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

Leucine amino peptidases (LAP; EC 3.4.11.1) constitute a diverse set of exopeptidases that catalyze the hydrolysis of leucine residues from the amino-terminal of protein or peptide substrates, (LAP) are present in animals, plants, and microbes. In this study, leucine amino peptidase was purified partial from Arachis hypogaea seeds by using gel filtration chromatography Sephadex G-100. The enzyme was purified 3.965 fold with a recovery of 29.4%. Its pH and temperature optimum were(8.7) and (37oC), respectively. The results show novel properties of LAP from Arachis hypogaea L. or peanut. The Km value for LAP (77 mM), with V max (1538 m mole min-1). We recommend a separate isoenzymeof the enzyme (LAP) from Arachis hypogaea on L. peanut seeds and study the kinetic qualities of each of them Keyword: Leucine amion peptidase (LAP), Arachis hypoaea L. seeds, Purification.


INTRODUCTION
Arachish ypogaea 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 Arachish ypogaea 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 oppositepairs; 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 amino peptidase is Leucin amino peptdase (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 amino peptidases (LAP s ) have been reported for most plant species 6 .The first group has ther molabile amino peptidases 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), homohexamer icmet all opeptidases that contain ethylene diaminetetraacetic 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).

Enzyme Extraction
Arachis hypogaea L. (Imported from Turkey )was obtainedfrom a local super market in Baghdad, Iraq.Homogenizing 1 g homogenate at 18000 g for 30 of Arachis hypogaea seeds in 5 ml 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 Folincicalteau 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).

Determination of LAP activity
h ydrolysis 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 o 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 MT ris-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: Theeffect of temperature on LAP activity was firmed in different temperature (20, 24, 37, 40, 45 o C) with Tris-HCl buffer pH 8.5.Then LAP activity was measured.

Different concentration of substrate
Different concentration have been prepared (45, 85, 125 , 165, 205 ) m M /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 1486.0Unit/mL and specific activity of 413.23 Unit/mg was considered as crude enzyme solution.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 1638.75Unit/mg.as shown Figure .(2).
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 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 LAP at 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 pH 7.2 15 and from wheat pH  9 showed that maximum activity was obtained at alkaline pH (8.0-11.0).
The optimum temperature for the activity of LAP from Arachis hypogaea L. seeds was determined to be 37 o C. as shown in Fig. (4).This result like the value obtained from pea shoots 37 o C 16,17 .In the other studies kiwifruit AP was most

CONCLUSION
In conclusion , in the present study,it was found one peak of the Arachishypogaeaon L.peanut enzyme (LAP) by chromatography(gel filtration ).The Arachi shypogaeaon 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 Arachis hypogaeaon L. peanut seeds and study the kinetic qualities of each of them .Note that the enzyme was purified for the first time and have got a successful and appropriate way because we got a good yield.