A Novel Class of Protease from Choerospondias axillaris (Lapsi) Leaves

Sudeep Karki1, Rupendra Shakya1 and Vishwanath P.Agrawal1,2

1Universal Science College and 2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal.

Abstract

A novel protease from leaves of Choreospndias axillaris has been reported. C. axillaris , locally called Lapsi is dioceous, deciduous fruit – bearing large tree, having multiple daily uses. In an attempt to find method for determining sex of Lapsi at seedling stage , we stumbled upon a unique protease that has thwarted our effort to find sex - related protein. Thus protease is highly thermo – stable and acid resistant. Its preparation can be autoclaved without significant loss in activity. Its activity can be repeatedly precipitated by trichloroacetic acid. It possesses a Km value of 29 µM and Vmax 52.63 pmoles/min for bovine serum albumin as the substrate. It is catalytically so powerful that the level of soluble protein in leaf is below 20 µg per g dry weight. Lapsi leaf protease is an specific endopeptidase attacking peptide bonds that have phenylalanine, tyrosine, alanine and threonine / aspartic acid residues.

Introduction

Choerospondias axillaries:(Locally called “lapsi”), is a large, deciduous fruit-bearing tree of the family Anacardiaceae.A native of the Nepal hills (850–1900m) Lapsi wood is used as light construction timber and fuel wood; seed stones are used as fuel in brick kilns and the bark has medicinal value. Nepal is unique in processing and utilizing lapsi fruits. The fruits are rich in vitamin C content.

Agrawal and Kesari (1992) were first to observe strong proteotytic activity in Lapsi leaves. Lapsi Protease was found to be active even under autoclave condition. Dekhang and Sharma (2006) reported optimium pH of 7 for the protease. The protease is not inhibited at all by phenylmethanesulfonylfluoride ( PMSF ) and 20-30% inhibited by sodium iodoacetic acid, thus revealing that protease is not a serine protease. No smaller proteolytic products of BSA could be seen in SDS-PAGE using silver staining indicating that the protease is not exopeptidase, Protease activity can be repeatedly precipitated by 0.2 M tricloroacetic acid TCA (Singh and Giri 2007). In order to find an insight into the mechanism of protease action, the present research was carried out

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The research was done for the fulfilment of degree requirement for B. Sc. Biochemistry (SK and RS)

Materials and Methods

Preparation of Lapsi Leaf Powder : Dried leaves of lapsi were blended in a glass blender to get fine powder.

Partial purification and concentration of protease : It involved following steps.

1. Washing of the lapsi powder (5gm) with 50 ml acetone
2. Extraction of acetone washed powder with 10 ml phosphate buffer (0.1M of pH 7).
3. Heat treatment of the extract at 70. ºC for 30min
` 4. TCA precipitation of protein in the extract by adding TCA (2.45M) to heat treated solution to a final concentration of 0.2M followed by keeping in freeze for 10min, centrifuging at 10000rpm/10min and dissolving the pellet dissolve in 200μl of phosphate buffer.

Determination of proteolytic activity :

A typical reaction mixture cntaining 28.75μg of protein and 50 μg BSA in the total volume of 200μl of 0.1M pH 7 phosphate buffer was incubated for 30 min . The proteolytic activity is measured by 3 following methods.

1. Direct Method : The reaction was stopped by adding Bradford reagent for determination of BSA ( Bradford 1976, Saleemudin 1980 ).

2. Indirect Method : The reaction was stopped by adding 2.45M TCA to a final concentration is 0.2M, centrifuged at 10,000rpm/10min and pellet dissolved in 200μl of phosphate buffer (0.1M of pH-7) and protein determined by Bradford method.

3. Determination of amino acid produced in reaction .by Ninhydrin method :
To the reaction mixture (100μl), 0.9ml distilled water and 4ml Ninhydrin (0.5% in ethanol) reagent were added and heated at 80ºC for 10 min, cooled and absorbance measured at 570nm. Alanine was used as standard.

Detection of amino acids by paper chromatography :
Ascending paper chromatography of the reaction mixture (60μl 0.1% isopropanol) was done on Whatman paper 1 using Butanol: Glacial acetic acid: water (40:10:50 V/V) as the solvent system. Chromatogram was dried in air for 6 hrs.Amino acid spots were made visible by spraying the chromatogram with 0.2% Ninhydrin reagent. Then the purple coloured zones were marked and Rf value calculated.


Determined RF value of Standard Amino acid used.

Amino-Acids Rf-value Amino-Acids Rf-value
Alanine 0.35 Threonine 0.27
Tryptophan 0.63 Valine 0.8
Leucine 0.89 Aspartic acid 0.25
Iso-leucine 0.86 Serine 0.15
Phenylalanine 0.85 Cystine 0.05
Glycine 0.1 Arginine 0.07
Histidine 0.08 Glutamic acid 0.2
Tryrosine 0.45 Lysine 0.06

Results and Discussion

In order to measure the effect of enzyme concentration on protease activity, the double TCA precipitated enzyme (50μl) preparation (5.75ug/10μl) was used. It was noticed that enzyme activity was linear up to 28.75μg of protein. In order to determine the effect of time of incubation on protease activity, 50μl of double TCA precipitation enzyme (28.75μg) of protein and 50μg of the substrate (BSA) were used and incubated for various periods. Result obtained showed that the proteolytic activity was linear only up to 30min. Therefore, further experiments were carried out using 28.75μgm of protein and 30mins of incubation.In order to determine the Km and Vmax by Lineweaver-bulk plot, the effect of BSA concentration on enzyme activity was measured.. It was found that protease has Km of 29.1μM for BSA and Vmax of 52.63pmoles/min.A low value of Km indicates that the substrate is tightly bound to enzyme.

Since BSA has 607 amino acids , 607nmoles of amino acids should be produced per nmoles of BSA degraded. In order to check this equivalency, the amino aid contents of reaction mixture were analysed ( Table 1 ).

Table 1 : Comparison of experimental and the theoretical values of amino acid content of reaction mixture

BSA (μg)
a BSA degraded(μg)
b Theoretical value of amino acids produced
( nmoles)
c Experimental value of amino acids produced ( nmoles)
D c/d Calculated no. of free amino aacids
10 5.5 48.207 13.6 0282 171
20 12 105.180 28 0.266 161
50 26.8 234.904 65 0.277 168
100 60.4 529.410 145 0.273 166

*For BSA degradation, reaction mixture containing 28.75μg of enzyme and equired amount of BSA in total volume of 200μl of 0.1 M pH 7 phosphate buffer .After 30 min of incubation 100 μl of the mixture was used for protein determination using direct method.
*molecular weight of BSA 69323.4 Da, 1 mole of BSA contains 607 moles of amino acids
( Hilger et al. ) ; 50μg of BSA contains 0 .722 nmoles of BSA.
*For amino acid determination, to the reaction mixture (100 μl ) 0. 8.ml water was added and ninhydrin method used.

It was observed that upon proteolysis one molecule of BSA yielded only 161 -171 molecules of amino acids ( Table 1 ) suggesting that in addition to free amino acids 70 – 75 % BSA is degraded to smaller peptides which cannot be observed in SDS-PAGE as well as not amenable to the Ninhydrin method.

In order to determine the identity of released amino acids during proteolysis of BSA ,we embarked upon the paper chromatography of the reaction mixture . In paper chromatogram, only 4 spots could be seen. Comparing with the Rf-values of standard amino acids ( Fig. 1 ) we concluded that amino acids produced by degradation of BSA are phenylalanine, tyrosine, alanine and threonine / aspartic acid.

Table 2. Rf valuue of amino acids

Sample(enzyme) Rf-value Amino acid
Spot A 0.26 Thr , Asp
Spot B 0.35 Ala
Spot C 0.44 Tyr
Spot D 0.85 Phe

So, we conclude that lapsi protease is very special type of protease. It’s not a exopeptidase, it is an endopeptidase with very specific activity attacking peptide bonds containing phenylalanine, tyrosine, alanine, threonine / aspartic acid residues. A serious literature search shows that such type of protease has not been reported in the literature.

References

Agrawal VP, Keshari and Singh D, (1992) Study of Lapsi (Choreospondias axillaris) Protease Unpublished result.

Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.

Dekhang RN and Sharma G (2006) Study of Protease from the leaves of Choreospondias axillaries (Lapsi). Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of . B. Sc. Biochemistry.

Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F ( 2001 ) Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. J. Clin. Exp. Immupol. 123 (3), 387-394. http://www.ncbi.nlm.nih.gov/protein/3336842?report=genpep

Saleemudin M, Ahmad H and Hussain A. (1980) A Simple, Rpapid and Sensitive Procedure for the Assay of endoprotease using Coomassie Brilliant Blue G-250. Analytical Biochemistry 105: 202-205.

Singh R and Giri S (2007) Characterization and purification of protease from leaves Choerospondias Axillaris (Lapsi).Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of B.Sc. Biochemistry.