Purification and characterization of a serine protease (CESP) from mature coconut endosperm
© Panicker et al; licensee BioMed Central Ltd. 2009
Received: 18 September 2008
Accepted: 09 May 2009
Published: 09 May 2009
In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm.
We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut.
Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies.
Proteases, being major regulatory enzymes, play a prominent house-keeping role in the cell physiology of all living systems. In plants, these enzymes execute an important role in the overall process of protein turnover in all stages of its life [1–6]. Seed development is an intricate process by which the seeds synthesize and store extensively a number of proteins, carbohydrates and lipids for subsequent use during seedling growth. Different types of proteases, which are active during seed development, are very scarcely studied, even though their importance in proteolytic processing of pre-proteins and regulation of intracellular protein levels are very well identified .
Coconut (Cocos nucifera L.), which belongs to the family of Palmae, is widely distributed in tropical countries. The properties of protein extracted from desiccated coconut showed that its protein content is comparable to soy protein in terms of composition and amount . However, the research pertaining to the biochemical aspects of endosperm development are still lacking except few studies, which shows the chemical constituents of coconut endosperm and biochemical changes happening during the germination of coconut seeds suggests a metabolically active stage during seed development [9–14]. While investigating the presence of proteases in the developing endosperm, we have identified three types of these enzymes . In the present paper, we report the purification and characterization of a serine protease from mature coconut endosperm (CESP), which possessed the BAPNA hydrolyzing activity. We also detected CESP in coconut endosperm during seed development and in different varieties of coconut using immunochemistry.
For the purification of CESP, eighty grams of fully matured coconut kernel was used and the entire purification procedure was carried out at 4°C and the total enzyme activity and protein concentration at each level of purification was estimated.
(i) Preparation of crude extract
Kernel was cut into small pieces, frozen in liquid nitrogen, powdered, homogenized in four volumes of ice-cold 20 mM TBS at pH 7.8 containing 150 mM NaCl (buffer A) using a wet grinder, strained through muslin cloth, centrifuged at 14,400 × g for 25 minutes, removed the upper creamy layer of fat and the clear crude extract was obtained by passing through glass wool.
(ii) Ammonium sulfate fractionation and gel filtration
The crude extract was subjected to ammonium sulfate fractionation (30–60%) and the pellet was dissolved in a minimum volume of ice-cold buffer A and then loaded on to a Sephadex G-200 gel filtration column (105 cm × 2.3 cm) equilibrated with the buffer A. The fractions (25 to 35) with high specific activity were pooled.
(iii) Phenyl-Sepharose Chromatography
Solid ammonium sulfate was added to the pooled fractions from the above step to yield a final concentration of 0.4 M (NH4)2SO4 and was then subjected to phenyl-Sepharose column (5 cm × 1.6 cm) pre-equilibrated with buffer A containing 0.4 M (NH4)2SO4. The bound enzyme was eluted using a gradient of buffer A containing 0.4 M to 0.0 M (NH4)2SO4. Fractions of 1 ml with high protease activity were pooled and dialyzed against buffer A without 150 mM NaCl (buffer B).
(iv) DEAE-cellulose Chromatography
The pooled fractions were passed through DEAE-cellulose column (4.5 cm × 1.5 cm) equilibrated with buffer B. The enzyme was eluted using a gradient of 0 – 0.5 M NaCl in buffer B and the fractions with high protease activity were concentrated by dialyzing against buffer B using Centricon 10.
(v) Arginine-Sepharose Chromatography
The dialysate from the previous step was loaded on this column (7 cm × 0.9 cm) equilibrated with buffer B and the bound enzyme was eluted using a gradient of (0–0.3) M NaCl in the same buffer.
(vi) Biogel P60 gel filtration chromatography
Selected fractions from the previous step was pooled and concentrated to a volume of 0.5 ml and subjected to Biogel P60 column (50 cm × 1 cm), equilibrated with buffer A.
Enzyme activity assay
Protease activity was assayed during the purification process using a synthetic peptide N∞-Benzoyl DL-arginine p-nitroanilide (BAPNA) as the substrate. The reaction mixture contained 100 mM Tris HCl (pH 8.5), enzyme solution and 0.5 mM substrate in a total volume of 500 μl. Reaction was carried out at 37°C for one hour and stopped by an addition of 500 μl of ethanol. The liberated colored p-nitroaniline was measured at 405 nm using spectrophotometer (Hitachi U 2000). One unit of enzyme activity (EU) was defined as 1 μmole of p-nitroaniline liberated per hour under the conditions of assay. The enzyme activity was calculated using a p-nitroaniline molar extinction coefficient of 10,500 M-1cm-1 at 405 nm.
Preparation of Antibody
Protein bands corresponding to the CESP proteolytic products of 26.7 kDa (CESP2) and 41.6 kDa (CESP1) were cut from the gel after SDS-PAGE and eluted using electro-eluter from Bio-Rad. Two rabbits were immunized independently (rabbit 1–26.7 kDa, rabbit 2–41.6 kDa) by giving three subcutaneous injections, for a total of 700 μg proteins into respective rabbits. After 7 days of the booster dose, rabbits were bled through the ear vein and tested for the presence and specificity of antibody by immuno-diffusion  and immuno-blot analysis . Total IgG from the immune sera directed against 26.7 kDa polypeptide was isolated by 33% ammonium sulphate fractionation and DEAE cellulose ion-exchange chromatography.
SDS-PAGE was carried out as described by Laemmli  on 12% polyacrylamide gel and non-denaturing PAGE on 7% polyacrylamide gel. Western blot analysis was done as described by Towbin  using immunized serum 1: 200. The detection of proteolytic activity on 12% polyacrylamide gel containing SDS and 0.25% gelatin was carried out as described by Heussen and Dowdle  except that Triton X-100 (1%) in Tris buffer pH 8.5 used for re-naturation of the gels. Gels were then incubated in the same buffer with out Triton X-100 for over night at room temperature.
Absorbance at 280 nm was used for monitoring the protein during chromatographic elution. Total proteins in the pooled fractions were estimated using the Coomassie dye-binding assay . Arginine-Sepharose and IgG-Sepharose affinity matrices prepared using cyanogen bromide method .
For the detection of CESP in different varieties (endosperms stored at -80°C about an year) and different developmental stages of coconut (fresh), crude extract was prepared from the endosperm of the coconuts collected from the identified palms.
Results and Discussion
Purification of CESP from the coconut endosperm
Summary of Purification of CESP from coconut kernel.
Total Protein (mg)
Total Activity (EU)
Specific Activity (EU/mg)
1. Crude extract
2. Sephadex G 200
6. Biogel P60
Homogenity and molecular size of the purified CESP
The SDS-PAGE analysis of the purified CESP immediately after Biogel P60 gel filtration step showed a single band at 68 kDa region (Fig. 2(II) A) and under non-denaturing conditions, gave a single broad protein band as shown in Fig. 2(II) B.
The molecular weight 68 kDa as determined by SDS-PAGE analysis (Fig. 2(II) B) and 69.5 kDa by gel filtration on Sephadex G-100 (see Additional file 1, Fig S1) indicate that CESP is a single polypeptide protease. The molecular weights of the serine proteases of cucumisin family and the latex peptidases also have been reported to be single polypeptide enzymes with molecular weight ranging between 60 and 80 kDa [25–28]. Molecular weights of the minor protein bands were estimated as 41.6 kDa and 26.7 kDa using SDS-PAGE analysis, suggesting that these might have arrived from the 68 kDa CESP.
Substrate specificity and pH dependence of the purified CESP
Hydrolysis of fluorogenic peptide substrate by the purified CESP
The effect of protease inhibitors, metal ions and complexes on CESP
Effect of protease inhibitors, metal ions, chelators and sulfhydryl reagents on the activity of the purified CESP
Zymogram analysis of purified CESP on gelatin polyacrylamide gel
Zymogram analysis of the purified protease showed gelatin hydrolysis at positions that correspond to 68 and 26.7 kDa protein bands (Fig 3E). As the zymogram of the of crude extract prepared from fresh coconut, showed only one band at 68 kDa , it is possible that 26.7 kDa band is an autolysed product of the protease that has retained protease activity. The cleavage of gelatin into small peptides indicates CESP to be an endopeptidase, which can cleave the protein substrate at different locations to generate small peptides [26–29].
Characterization of the polyclonal antisera raised in rabbits against (41.6 kDa and 26.7 kDa) polypeptides
Polyclonal antisera were raised in rabbits against the 41.6 kDa (CESP1) and 26.7 kDa (CESP2) polypeptides. Specificity against peptides was verified. Western blot analysis of step purification using these antisera recognized 68 kDa protein (see Additional file 1, Fig. S2 and Fig S3), Analysis of the purified CESP after storage for one week, gave two distinct bands in each case, one represented the intact CESP and the other represented either 41.6 kDa or 26.7 kDa polypeptides corresponding to CESP1 and CESP2 depending on the antisera used (see Additional file 1, Fig. S4).
Zymogram and western blot analysis of the purified CESP after PAGE on gelatin gels
CESP in different varieties of coconut (Cocos nucifera) and distribution during different stages of endosperm development
In general the involvement of proteases in the synthesis or degradation of proteins are clear. However, at this point in our studies, we do not know what is the function of CESP. There might be a possibility for a developmental specific function for CESP, as an endopeptidase more likely towards the processing of immature proteins and for the removal of regulatory proteins as and when it is needed during development.
Identified enzyme (CESP) is a single polypeptide serine protease with approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. CESP is present in all varieties of coconut studied and in all stages of coconut endosperm development with maximum amount in fully matured coconut. But the physiological role of this alkaline protease is unknown. However, these finding may open up a lot of interest in the involvement of this protease in the area of development or in the storage and shelf life of coconut and its products.
List of abbreviations used
- BAPNA :
N∞-Benzoyl DL-arginine p-nitroanilide
- TLCK :
N∞-p-Tosyl-L-Lysine chloromethyl ketone
- TPCK :
N∞-p-Tosyl-L-Phenylalanine chloromethyl ketone
- SBTI :
Soybean trypsin inhibitor.
Authors acknowledge the financial support received by LM Panicker from Central Plantation Crops Research Institute (Indian Council of Agricultural Research (ICAR)), Govt. of India.
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