L-ASPARAGINASE FROM PHYLLANTHUS EMBLICA (AMLA): A NOVEL SOURCE

L-ASPARAGINASE FROM PHYLLANTHUS EMBLICA (AMLA): A NOVEL SOURCE

Apoorva Singh ¹, Neelam Verma ¹ and Kuldeep Kumar ^{* 2}

Department of Biotechnology ¹, Punjabi University, Patiala - 147001, Punjab, India.

Department of Biotechnology ², Multani Mal Modi College, Patiala - 147001, Punjab, India.

ABSTRACT: The biologically active compounds in plants reckoned their application in medicines from ancient times. Among the biologically active compounds enzymes have evolved as a potential therapeutic agent. L-asparaginase a therapeutic protein is extensively employed in medical sector to diagnose and treat leukemia. Bacteria, fungi, yeast and plants have been identified as potential sources of L-asparaginase. The side effects associated with microbial L-asparaginase restricted their utilization in treatment of leukemia and bought medicinal plants as source into focus. In the present study, different species of Phyllanthus genera have been screened for presence of L-asparaginase in different seasons. The leaves of Phyllanthus emblica with highest enzyme activity (20.3 IU/ml) and specific activity (5.2 IU/mg) emerged out as potential source for L-asparaginase production. Phyllanthus amarus and Phyllanthus niruri also contained appreciable amount of L-asparaginase. This is the first report of identifying presence of L-asparaginase in Phyllanthus genera. L-asparaginase from Phyllanthus emblica was further purified to homogeneity and the molecular mass of enzyme was found to approximately 85kDa. The yield of purified enzyme was 63.54% with 8.5 fold increase in specific activity. Kinetic parameters, K_m and V_max of enzyme, were found to be 3.83 mM and 292.141mM/min, respectively.

L-asparaginase, Phyllanthus genera, Purification, Characterization

INTRODUCTION: Among the biologically active compounds present naturally, enzymes due to their high affinity, specificity and catalytic efficiency has evolved as potential therapeutic agent and has found application in food and textile industries. L-asparaginase from amidohydrolase family catalyzes the breakdown of L-asparagine to L-aspartic acid and ammonia. This action mechanism of L-asparaginase unwrapped various applications of enzyme in food and medical sector.

L-asparaginase enzyme has evolved as anti-leukemic and anti lymphoma agent and reckoned as the largest group of therapeutic enzyme ¹. Unlike normal cells tumor cells rely on asparagine from body fluids for their growth. The asparaginase when injected as anti leukemic agent reduces the free circulating asparagine, leading to starvation of asparagine in susceptible tumor cells ^{2, 3}.

L-asparaginase is also used in food processing industry to avoid the formation of acrylamide (tumor causing agent) during production of starchy food products and as quality assurance parameter for fruit juices ⁴ and vegetables ⁵. L-asparaginase is widely distributed among bacteria, yeast, fungi, actinomycete, plants and rodent serum. Microorganisms such as bacteria, fungi, yeast, actinomycetes and algae have been identified as proficient sources of L-asparaginase. Commercially available L-asparaginase from Escherichia coli and Erwinia carotovora has been extensively acquired as a chemotherapeutic agent in treatment of leukemia ⁶. The L-asparaginase from microbes has low specificity to asparagine leading to sensitivity and toxic side effects in patients treated with enzyme. The side effects associated with microbial L-asparaginase turned the focus of research for screening of other source for L-asparaginase production.

L-asparaginase has been extracted from various plant species Lupinus leuteus, Dolichos lablab seedlings ⁷, Capsicum annum, Tamarindus indica ⁸, Pisum sativum ⁹, Lupinus arboreus, L. angustiplius ¹⁰ and in the root soil of Pinus pinaster and Pinus radiate ¹¹. L-asparaginase encoding cDNA sequence has been isolated from soybean leaves ¹². Withania somnifera has been reported as a potential source of L-asparaginase and its different cytotypes were compared ^{13, 14}. L-asparaginase was also found in Citrus lemon ¹⁵ and Solanum nigrum ¹⁶. Plants have emerged as competent source of enzymes as plant enzymes are easy to handle, have fewer chances of pathogenicity and can be used crude to develop drug formulations which saves cost and time. Also according to literature reports the utilization of L-asparaginase from plants for treatment of acute lymphoblastic leukemia has been studied less ¹⁷. Different species of Phyllanthus genera have been utilized as traditional medicinal value plants from ancient time and have been involved in treatment of common ailments like diabetes, ulcers and urinary treatments ¹⁸. The current work is focused towards screening of different species of Phyllanthus genera for presence of L-asparaginase, followed by purification and characterization of  isolated L-asparaginase which could act as potential anti tumor agent with negligible side effects due to immunological responses.

MATERIALS AND METHODS:

Plant Material: Different plant parts of different species of Phyllanthus genera were collected in different seasons in sterile polythene bags Table 1. The plants are cultivated and well documented by the Department of Botany, Punjab Agriculture University, Punjab.

TABLE 1: SEASON AND PHARMACOLOGICAL RELEVANCE OF SCREENED SPECIES OF PHYLLANTHUS GENERA

Extraction of L-asparaginase: Different parts of selected species of Phyllanthus genera were collected and washed with distilled water. The parts were crushed and homogenized with 0.1M KCl buffer (pH 8.6). It was then centrifuged at 8000 rpm for 20 min at 4 ºC and filtered. The supernatant thus obtained was taken as crude enzyme ⁸.

Enzyme Assay: Nessler’s method based on estimation of ammonia released on breakdown of asparagine by the enzyme was adopted for L- asparaginase assay ²⁷. The reaction between Nessler’s reagent (K₂HgI₄) and ammonia leads to production of pale yellow color. The color intensity is directly proportional to the amount of ammonia present. The standard graph of ammonium sulphate was plotted. Further the enzyme activity of crude enzyme was determined by Nessler’s method and the intensity of pale yellow color was determined by taking absorbance at 480 nm. The micromole of ammonia produced was determined from ammonium chloride standard curve.

Protein Estimation: In order to determine the specific enzyme activity the crude enzyme was subjected to protein estimation by Folin-Lowry’s method with bovine serum albumin (BSA) as standard ²⁸.

Purification of L-asparaginase: L-asparaginase enzyme purification was carried out sequentially according to steps proposed by Scopes ²⁹.

Ammonium Sulphate Precipitation: The crude enzyme was precipitated out by varying the ammonium sulphate saturation from 20% to 100% at 4 °C under mild stirring conditions. The sample was kept for 12 h at 4 °C without stirring. When the precipitation occurred, the sample was centrifuged for 20 min (5000 rpm at 4 °C) and pellet was dissolved in phosphate buffer saline.

Dialysis: The precipitate obtained after ammonium sulphate precipitation was poured in activated dialysis membrane tube and placed in buffer solution (0.01M Na borate buffer). Enzyme activity and protein content of the dissolved pellet and supernatant was quantified after the dialysis.

Q-Sepharose Strong Anion Exchange Chromatography: The dialyzed sample was loaded onto Q Sepharose chromatographic column (1.2 × 8.2 cm) pre-equilibrated with 0.01M sodium borate buffer (pH 8.6) at a flow rate of 1 ml min^-1. The column was washed with 0.01M sodium borate buffer (pH 8.6) to remove the unbound proteins.

Further the elution was carried out by raising the NaCl salt gradient from 0.1M - 0.6M and 1.5 ml fractions were collected at flow rate of 1 mL/min. The fractions were evaluated for enzyme activity and protein concentration.

Molecular Weight Determination: Fractions with highest specific activity along with protein marker were consequently loaded along with loading sample into polyacryl amide gel (10%) as supporting medium to determine the molecular weight and purity of L-asparaginase. Electrophoresis was carried out at 100V. The gel was washed with deionised water and thereafter stained with bromophenol blue and visualized with comassie brilliant blue.

Kinetic Characterization of L-asparaginase: Further L-asparaginase kinetics was studied to determine the effect of experimental parameters like pH, temperature and substrate concentration on the rate of reaction. In order to determine the effect of pH on enzyme activity, the crude enzyme was incubated in 0.01 M sodium borate buffer of different pH range (5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5). The reaction mixture was incubated at different temperature range (4-80 ºC) for 15 min in 0.01 M sodium borate buffer (pH 8.6) to determine the effect of temperature on reaction rate. The determination of effect of substrate concentration was achieved by Lineweaver-Burk’s plot ³⁰ using different L-asparagine concentrations (1.0 to 10.0 mM).

RESULTS AND DISCUSSION:

Potential Plant Source of L-Asparaginase: Different plant parts of selected species of Phyllanthus genus in different seasons were screened for presence of L-asparaginase Table 2. Among all plants, maximum L-asparaginase activity was observed in Phyllanthus emblica (HS16497) leaves during the non fruiting season. Appreciable amount of L-asparaginase was also observed in Phyllanthus amarus, Phyllanthus niruri and Phyllanthus urinari. In all the plants, leaves were identified as potential source for extraction of enzyme in comparison to other plant parts and maximum enzyme activity in leaves was observed during the non fruiting season. Therefore, the results inferred that maximum concentration of L-asparaginase is found in plants at the time of synthesis of new proteins i.e. before the fruiting season. Similar results with high L-asparaginase activity in different medicinal plants before fruiting season has been reported ³¹.

Protein Estimation: The leaves of screened plants showed maximum enzyme activity. The protein estimation and specific activity of the leaves of screened medicinal plants was estimated. The leaves of Phyllanthus emblica showed maximum specific activity of 5.2 IU/mg for L-asparaginase enzyme. Considerable enzyme specific activity was observed in Phyllanthus amarus and Phyllanthus niruri Table 3.

TABLE 2: COMPARATIVE SCREENING ACCOUNT OF SELECTED SPECIES OF PHYLLANTHUS GENERA FOR L-ASPARAGINASE PRODUCTION

TABLE 3: PROTEIN CONTENT AND SPECIFIC ACTIVITY OF L-ASPARAGINASE ISOLATED FROM DIFFERENT SPECIES OF PHYLLANTHUS GENERA

Purification: L-asparaginase from leaves of Phyllanthus emblica with maximum specific activity was further purified by ammonium sulphate precipitation and Q-sepharose anion exchange chromatography. The specific activity of the crude extract improved from 5.2 units/mg to 16.6 units/mg after purification. 40-60% saturation of ammonium sulphate fractionation showed 3.19 fold purification of enzyme with 73% recovery.

TABLE 4: PURIFICATION SCHEME OF L-ASPARAGINASE FROM PHYLLANTHUS EMBLICA LEAVES

Further, the extract was pooled through Q-sepharose anion exchange chromatography. Activity was increased by 8.5 fold when eluted with 0.5 M NaCl solution with 63.54% recovery. The results for purification of L-asparaginase are summarized in Table 4. Comparative purification results have been reported in plants like green chilli ⁸, pea ³² and Withania somnifera ³³.

Single band in the electrophoresis study further confirmed the purity achieved through purification steps. Approximate molecular weight of purified enzyme was found to be 85kDa, as determined by comparing it against protein marker ladder on gel Fig. 1. The findings are in comparison with L-apsarginase isolated from Withania somnifera with molecular weight of 72 KDa ¹³ and from Phaseolus vulgaris seeds of molecular weight 79 KDa ³⁴.

FIG. 1: MOLECULAR WEIGHT DETERMINATION OF L-ASPARAGINASE FORM PHYLLANTHUS EMBLICA LEAVES. (1) Broad range (10-250 kD) Protein marker (2) Ammonium sulphate precipitation (3) Q-Sepharose anion exchange chromatography (4) and (5) Crude enzyme

Kinetic Characterization of L-asparaginase: Kinetic parameters of purified L-asparaginase from Phyllanthus emblica leaves was determined by varying the pH, temperature and substrate concentration. Using Lineweaver-Burk plots Fig. 2 K_m and V_max for enzyme were found to be 3.83 mM and 292.141mM/min respectively. The K_m obtained is found to be comparable to L-asparaginase purified from green chilli (3.3 mM) ⁸ and lower  in comparison to L. arboreus and L. angustifoliusw K_m value of 6.6 and 7.0mM respectively ³⁵ .The K_m value was also found to be comparative with K_m value of L-asparaginase from Escherichia coli (3.5 mM) and Erwinia carotovora (7.14mM) ^{36, 37}. L-asparaginase from Phaseolus vulgaris seeds also showed higher K_m value of 6.72mM ³⁴.

FIG. 2: LINEWEAVER–BURK PLOT FOR THE DETERMINATION OF K_M AND V_MAX FOR L-ASPARAGINASE FROM PHYLLANTHUS EMBLICA

The extract from Phyllanthus emblica showed maximum activity at 37ºC Fig. 3 and 8.5 pH Fig. 4. The optimum pH and temperature were found to be comparative with that of L-asparaginase extracted from microorganisms like Erwinia carotovora ³⁷ and Pectobacterium carotovorum ³⁸. Similar optimum pH and temperature have been reported for L-asparaginase extracted from various plants like Vigna unguiculata ³⁹, Withania somnifera ³³, Solanum nigrum ¹⁶ and Phaseolus vulgaris seeds ³⁴.

CONCLUSION: Four species of Phyllanthus genera were explored for presence of L-asparaginase with highest activity in Phyllanthus emblica. Purified L-asparaginase from Phyllanthus emblica showed comparative similarities with bacterial L-asparaginase. The low K_m value of 3.83 mM and high V_max of 292.141mM/min illustrated high affinity of enzyme towards the substrate. Moreover, the enzyme showed stability over a range of temperature and pH. Furthermore, plant enzymes have lesser chances of pathogenicity and side effects, thus L-asparaginase extracted from Phyllanthus emblica could act as potential therapeutic protein and could be widely further explored for its anti leukemic activity.

ACKNOWLEDGEMENT: The authors wish to thank Modi Education Society and Dr. Khushvinder Kumar, Principal, M. M. Modi College, Patiala, India for encouragements.

CONFLICT OF INTEREST: There are no conflicts of interest.

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    How to cite this article:

    Singh A, Verma N and Kumar K: L-asparaginase from Phyllanthus emblica (amla): A novel source. Int J Pharm Sci & Res 2018; 9(12): 5394-00. doi: 10.13040/IJPSR.0975-8232.9(12).5394-00.

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