Leucine Aminopeptidase from Arachis hypogaea L. Seeds Partial Purification and Characterization

Introduction

Arachis hypogaea L. (Peanut)  is one of seeds that has great quantity of oil and protein , therefor, it is considered as very important food legume to human nutrition in the world . We can find Arachis hypogaea L. (Peanut) widely distributed in the tropical and subtropical areas of the world  [1]. It is an annual herbaceous plant growing (30-50) cm (1.0-1.6) feet tall. It has opposite  leaves  , pinnate with four leaflets (two opposite pairs; no terminal leaflet). The flowers of   Arachis hypogaea L. are similar to typical pea flowers in shape, its  color is yellow with reddish veining [2] .

Biochemical, physiological, chemotaxonomy and gentic variability investigation studies make use of qualitative and quantitative isoenzymatic analysis, these studies benefit of a large number of plant population, cultivars and species [3].

Aminopeptidase (EC 3.4.11) is one of the group of proteases, important enzymes that play major role  in many different life processes. The hydrolysis of amino acids  can be catalyzed by using  Aminopeptidase  which found in the N-terminal of peptide and are involved in proteins degradation to free amino acids [4]. The major and greatest studied group of aminopeptidase is Leucin aminopeptdase (LAP, EC 3.4.11.1) , especially in microorganisms and animals, therefor recognize it is  x-ray crystal structure   and their nucleotide sequences [5].

Generally, two classes of leucyl aminopeptidases (LAP_s) have been reported for most plant species [6]. The first group has thermolabile aminopeptidases with molecular weight of approximately 60-90 KDa and a neutral pH optimum. The  second group are enzymes like plant LAP_s but isolated from animals. With  large (250-330 KDa), homohexameric metallopeptidases that contain ethylene diamine tetraacetic acid (EDTA) and be statin. They are heat stable and possess on alkaline pH optimum. LAP_s with alkaline pH optimum have been biochemically purified from a number of plants [7].

In the present study, we purified major LAP from Arachis hypogaea L. seeds and characterized its enzymoloical properties (pH and temperature optimum).

Material and Methods

Enzyme Extraction

Arachis hypogaea L. (Imported from Turkey )was obtained from a local super market in Baghdad, Iraq. Homogenizing 1g homogenate at 18000 g for 30 of Arachis hypogaea seeds in 5mL of phosphate buffer, pH 7.8 use a Teflon pestle homogenizer were used for preparing crude extract. The supernatant was separated as crude after getting rid of insoluble debris  by centrifuging min at 4°C  .

Detrminations of protein concentration

This  method is based on the reaction of proteins with the alkaline sulphate followed by Folin-cicalteau reagent which produces a blue color complex due to the reaction of alkaline copper sulphate with the protein, the intensity of the color depends upon   the quantity of the protein detected [8]. As shown in figure (1).

Figure 1: Protein standard curve. Click here to View figure

 

hydrolysis of the peptide bond of leucinamide is measured according to Mitz and Schlueter  method spectrphotometrically at 238 nm  .Unit of enzyme activity   like one micromole of L- lucinamide hydrolyzed per minute at 25˚C and pH 8.5 [9].

Parital purification of LAP

Gel filtration chromatography using sephadex G-100 column was used for purification of partially purified enzyme. The height of 120 cm in a glass column with an internal diameter of 2.0 cm were used for packing the  column ,then equilibrated with 0.1 M Tris-HCl buffer pH 8.5 .The flow rate was at 0.5 mL min^-1. Forty fractions had been collected for each 2 mL and both the   protein content and the enzyme activity were determined for each separate fraction, as pointed out in the previous section.

Effect of pH on LAP Activity

The effect of pH on LAP activity in the purified extract was  firmed in different pH (8.1, 8.3, 8.5, 8.7, 8.9) with Tris-HCl buffer pH 8.5. Then LAP activity was measured

 Effect of temperature on LAP Activity

The effect of temperature on LAP activity was firmed in different temperature (20, 24, 37, 40, 45˚C) with Tris-HCl buffer pH 8.5. Then LAP activity was measur

Different Concentration of Substrate

Different concentration have been prepared (45, 85, 125 , 165, 205 ) mM /L of substrate (leucinamide) in buffer [9] Km and Vmax for enzyme  to substrate were determined by using the Lineweaver-Burk plot [the relationship between 1/V versus 1/[S].

Result and Discussion

Table 1 summaries the result of partial purification of LAP from Arachis hypogaea. The supernatant with LAP activity of 1486.0 Unit/mL and specific activity of 413.23 Unit/mg was considered as crude enzyme solution.

Table 1: Purification summary of aminopeptidase Arachis hypogaea. Click here to View table

 

This crude enzyme solution fraction was made up to a known volume, partially purified LAP was obtained from this crude enzyme solution fraction and loaded on sephadex G-100 gel filtration column.Gel filtration was purified to the enzyme 3.965 fold with a yield 29.4% and specific activity of 1638.75 Unit/mg. as shown Figure (2).

Figure 2: Atypical elution profile for the chromatography leucin-   aminopeptidase from Arachishypogaeaon L.  Click here to View figure

The exact role of LAP_s in plants is not known, while it is enzymes take part in some important processes, such as protein mobilization from cotyledons after germination, and protein turnover required for cell maintenance in vegetative and reproductive organs[10]. LAP_s are involved in rapid turnover of protein required in wounding or wounding or pathogen attack [11].

The highest enzyme activity in the range range of  pH (8.1 – 8.9), with optimum activity in Tris-HCl buffer at pH 8.7 was shown as in figure (3). This result was agree with  the value got from Fasciola gigantic LAPat  pH 8.0 [12], from Kiwifruit pH 9.0 [13]. In comparison with , aromatic aminopeptidase which was isolated from grapes  gave a result which was  pH 7.0 [14],  from barley  pH7.2 [15] and  from wheat pH 7.6 [5].  While Matsui et al 2006 [9] showed that maximum activity was obtained at alkaline pH (8.0-11.0).

Figure 3: Effect of pH on LAP activity. Click here to View figure

 

The optimum temperature for the activity of LAP from Arachis hypogaea L. seeds was determined to be 37˚C. as shown in figure (4). This result like  the value obtained  from pea shoots 37˚C [16,17]. In the other studies kiwifruit AP was most active at 37˚C [13]. Unlike stability of  aromatic aminopeptidase, lucine aminopeptdases  remained  even at temperatures over 60˚C, an optimum temperature of 60-70 ˚C was used for characterizing  their  activities   [9]

Figure 4: Effect of temperature on LAP activity. Click here to View figure

 

Figure 5: Activity of LAP with different concentration of substrate . Click here to View figure

 

Figure 6: Determination of Km and Vmax in partial purified enzyme. Click here to View figure

 

A linear relationship was obtained a Vmax [1538 U/mL] and Km value of [77 mM].An enzyme with low Km has a more affinity for its substrate .

Conclusion

In conclusion , in the present study,it was found one peak of the Arachishypogaeaon L.  peanut enzyme (LAP) by chromatography(gel filtration ).The Arachishypogaeaon L.  peanut enzyme (LAP) reach optimum activity at pH is 8.7 and temperature at 37 o C . The purified enzyme indicated maximum activity(Vmax) of 1538 m mole min ^-1 with its parallel Km value of 77  Mm .We recommend a separate isoenzyme of  the enzyme (LAP)from Arachishypogaeaon L.  peanut seeds and study the kinetic qualities of each of them . Note that the enzyme was purified for the first time and have been a successful and appropriate way because we get a good   find yield.

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