completely inhibited by 10 mM EDTA.

Table 3:

Effect of various metal ions on the purified amylases from Ok-Rong mango.

Treatment	Relative activity (%)
	Isoform I	Isoform II
EDTA	0± 0.0	0± 0.0
Triton-X 100	103± 2.5	109± 0.6
β-mercaptoethanol	84± 3.9	88± 0.9
Hg2+	0± 0.0	0± 0.0
Na+	94± 1.3	102± 6.0
Co2+	84± 4.2	94± 0.5
Zn2+	99± 2.6	97± 2.5
Ca2+	106± 6.5	107± 2.4
Non	100	100

Values are expressed as the mean±standard error of three replicates.

Substrate specificity

Limit-dextrin was relatively found to be the best substrate (Table 4). Amylopectin and soluble starch were good substrates for α amylase from Ok-Rong mango.

Table 4:

Substrate specificity of the amylases from Ok-Rong mango.

Substrate	Relative activity (%)
Limit- dextrin	100
Amylopectin	84±1.1
Starch	82±2.0
Glycogen	20±1.7
Maltose	21±2.8
β-cyclodextrin	0±0.0

Values are expressed as the mean±standard error of three replicates.

Discussion

The finding for novel enzyme still be necessary in the field of enzyme technology for the best one. Plant is one of the most common sources for amylase enzyme studies due to its important role for basic metabolism including rapid degradation of starch during germination and fruit ripening. Mangos, M. indica, normally accumulated starch in pulp as the main storage. Recently, there are one report described that the saliva amylase was able to degraded mango starch. It showed obvious pits and cracks due to the action of amylase in the stomach under the observation by optical microscopy [12]. For amylases in mango fruits, not only pulp, mango peel also exhibited α amylase activity during ripening stage [13]. However, strains and varieties vary their basic biochemical properties. For Alphonso mango, the hydrolase including α amylase showed relatively high activity and gradually increased with maximum levels of activity around climacteric stage [14]. It was observed to be increased in the middle stage of ripening process in Ashwina hybrid mango, as well [15]. This related with an increase in amylase activity only on the 6^th day after harvesting in Haden Mango [16]. The α-type amylase has been detected to bind and degrade starch granules in the early stage of ripening, but it degrade soluble starch for all the period of ripening stage in Keitt mangos [17]. Whereas the β-type amylase exhibited significantly higher activity than the α-type for the early stage just only for starch granule degradation. For application in post-harvesting technology, mango have to be carefully harvested and kept to prevent from any texture damages. Those including spongy tissues in Tommy Atkins mango resulted in three times amylase activity lower than that of healthy pulp [18] and the remaining of unhydrolyzed starch. Our previous study revealed that amylase in Ok-Rong mango pulp showed significantly high activity among Thai fruits during ripening stage (data not shown). Therefore, this study started from the purification of amylase using β-cyclodextrin affinity chromatography. This column was successful for purifying many plant α amylases in just one step [19, 20]. Bands from SDS-PAGE and zymographic analysis were 60 and 64 kDa with apparent α amylase activity. For plant amylases, masses in range 45–70 kDa with pI 4.5–5.5 are quite common (Table 5). They were classified into the low-pI group α amylases [26].

These two isozymes in this study were identified as members of Family 1 of α amylases. Those are found in all plants especially cereal grain endosperm [27] with varieties structure and properties (isozymes) even in one individual organisms. There are five α amylase isozymes in apple [28], two isozymes in root beet [29], and two isozymes in azuki bean [25]. Pea (Pisum sativum) seedlings contained two to seven isozymes [30, 31]. For Keitt mango, at least three bands of amylolytic activity were observed. They became gradually higher in fruit extract at 3 and 5 days after harvesting [17].

Many α amylases in plants showed activity toward starch over a broad range of temperatures, 40°C–70°C (Table 5). The medium optimal temperature, at 70°C, also found for two α amylase isozymes from azuki bean including two isozymes of this report. These enzymes can be classified as mesophilic α amylases, active at 50°C–70°C. Optimal pH of the two Ok-Rong amylase isozymes were four. These corresponded with the increasing of hydrolases activity for carbohydrate degradation as ripening progresses where pH was increasing from 3.4 to 6.0 [32].

α Amylase is a metalloenzyme that contains at least one activating and stabilizing Ca²⁺ ion [33]. Calcium plays an important role in maintaining and stabilizing the structure of α amylase by interacting with negatively charge amino acid residues, such as aspartic and glutamic acid, especially those in their catalytic triad [34]. In previous reports, α amylases from Haden mango, P. sativum, Vigna mungo, V. radiata and V. angularis were shown to be inhibited by 5–10 mM EDTA [16, 19, 20, 23, 25, 35]. Related to this study, both isoforms of α amylase were completely inhibited by 10 mM EDTA. The effects of EDTA indicate that the metal ion is required for this enzyme. The addition of 10 mM Zn²⁺, a known inhibitor of α amylase from many sources [36], showed no effects on the enzyme. α Amylase from Chokanan mango exhibit 61%remaining activity in 5 mM Zn²⁺ solution [13]. α Amylase from Pachyrhizus erosus L. tuber has 42.5% activity after the addition of 10 mM Zn²⁺, whereas 100 mM Zn²⁺ loss 90% activity [37]. Hg²⁺ completely inhibited the activity. Ranwala and Miller reported the complete inhibition in tulip by 2 mM Hg²⁺ and Ag⁺, as well [38]. The addition of β-mercaptoethanol, in this study, slightly affected the activity of the α amylases. The study of the disulfide bridges in cold-active α amylase demonstrated that none of disulfide bonds are important in stabilizing the native structure of the enzyme [39]. However, charged amino acids play an important role in the conformation of amylases. Salt-bridges are often abundant in thermostable proteins predicting of their role in enzyme stability [40].

Limit-dextrin, amylopectin and starch were suggested to be good substrates for Ok-Rong mango α amylases. The enzymes showed low activity toward glycogen, a high branch-point polysaccharide. These results related to the endo-acting property of α amylase.

Conclusions

α Amylases from Ok-Rong mango (M. indica) were purified by β-cyclodextrin affinity chromatography for the first time with a final specific activity of 59.27 U mg⁻¹. The two isozymes of α amylase from Ok-Rong mango were characterized. Both were major enzymes and classified as members of the low-pI group of amylases with identical structure, properties and functions. They are mesophilic. It is highly possible that these enzymes could be applied for many purposes, including industries where a variety of enzymatic reactions is needed.