Optimization of pectinase extraction from mango (Mangifera indica cv. Chokanan) peel using response surface methodology

Today pectinases (EC 3.2.1.15) have become an integral part of the food and feed industry and plant peel could be a potential source of pectinase. Thus, the main objective of the study was the optimization of pectinase extraction from mango (Mangifera indica cv. Chokanan) peel. For this purpose, response surface methodology (RSM) was employed to optimize the extraction conditions and the effect of independent variables, namely temperature (-25 to +25°C), mixing time (2–10 min) and pH of buffer (1–8), on specific activity, storage stability, temperature stability and surfactant agent stability of pectinase from mango peel was investigated. The study demonstrated that using optimum temperature, mixing time and pH of buffer, protected pectinase during extraction, as indicated by low activity and low stability loss. It was found that the interaction effect of mixing time and buffer content improved the pectinase stability, and pH of buffer had the most significant effect on specific activity of the pectinase. The ideal condition of 2.5°C temperature, 6 min mixing time at pH 4.5 was established for pectinase extraction from mango peel. The result indicated that the optimized extraction of pectinase from mango peel provides high activity and stability of pectinase in harsh conditions, which makes the enzyme suitable for use in various types of industry and biotechnological applications. Furthermore, there was not any significant (p>0.05) difference between the experimental and predicted values. This ensured that the response surface models used to indicate property changes of pectinase as a function of enzyme extraction conditions were sufficient

Characterization of serine protease from mango (Mangifera indica cv. Chokanan) peel

Mango (Magnifera indica cv. Chokanan) is one of the most popular tropical fruits in the world and currently ranked 5th in total world production among the major fruit crops. Mango peel is one of the major wastes of food and beverage industries, however, it can be used as a valuable, economic and available media sources for commercially producing the natural enzymes. This study aimed at characterizing serine protease which was previously purified from mango peel by alcohol salt aqueous two-phase system (ATPS). The molecular weight of purified protease was determined with sodium dodecyl polyacrylamide sulfate gel electrophoresis (SDS-PAGE) to be 65 kDa, it showed maximum activity (> 80%) at pH 4-10 and exhibited high thermal stability (>90%) for 60 min at 65°C with the highest activity at 70°C and at pH 8. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The activity of the protease was activated by Ca2+and Mg2+ while Li+, Na+, K+ and Sn2+ had no effect on the protease activity. However, reduction in the activity of protease was observed in the presence of Ba2+, Zn2+, Pb2+, Co2+, Mn2+ and Cu2+. The enzyme was resistant to denaturation by sodium dodecyl sulfate and the non-ionic surfactants such as Tween 80 and Triton X-100. The properties of serine protease extracted from mango peel and discussed in this study made it applicable in industrial processes that are performed under high temperature, in alkaline medium, or in the presence of denaturants and surfactants

Optimization of extraction of novel pectinase enzyme discovered in red pitaya (Hylocereus polyrhizus) peel

Plant peels could be a potential source of novel pectinases for use in various industrial applications due to their broad substrate specificity with high stability under extreme conditions. Therefore, the extraction conditions of a novel pectinase enzyme from pitaya peel was optimized in this study. The effect of extraction variables, namely buffer to sample ratio (2:1 to 8:1, X1), extraction temperature (−15 to +25 °C, X2) and buffer pH (4.0 to 12.0, X3) on specific activity, temperature stability, storage stability and surfactant agent stability of pectinase from pitaya peel was investigated. The study demonstrated that the optimum conditions for the extraction of pectinase from pitaya sources could improve the enzymatic characteristics of the enzyme and protect its activity and stability during the extraction procedure. The optimum extraction conditions cause the pectinase to achieve high specific activity (15.31 U/mg), temperature stability (78%), storage stability (88%) and surfactant agent stability (83%). The most desirable conditions to achieve the highest activity and stability of pectinase enzyme from pitaya peel were the use of 5:1 buffer to sample ratio at 5 °C and pH 8.0

Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%

Characterization of polyphenol oxidase from mango (Mangifera indica L. cv. Chokanan) peel

Plant polyphenol oxidase showed positive effect in the production of coca, black tea and flavonoid-derived colorants and antioxidants. High activity and stability in a wide range of pH and temperature of plant enzyme make it suitable and also inexpensive for use in industry. For these reasons, there is growing interest in seeking more plant sources of polyphenol oxidase. Mango (Mangifera indica L. cv. Chokanan) peel can be a potential source of polyphenol oxidase, which has been extracted and purified from peel of mango using the aqueous two-phase system (ATPS). In the present study, the effects of different temperatures, pH, inhibitors and metal ions on the stability and activity of polyphenol oxidase from mango peel were investigated. In addition, the molecular weight of this enzyme was estimated at 133 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The highest enzyme activity of polyphenol oxidase to catalyze catechol in sodium phosphate buffer was achieved at 55°C at pH 5.5. Furthermore, the enzyme was stable at temperatures of 10 to 60°C and pH 3 to 6. Beta-mercaptoethanol, ascorbic acid, l-cysteine and pyrogallol were effective inhibitors of the enzyme. Also, activity of polyphenol oxidase was increased in the presence of some metal ions such as Ca2+, Mg2+ and Cu2+ which implies that the enzyme involved metal ions. Therefore, polyphenol oxidase extracted from mango (Mangifera indica L. cv. Chokanan) peel has potential applications in various industries because it is thermostable under high temperatures in either acidic medium, or when there is the presence of metal ions

Characterization of pectinase from mango (Mangifera indica Cv. chokanan) peel

Today, pectinase has emerged as an integral part of the food and feed industries. Plant peel could be a potential source of pectinase, which has been extracted and purified from mango (Mangifera indica cv. Chokanan) peel using the aqueous two-phase system (ATPS). In the present study, the effects of temperature, pH and metal ions on the stability and activity of pectinase were investigated. In addition, the molecular weight of this enzyme was determined as 31 kDa with SDS-PAGE. Pectinase showed the highest enzyme activity at 60ºC for 30 min after incubation at different temperatures (20 to 80ºC). Also, this enzyme has been shown to be thermostable because more than 90% of residual enzyme activity was retained at temperatures of 20 to 60ºC for 30 min. Pectinase was incubated in different pH from 3 to 9 and the highest enzyme activity was achieved at pH 8. Furthermore, the enzyme was stable at pH 5 to 9 after enzyme incubation at different pH for 24 h at 4ºC. Activity of the enzyme was significantly decreased at pH 3 and 9 due to the protein denaturation. Pectinase activated by Ca2+ showed that this cation has an important effect on activity and stability of the enzyme; but Li+, Na+ and K+ had no effect on its activity. Also, the reduction in the activity of pectinase was observed in the presence of Fe2+, Cu2+, Mn2+, Zn2+ and Al3+. Therefore, pectinase extracted from mango peel has potential applications in various industries like food and feed because it is thermostable under high temperatures in either alkaline medium or when there is the presence of metal ions

Purification of a novel protease enzyme from kesinai plant (Streblus asper) leaves using a surfactant–salt aqueous micellar two-phase system: a potential low cost source of enzyme and purification method

Serine protease from kesinai leaves was purified for the first time by a surfactant–polymer aqueous micellar two-phase system. The effectiveness of different types and concentrations of non-ionic surfactants (Pluronic series and X-114) on the partitioning behaviour of the protease was evaluated. The results showed that the enzyme preferentially partitioned into the bottom surfactant-rich phase, while the hydrophilic amino acid preferred the top aqueous phase. This distribution of the enzyme is due to the hydrophobic interaction of the serine protease with the hydrophobic lid of the micelle core in the bottom phase. The influence of different types of salts (K2SO4, KH2PO4, KCl and KNO3) on the purification and selectivity of the enzyme was determined. The protease partitioning in the bottom phase increased in the presence of KNO3, which confirmed that the salt was able to improve the protein solubility in bottom phase and increase the hydrophobic interaction between the two phases. In addition, the protease from the bottom phase was re-extracted to a new aqueous phase solution to remove and recycle the surfactant. Addition of potassium thiocyanate led to the partitioning of the enzyme in top aqueous phase due to high ionic strength of SCN−, which forced the lighter micellar phase toward the upper position of the system. A high purification factor (10.3) and yield of 92 % of the enzyme were achieved in a solution of 31 % of Pluronic L61 using 0.3 % KNO3 and 50 % crude feedstock at pH 7.0

Purification and recovery of serine protease from mango (Mangifera indica cv. Chokanan) waste using aqueous two-phase system: potential low cost of enzyme and purification method

Serine proteases are one of the most important groups of protease enzymes which have been used widely in different types of industries and biotechnological applications. Plant peel could be a potential source of proteases due to easy purification methods, low levels of interfering substances during purification and good yield of proteases. Thus, in this study, serine protease as valuable component for the first time was purified and recovered from mango (Mangifera indica cv. Chokanan) waste using aqueous two-phase system based on polyethylene glycol and salt. Response surface methodology (RSM) with a central composite design (CCD) was employed to optimise the aqueous two phase system. The effectiveness of important parameters such as different molecular weight of polyethylene glycol (3000-8000 g/mol, X1), tie lie length (23-34% w/w, X 2) and NaCl (0-10% w/w, X3) on purification factor (Y1), yield (Y2), partition coefficient (Y3) and selectivity (Y4) of serine protease from mango waste was determined. Purity and molecular weight of the enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The partition coefficient of enzyme was decreased by increasing of the polyethylene glycol molecular mass. In addition, the phase composition showed a significant effect on yield and partition coefficient of the enzyme. Based on this system, the purification factor of serine protease from peel of Mangifera indica cv. Chokanan was increased to 12.51 with a high yield of 89%. Therefore, this study proves that aqueous two-phase system canbe an inexpensive and effective method for purification of serine protease from mango peel

Characterization of novel amylase enzyme from mango (Mangifera indica cv. Chokanan) peel

Amylase is one of the important industrial enzymes used in different types of industries such as food, detergent, pharmaceutical, pulp and paper. Mango peel could be a potential source of amylase, which has been extracted and purified from mango (Mangifera indica cv. Chokanan) peel using alcohol/salt, aqueous two phase system. In this study, the effect of temperature, pH, metal ions, inhibitors and surfactant agents on amylase activity and stability were investigated. In addition, purity and molecular weight of amylase was determined using sodium dodecyl sulphate gel electrophoresis. Amylase showed the highest activity and stability at 50°C for 20 min after enzyme incubation at different temperatures (20 to 90°C) in interval time. Amylase from mango peel is thermostable because more than 85% of enzyme activity was retained at temperatures of 20-55°C for 20 min. The amylase was incubated at pH 3-10 and the highest enzyme activity was obtained at pH 7.0. The enzyme activity was significantly decreased at pH 3.0 and 10 because of protein denaturation. Molecular weight of amylase from Mangifera indica L. cv. Chokanan was 42 kDa. Activity of amylase was significantly (p < 0.05) increased in presence of Ca2+ but Zn2+ and Cu2+ reduced the enzyme activity due to replacing of calcium cation from the binding site of amylase. In addition, the effect on amylase activity was investigated at a concentration of 5 mM. The enzyme was completely deactivated in presence of carbodimine and p-chloromercuribenzoic acid whereas iodoacetamide did not show any significant (p <0.05) effect on amylase activity. Thus, amylase from mango peel with this unique characteristic has potential application in various kind of industries such as food, detergent, pharmaceutical and biotechnological applications

Purification and characterization of a novel amylase enzyme from red pitaya (Hylocereuspolyrhizus) peel

An amylase enzyme from red pitaya (Hylocereuspolyrhizus) peel was purified 234.2 folds with 72.1% recovery using ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Gel filtration chromatography and SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 42.1 kDa. The apparent Kmand Vmaxof the amylase were 2.7 mg/ml and 34.30 u/min/mg of protein, respectively. The enzyme was highly active and stable over a wide pH range from pH 3 to pH 11.0, with optimum activity being observed at pH 5.0. The enzyme was highly selective for soluble starch, amylopectin, glycogen and pulullan. The purified amylase did not require calcium and displayed extreme stability with regard to surfactants and oxidizing agents. EDTA, a powerful chelating agent, did not have any significant effect on the stability of the enzyme. Such characteristics have not been previously reported for this type of enzyme from fruit peel. This enzyme, which possesses unique properties, could be widely used in different types of industries, especially in food and biotechnological applications