Extraction and characterization of Arylesterase enzyme from (peel and pulp) of Annona muricata fruit

The extraction of arylesterase from the aqueous extract of (peel and pulp) of Annona muricata fruit was conducted using different biochemical techniques. It was shown that, max activity was obtained in the pulp than in peel, and by using gel filtration chromatography on sephadex G-75 for the pulp part, the solution of the proteinous precipitate produced by acetone precipitation, contained three proteinous peaks. The activity for peak A (1653) and peak B(2310.9) but the third peak C was very low. while maximum specific activity was obtained in the second peak (B) which showed (23107), (18366 ) IU/ml /mg for (A) and very low for the third one (C), and (12.58),(26.531) folds of purification for B and A peaks respectively .Furthermore, the comparative molecular weight of the partially purified isoenzymes arylesterase Kirkuk University Journal /Scientific Studies (KUJSS) Volume 12, Issue 2, March 2017 ISSN 1992 – 0849 Kirkuk University Journal /Scientific Studies (KUJSS) Volume 12, Issue 2, March 2017 ISSN 1992 – 0849 (peaks B and A) using gel filtration were found to be (92,129), (86,895) Dalton respectively. The optimum conditions of arylesterase were determined as maximum activity was obtained using (9 mM) Tris – HCl as a buffer at pH (7.2), with incubation temperature (37oC), incubation time (25min) and (4mM) of phenyl acetate as a substrate. Using Linweaver–Burk plot, it was found that maximum velocity (Vmax) and Michaela’s constant (Km) had the values of (211UI/ml) and (2.8 mM) respectively.

, [4] and other living organisms including humans [5], [6], [7]. This enzyme catalyses the hydrolysis of toxic metabolites (e.g. paraoxon) and so it is called paraoxonase., and it is involved in the degradation of plastics and hydrolysis of organophosphates [8], phenylacetate, estrogenester, The health benefits of green tea have been extensively studied in the past few decades. Nowadays, tea is considered as a source of dietary constituents endowed with biological and pharmacological activities with potential benefits to human health Results of the current study revealed that green tea extract reversed the elevation of liver enzymes, lipid peroxidation and Advanced oxidation protein products in addition to attenuating the reduction of SOD,CoQ10 and paraoxonase (arylesterase) levels [9][10]. difficult childbirth, asthma, hypertension, parasites and annona: a novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism [18]. Inhibit of annonaceous acetogenins on class II 3hydroxy-3-methylglutaryl coenzyme A reductase from Streptococcus pneumonia, have an antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria [19] [20].
The aim of this research is to extract and characterize arylesterase enzyme from medicinal plant(( peel and the pulp parts ) of the common names of the fruit( Graviola, soursop, Brazilian paw paw,Annona muricata.) due to its medical , biological applications.

Raw Material
Annona muricata (Custand apple) fruits (the Indian source) were taken from locally market in Erbil, it was restored at 25°C.

Extraction of Enzyme
Arylesterase extracted from (peel and pulp) of the Annona Muricata by the following method 120g of the pulp and 16 g of the peel are used separately, were mixed with 400 and 70 ml of distilled water using a blender respectively for 10 min. at 4°C in water bath. Extraction was performed using high speed refrigerated centrifuge for 15min.
at4000xg speed and 4°C temp, (this experiment was carried in duplicate).

Enzyme activity assay
Arylesterase was assayed by using phenyl acetate as substrate,

Molecular weight determination
The molecular weight of peak as source of arylesterase was determined by the elution volume from sephadex G-75 column

Characterization of Arylesterase.
To develop assay conditions where arylesterase shows a maximum activity, a series of experiments were performed these included changing the enzyme concentration, the incubation time, the incubation temperature, buffer concentration, the pH of the conditions and the substrate concentration.

Results and Discussion.
The activity of Arylesterase from the pulp part of the fruit was shown in

Molecular weight determination.
The molecular weight of unknown compound separated by the column chromatography could be determined from the standard curve.  The role of enzyme-catalyzed reactions, like most chemical reactions, increases with temperature. The initial reaction rate will rise with temperature until it becomes impossible to. In practice, most enzymes are completely in activated above (80 ºC) [25]. In this study used (0, 5,10,15,20,25,30,37,40,45) ºC, it has been found that as the temperature increased, there was a concave up increase in the enzyme activity until it reached a maximum value at a temperature of (37ºC ) and the acivity was dropped to less than 37% at 70 ºC as shown in figure (5).   [2] R.C. Chang; J.C. Chien; J.F Shaw"Vibrio mimicus arylesterase has thioesterase cholinesterase inhibition in four species of wild birds" Pesticide Biochemistry and Physiology, 11(1995):294-300.