Purification and biochemical characterization of a chymotrypsin-like serine protease from Euphorbia neriifolia Linn.

Process Biochemistry 46 (2011) 1654–1662

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Purification and biochemical characterization of a chymotrypsin-like serine protease from Euphorbia neriifolia Linn. Ravi Prakash Yadav, Ashok Kumar Patel, M.V. Jagannadham ∗ Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

a r t i c l e

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Article history: Received 8 December 2010 Received in revised form 10 May 2011 Accepted 23 May 2011 Keywords: Euphorbia neriifolia Neriifolin Euphorbiaceae Serine protease Chymotrypsin

a b s t r a c t Neriifolin, a chymotrypsin-like serine protease, has been purified from the latex of Euphorbia neriifolia Linn. by ammonium sulfate precipitation, cation exchange chromatography and gel filtration. The molecular mass of the enzyme is 35.24 kDa, with an isoelectric point of pH 5.7. The enzyme consists of 18 tryptophan, 25 tyrosine and 9 cysteine residues with 4 disulfide bridges. The extinction coefficient (ε1% ) is 38.28. The Km values are 1.39 ± 0.08 mM and 1.94 ± 0.17 mM, with N-succinyl-l280 nm Phe-p-nitroanilide and ␣-leucine-p-nitroanilide as substrates, respectively. Neriifolin retains proteolytic activity over a wide range of pH and temperature value, with pH optima of 8.5 and an optimal temperature of 55 ◦ C. Inhibition of enzyme activity by chymostatin and amidolytic activity against synthetic substrates specific to chymotrypsin indicates that the enzyme belongs to chymotrypsin-like serine protease class. Polyclonal antibodies specific to neriifolin and immunodiffusion reveal that the enzyme has unique antigenic determinants. The amino terminal sequence of the first 14 residues of neriifolin is D–F–P–P–N–T–H–I–G–I–P–N–G–Y. A high ratio of milk-clotting activity to proteolytic activity as well as stability against variations in pH and temperature, surfactants, oxidizing agents and compatibility with detergent additives make neriifolin an excellent candidate for industrial applications. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Proteases are the single class of enzymes that occupy a pivotal position with respect to their applications in both physiological and commercial fields. They represent one of the three largest groups of industrial enzymes and account for approximately 60% of the total worldwide sale of enzymes [1]. Proteolytic enzymes from plant sources have received special attention because they are active over wide ranges of temperatures and pH, as well as in the presence of surfactants, organic solvents and denaturing agents. This stability enables their use in processes that restrict the use of conventional enzymes for industrial applications. They are extensively applied

Abbreviations: BLAST, basic local alignment search tool; BAPA, NRbenzoylarginine-p-nitroanilide; BSA, bovine serum albumin; DFP, diisopropylfluorophosphate; DMSO, dimethyl sulfoxide; DTNB, 5,5 ␮-dithiobis (2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GuHCl, guanidine hydrochloride; GuSCN, guanidine isothiocyanate; HgCl2 , mercuric chloride; IAA, iodoacetic acid; LGP, latex glycoprotein; MALDI-TOF, matrix assisted laser desorption/ionization time-of-flight; NEM, N-ethyl maleimide; NCBI, National Center for Biotechnology Information; PMSF, phenyl-methanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SBTI, soybean trypsin inhibitor; Tris, tris (hydroxymethyl) aminomethane; TCA, trichloroacetic acid. ∗ Corresponding author. Tel.: +91 542 2367936; fax: +91 542 2367568. E-mail addresses: [email protected], [email protected] (M.V. Jagannadham). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.05.013

in various industries such as the production of food, detergents, pharmaceuticals and leathers. The largest applications of proteases are in most likely the food industry and in laundry detergents. In food industries, the use of proteases has a long history, as in the production of flour, milling and baking [2]. The hydrolytic property of proteases is exploited for degradation of the turbidity complex that results from protein in fruit juices and alcoholic liquors, quality improvement of protein-rich foods, soy protein hydrolysis, gelatin hydrolysis, casein and whey protein hydrolysis, meat protein recovery and meat tenderization [3]. In addition to their industrial and medicinal applications, some proteases have also been used as model systems to improve our understanding of structure–function relationships and protein folding pathways in basic research [4–6]. Selective peptide bond cleavage of some proteases has also been used in the elucidation of structure–function relationships, in peptide synthesis and to sequence proteins [7]. The vast diversity of proteases, in contrast to the specificity of their action, has attracted worldwide attention in attempts to exploit their physiological and biotechnological applications. Commercial use of proteases having different origins for hydrolysis of food proteins appears to be very promising due to the biological origin of enzymes [8]. Most of these enzymes come from microbial sources, but some plant cysteine proteases, namely, papain, bromelain, and ficin, are still preferred in a number of

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processes. The major drawback in the use of cysteine proteases is that their activity is readily reduced by air oxidation and metal ions. Therefore, applications using these enzymes require reducing and chelating agents that are not economical or convenient to include. By contrast, plant serine proteases are both stable and active under harsh conditions of temperature and pH, as well as in the presence of either surfactants or oxidizing agents. Thus, they are more useful and economical for industrial applications [9]. Serine proteases are one of the largest groups of proteolytic enzymes that catalyze the hydrolysis of specific peptide bonds in their substrates. Their activity is dependent on a set of three amino acids (serine, histidine and aspartate) in the active site of the enzyme. These residues are referred to as the “catalytic triad.” Serine proteases are involved in numerous regulatory processes and are the best-characterized groups of proteolytic enzymes in mammals (e.g., the trypsin/chymotrypsin family) and microorganisms (e.g., the subtilisin family). In plants, they are widely dispersed among different taxonomic groups and are found to be involved in a number of physiological processes, such as protein degradation and processing, microsporogenesis, symbiosis, the hypersensitive response, signal transduction, differentiation and senescence [10]. Despite being the largest class of proteases in plants, the functions and regulatory roles of plant serine proteases are poorly understood, probably due to a lack of known physiological substrates. Therefore, the search for new plant serine proteases continues to identify industrially applicable and cost effective enzymes, as well as to understand their physiological role in plants. Euphorbia neriifolia Linn. (popularly known as common milk hedge), belonging to family Euphorbiaceae, is a commonly occurring plant in the dry, hilly, rocky grounds of north and central India. The entire plant, including the leaves and roots, is used to treat abdominal discomfort, bronchitis, tumors, leucoderma, piles, inflammation, spleen enlargement, anemia, ulcers, fever and common coughs and colds [11]. Phytochemical studies have led to the isolation of diterpenes (such as antiquorin), triterpenes (such as nerifolione), anthocyanins (such as dolphin) and tulipanin from different parts of the plant. A pharmacological activity study reported the efficacy of E. neriifolia leaf extract as an antioxidant and immunomodulator in vitro and demonstrated that the latex is cytotoxic, anti-arthritic, anti-inflammatory, wound-healing and possesses antitumor activity [11]. However, the proteins and other biochemical constituents of the latex have not been investigated in detail. In view of such medicinal importance we conducted a search for biochemical constituents in the plant latex. During the course of screening for biochemical constituents, a substantial amount of proteolytic activity was observed in the latex of the plant. This manuscript describes the identification and purification, as well as the biochemical and immunological characterization, of a chymotrypsin-like serine protease isolated from the latex of E. neriifolia Linn. 2. Materials and methods 2.1. Materials SP-Sepharose and Sephacryl S-200 HR were purchased from GE Healthcare. Acetonitrile, agarose, ampholine carrier ampholytes, BSA, ␤-mercaptoethanol, casein, chymostatin, Coomassie brilliant blue R-250, Coomassie G-250, DFP, DTNB, EDTA, Freund’s complete and incomplete adjuvant, GuHCl, GuSCN, glycerol, hemoglobin, HgCl2 , hen egg white, IAA, lysozyme, o-phenanthroline, PMSF, ribonuclease A, SBTI, urea and all synthetic substrates were obtained from Sigma Chemical Co. (United States). 2.2. Methods 2.2.1. Collection of latex Latex was collected in 10 mM sodium acetate buffer at pH 4.5 by superficial incisions on stems of E. neriifolia plants, and the latex was then frozen at −20 ◦ C for 24 h. The latex was thawed and centrifuged at 24,000 × g for 45 min to remove

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the gum and other insoluble materials. The clear supernatant was subjected to 80% ammonium sulfate precipitation at 4 ◦ C, and the precipitate was recovered by centrifugation at 24,000 × g for 30 min at 4 ◦ C, dissolved in 10 mM sodium acetate buffer at pH 4.5 and dialyzed overnight against the same buffer. The protease activity and protein content was then measured. 2.2.2. Cation exchange chromatography The dialyzed suspension after ammonium sulfate precipitation from the above step was subjected to cation exchange chromatography on SP-Sepharose fast flow column pre-equilibrated with 10 mM sodium acetate buffer at pH 4.5. The column was washed thoroughly with the same buffer until no protein was detected in the eluate. The bound proteins were eluted with the same buffer using a linear gradient of NaCl from 0 M to 0.8 M. Fractions of 3 mL volume were collected at a flow rate of 3 mL/min. All of the fractions were assayed for protein content, proteolytic activity as well as homogeneity using absorbance at 280 nm, an enzyme assay and SDSPAGE, respectively. The active fractions from each peak were pooled, separately from the other peaks, and stored at 4 ◦ C.Gel filtrationActive and homogenous fractions from the cation exchange were pooled, desalted and concentrated using an Amicon membrane concentrator with a 10 kDa cutoff. The resulting enzyme preparation was subjected to gel filtration on a Sephacryl S-200 HR column (120 cm × 1 cm) pre-equilibrated with 25 mM phosphate buffer at pH 6.5 containing 0.5 M NaCl, and the column was eluted isocratically. All of the fractions were analyzed as described above. The active and homogenous fractions were pooled, concentrated and stored at 4 ◦ C for further use. The pure enzyme thus obtained was named neriifolin. 2.2.4. Protein concentration Protein concentration was determined spectrophotometrically (absorbance at 280 nm) as well as by Bradford’s method [12], using BSA as the standard. 2.2.5. Activity measurements The hydrolyzing activity of the protease was monitored using denatured casein and denatured hemoglobin, as described by Arnon [13]. For activity measurements, 15 ␮g of the enzyme in 0.5 mL of 50 mM Tris buffer at pH 8.0 was added to 0.5 mL of 1% substrate in the same buffer and the reaction was allowed to proceed for 1 h at 37 ◦ C. The reaction was terminated by the addition of 0.5 mL of 10% TCA and allowed to stand for 10 min. The resulting precipitate was removed by centrifugation at 10,000 × g for 10 min and the absorbance of TCA soluble peptides in the supernatant was measured at 280 nm. One unit of enzyme activity is defined as the amount of enzyme that, under the conditions described, gives rise to an increase of one unit of absorbance at 280 nm per min of digestion. The specific activity is the number of units of activity per milligram of protein. 2.2.6. Electrophoresis and zymography Assessment of the homogeneity of the enzyme, at different stages of purification, as well as molecular mass determination of the purified enzyme, was conducted using 12% SDS-PAGE under reducing and non reducing conditions according to the method of Laemmli [14]. The proteins were stained with Coomassie brilliant blue R-250. For zymography, 1.2% casein was added in the 12% polyacrylamide gel. The gel was run at 200 V for 1 h and soaked in 2.5% Triton X-100 to displace the SDS. Gels were then incubated in reaction buffer (50 mM Tris buffer at pH 8.0 and 1 mM CaCl2 ) for 15 h at 37 ◦ C and later stained with Coomassie brilliant blue R-250. The unstained region of the gel reveals the proteolytic activity of the neriifolin. 2.2.7. Mass spectrometry The molecular weight of the purified enzyme was determined by Micromass TOF Spec MALDI/TOF. Samples were dissolved at a concentration of 10 pmol/␮l in 1:1 (v/v) 1% aqueous formic acid and methanol. Positive ionization was used for the sample analyses with an electrospray voltage of 1.0 kV, a sampling cone voltage of 40 V and an MCP detector voltage of 2700 V. Nitrogen was employed as the API gas, and data were acquired over the appropriate m/z range at a scan speed of 3.0 s in continuum mode. An external calibration was made using horse heart myoglobin (MW 16,951.50 Da), and data were processed using the Mass Lynx suite of software programs supplied with the mass spectrometer. 2.2.8. Isoelectric focusing The isoelectric point of the purified enzyme was determined by isoelectric focusing on polyacrylamide disc gels as described by Patel et al. [15]. Electrophoresis runs was carried out with ampholine carrier ampholytes in the pH range of 5.0–8.0 at 300 V for 2 h using 0.1 M NaOH as the catholyte and 0.1 M orthophosphoric acid as the anolyte. The enzyme in the IEF-PAGE gel was visualized by Coomassie G-250 staining. 2.2.9. Carbohydrate content and glycostaining The carbohydrate content of neriifolin was determined using the phenol sulfuric acid method [16]. The gel was also stained with Schiff’s reagent, which is specific for glycoproteins [17]. 2.2.10. Tyrosine and tryptophan content The amounts of tyrosine and tryptophan in the enzyme were measured spectrophotometrically as described by Goodwin and Morton [18]. The absorbance

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spectra of the enzyme in 0.1 M NaOH was recorded between 300 and 220 nm using a Beckman DU 640 B spectrophotometer, and the absorbance values at 280 and 294.4 nm were deduced from the spectra. The standard formula given by Goodwin and Morton was used to estimate the tryptophan and tyrosine contents. To validate the current estimations, the amounts of tryptophan and tyrosine in papain, ribonuclease, BSA and lysozyme were also determined under similar experimental conditions.

definite amount of enzyme was incubated in the assay buffer with substrate concentration in the range of 0–40 mM, the initial rate of product formation is measured and initial velocity, vo were calculated and plotted on the Y-axis versus the concentration of substrate (X-axis) and a nonlinear regression analysis using a rectangular hyperbola model is performed and the value of Vmax and Km were calculated [22]. The value of the Kcat was obtained by dividing Vmax by the amount of enzyme in moles. The specificity constant was calculated by dividing Kcat by Km (Kcat /Km ).

2.2.11. Free and total cysteine content Free and total cysteine residues in the enzyme were estimated by Ellman’s method [19]. For free cysteine estimation, the purified enzyme was activated with 10 mM ␤-mercaptoethanol in 50 mM Tris buffer at pH 8.0 for 15 min and dialyzed against 100 mM acetic acid at 4 ◦ C for 24 h. For the estimation of total cysteine content, the enzyme was reduced in the presence of 6 M GuHCl for 15 min at 37 ◦ C and dialyzed against 100 mM acetic acid. To validate these estimates, the free and total cysteine contents of papain, ribonuclease, BSA and lysozyme were determined under similar experimental conditions.

2.2.18. Application of neriifolin as a detergent additive The suitability of neriifolin as a detergent additive was determined by measuring its stability in the presence of various surfactants and oxidants. The stability of the enzyme was measured against SDS, Triton X-100 and the oxidizing agent H2 O2 . The enzyme was incubated with different concentrations of surfactants as follows: SDS (0.05%, 0.1%, 0.2%, 0.4%), Triton X-100 (0.1%, 0.2%, 0.4%, 0.8%) and the oxidizing agent H2 O2 (1%, 2%, 3%, 4% 5%) for 60 min at 55 ◦ C, and the residual activity of neriifolin was measured as described earlier. The compatibility of neriifolin with commercial laundry detergents was studied using Ariel, Tide (Procter and Gamble Ltd.), Rin, Surf Excel, (Hindustan Lever Ltd.) and Henko (SPIC India Ltd.), which are widely used in India. For the compatibility study, these detergents were dissolved in distilled water at a concentration of 7 mg/mL [23]. The proteases present in these detergents were inactivated by incubating the detergent solutions at 70 ◦ C for 1 h prior to the addition of the enzyme neriifolin. After the addition of neriifolin to the detergent solution, the mixture was incubated at 55 ◦ C for 1 h, and the residual protease activity was determined. The enzyme activity of a control sample (without any detergent) was taken as 100%.

2.2.12. Extinction coefficient The extinction coefficient of the enzyme was determined by a spectrophotomet) = 10 (5690nw + 1280ny + 120nc )/M, with ric method [20] using the formula (ε1% 280 nm the following values: nw , ny and nc are the number of tryptophan, tyrosine, and cystine residues in the protein; M is the molecular mass of the protein; and 5690, 1280, 120 are the extinction coefficients of tryptophan, tyrosine and cystine, respectively. 2.2.13. Assay for amidolytic activity toward synthetic substrates The amidolytic activities of the enzyme were measured using BAPA, l-alanine-pnitroanilide, l-alanine-alanine-p-nitroanilide, N-succinyl-alanine-alanine-alaninep-nitroanilide, l-glutamyl-p-nitroanilide, N-succinyl-l-Phe-p-nitroanilide and ␣leucine-p-nitroanilide, as described by Arnon [11] with some modifications. The synthetic substrates (5–20 mM) were prepared by dissolving the required amount in the minimum volume of DMSO allowed and making up the final volume with 50 mM Tris buffer pH 8.0. The enzyme was incubated with the assay buffer and synthetic substrate at 37 ◦ C for 30 min. The reaction was terminated by the addition of acetic acid, and the liberated p-nitroaniline was measured by absorbance at 410 nm as described by Erlanger [21]. One unit of enzyme activity was defined as the amount of enzyme under given assay conditions that gave rise to an increase of one unit of absorbance at 410 nm per min of digestion. 2.2.14. Effect of various inhibitors on the protease activity of neriifolin The effect of increasing concentration of various protease inhibitors on the hydrolysis of casein by neriifolin was monitored to identify the mechanistic class of the enzyme. The inhibitors used were specific to serine proteases (DFP, chymostatin, PMSF, SBTI), cysteine proteases (IAA, HgCl2 , iodoacetamide, sodium tetrathionate, ␤-mercaptoethanol) and metalloproteases (EDTA, o-phenanthroline). In each case, 15 ␮g of enzyme was incubated in the presence of increasing concentration of the inhibitor in 50 mM Tris buffer at pH 8.0 for 30 min at 37 ◦ C and assayed for activity. A control assay for the enzyme activity was done without inhibitors and the resulting activity was taken as 100%. 2.2.15. Dependence of enzyme activity on pH and temperature The effect of pH on the enzymatic activity of the purified enzyme was determined within the range of pH 2.0–12.0. The buffers used were glycine–HCl (pH 2.0–3.5), sodium-acetate (pH 4.0–5.5), sodium-phosphate (pH 6.0–7.5), Tris (pH 8.0–10.0) and glycine (pH 10.5–12.0). Denatured casein or hemoglobin, dissolved in the corresponding buffer of required pH, was used as substrate to measure the activity of the enzyme. Denatured hemoglobin was used as substrate in the enzyme assays at low pH as casein is insoluble below pH 4.0. Activity measurements were conducted as described above. Similarly, an analysis of the effect of temperature on the caseinolytic activity of the enzyme was conducted to determine the temperature optimum. Enzyme samples were incubated at different temperatures in the range of 10–80 ◦ C for 15 min and an aliquot was used for activity measurement at the same temperature. 2.2.16. Stability The ability of the neriifolin to retain its activity under conditions of varying pH (2–12) and temperature (20–85 ◦ C), as well as in the presence of denaturants (urea, GuHCl and GuSCN), organic solvents (ethanol, methanol, isopropanol, butanol and acetonitrile) and metallic salts such as HgCl2 , CuCl2 , CaCl2 , MnSO4 and MgSO4 was examined. The enzyme was incubated under specified conditions of pH, chemical denaturant, organic solvent or metallic salts for 24 h, and the residual activity was determined. In the case of temperature stability measurements, the sample was incubated at the desired temperature for 15 min and the residual activity was measured as described earlier. 2.2.17. Enzyme kinetics The effect of increasing substrate concentration on the reaction velocity of the enzyme-catalyzed reaction was measured using synthetic substrates (N-succinyll-Phe-p-nitroanilide and ␣-leucine-p-nitroanilide) specific for chymotrypsin. A

2.2.19. Autodigestion Proteases in general are prone to autodigestion, and the extent of autolysis depends on the enzyme concentration, pH, duration of incubation, temperature and type of activator, if any. In the present study, to measure the extent of autolysis, neriifolin was incubated at room temperature in the concentration range of 0.05–0.5 mg/mL in different aliquots. After 30 min of incubation, 15 ␮g of enzyme was drawn from the aliquots and assayed for proteolytic activity, as described in the materials and methods section (Section 2.2.5) and this activity was considered 100%. Similarly, after 12, 24, 36 and 48 h of incubation, 15 ␮g of enzyme was drawn and assayed for the proteolytic activity, and the residual activities were calculated. 2.2.20. Milk-clotting activity Milk-clotting activity was measured by the method of Arima et al. [24], with slight modification and the amount of enzyme that clotted 1 ml of 10% skimmed milk containing 10 mM CaCl2 at pH 7.0 in 1 min at 37 ◦ C is defined as one milk-clotting unit. 2.2.21. Antigenic properties Antibodies to the purified enzyme were raised in a male albino rabbit (1 kg body mass) as described by Patel et al. [15]. The presence of antibody was confirmed by the Ouchterlony double immunodiffusion [25]. 1% agarose in phosphate-buffered saline containing 0.02% sodium azide was solidified in petri dishes and appropriate holes were punched into the agarose. A total of 40 ␮g each of the following antigens were included in peripheral wells: milin a serine protease from Euphorbia milii [26], carnein a serine protease from noxious weed Ipomoea carnea [15], indicain a subtilisin like serine protease from Morus indica [27] and the purified enzyme neriifolin. A total of 100 ␮l of antiserum against neriifolin was loaded in the central well and the plate was left at room temperature for 24 h. 2.2.22. N-terminal sequence The protein sample for sequencing was electrophoresed and transferred as a blot onto a PVDF membrane. The N-terminal sequence was determined on an Applied Biosystems Procise Sequencer by Edman automated degradation. The N-terminal sequence of neriifolin was compared with other plant serine proteases using NCBI -BLAST and CLUSTAL W [28,29].

3. Results and discussion 3.1. Purification of neriifolin A new serine protease from the latex of E. neriifolia Linn. was purified to homogeneity by a simple procedure using cation exchange chromatography and gel filtration. The elution profile from the cation exchange chromatography resolved into two peaks, I and II, as shown in Fig. 1a. The material that did not bind to the column and the buffer washes also exhibited some protein content with measurable proteolytic activity but were highly heterogeneous on SDS-PAGE (data not shown). The magnitude of activity as well as the purity of the fractions from peak II are higher relative to the pools from peak I and the majority of the total activity

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Table 1 Purification scheme for the protease neriifolin from the latex of Euphorbia neriifolia. Step

Total protein (mg)

Total activitya (units) (unit/mg) fold

Specific activity (unit/mg)

Purification fold

Yield (%)

Crude extract Ammonium sulfate precipitation SP-Sepharose Gel filtration on Sephacryl S-200

216.90 120.68 16.75 9.56

650.75 1045.60 320.65 235.86

7.61 8.66 19.14 24.46

1.00 1.14 2.51 3.24

100.00 63.34 19.42 14.28

a Definition of one unit: 1 unit of enzyme activity is defined as the amount of enzyme, under the assay conditions described, which gives rise to an increase of unit absorbance at 280 nm/min of digestion. Denatured casein was used as substrate.

loaded on the column was eluted in peak II. The fractions from peak II were pooled and subjected to further purification by gel filtration on Sephacryl S-200 (Fig. 1b). The homogeneous and active fractions were pooled, concentrated, dialyzed against 10 mM Tris buffer at pH 8.0 and stored at 4 ◦ C for further use. The purified protein is named neriifolin according to protease nomenclature and, the purification of neriifolin in a typical batch is summarized in Table 1. The specific activity of the purified enzyme is 24.46 U/mg of protein, as compared to the activity in the crude latex, which was 7.61 U/mg of protein using denatured casein as a substrate. The total recovery of the protease activity was 14.28%. The low recovery of the total protease activity may be due to the presence of multiple proteases in the crude latex. The purification protocol is highly reproducible, and the yield and specific activity of the enzyme were consistent

Fig. 1. Elution profile of crude latex after removal of gum (a) Cation exchange chromatography on SP Sepharose fast flow column pre-equilibrated with 10 mM sodium acetate pH at 4.5. The bound proteins were eluted with a linear salt gradient of 0–0.8 M NaCl in the same buffer. Fractions of 3 mL at the rate of 3 mL/min were collected. (b) Gel filtration on Sephacryl S-200 HR. Peak II fractions (55–90) from the SP elution profile were concentrated and loaded on to a Sephacryl S-200. All elution fractions from the two chromatography columns were assayed for activity () and protein content (䊉). Fractions 50–85 from the gel filtration elution profiles were pooled, concentrated and kept at 4 ◦ C for further use.

from batch to batch. The proteolytic nature of neriifolin was also confirmed by casein zymography, where digested casein appeared as white band corresponding to the position of the neriifolin in the gel (Fig. 3a).

3.2. Homogeneity and physical properties of neriifolin The purified enzyme migrates as a single band on SDS-PAGE under both reducing and non-reducing conditions, which confirms its purity (Fig. 2a). Further, the homogeneity of the enzyme was confirmed by mass spectrometry. The molecular mass of neriifolin estimated by SDS-PAGE and mass spectrometry (Fig. 2a and b) is 36 and 35.24 kDa, respectively. The molecular masses of the plant serine proteases known at present vary from 19 to 110 kDa, but the majority are between 60 and 80 kDa (10). The molecular mass of neriifolin falls within the low range of the molecular mass of plant serine proteases. Neriifolin shows a single band (Fig. 3c) by isoelectric focusing, with an approximate isoelectric point (pI) of pH 5.7. The reported euphorbian serine proteases are mostly acidic and rarely alkaline [30]. However, some of the euphorbian serine proteases were also reported to have high basic isoelectric points [31]. Some properties of euphorbian proteases compared to neriifolin are compiled and presented in Table 2. The extinction coefficient of the enzyme (ε1% ), as determined by spectrophotometric methods, 280 is 38.28, and this value was used for all experimental purposes.

Fig. 2. (a) SDS-PAGE of purified protease. Lanes 1, 2, 3 and 4 showing marker, crude, pure protein (reduced) and pure protein (non reduced), respectively. Phosphorylase b (97.40 kDa), Albumin (66.0 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (30.0 kDa), soybean tyrosine inhibitor (20.1 kDa), and chicken egg white lysozyme (14.4 kDa) were used as molecular weight markers. (b) Mass spectrometry of neriifolin by MALDI-TOF.

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Table 2 Summary of some properties of euphorbian proteases.a , b Enzyme

Neriifolin Milin Euphorbains lc Euphorbains y1 Euphorbains l Euphorbains y3 Euphorbains t1 Hevain b a b

Mr. (kDa)

35.24 51 70 67 43 67 74 58

pI

5.70 7.20 8.00 5.20 4.90 6.30 5.50 5.30

pH optima

8.50 8.00 8.30 7.01 7–7.5 7.01 7.50 6.30

Glycosylation

Yes Yes Yes Yes Yes Yes Yes No

Specific amino acid residue Cysteine

Tyrosine

Tryptophan

9 14 20 9 7 10 18 10

25 14 27 10 10 14 29 12

17 23 13 9 7 7 4 2

Lynn et al. [30]. Yadav et al. [26].

3.3. Carbohydrate content Neriifolin is a glycoprotein with a detectable amount of carbohydrate content, which was measured to be 5–7%. Furthermore, to confirm the glycosylation, an SDS-PAGE gel containing neriifolin was stained with Schiff’s reagent. The glycosylated enzyme produces a pink stained band on the gel (Fig. 3b) whereas the deglycosylated enzyme produces no color (data not shown). Most of the serine proteases reported from the Euphorbiaceae family have sugar moieties in their molecular architecture, but the roles of sugar residues in proteases are not very well defined. Generally, they play a role in protecting the protein from degradation, enhance thermal stability as well as solubility and enable transport inside the cell [32]. 3.4. Specific amino acid residues The number of tryptophan and tyrosine residues in neriifolin was 18 (measured value 18.4) and 25 (measured value 24.67), respectively. The total number of cysteine residues is 9 (measured

value 9.14), with 1 (measured value 1.12) free cysteine and the other 8 forming 4 disulfide bonds. Under similar experimental conditions, ribonuclease (GenBank: accession number CAB60003), papain (GenBank: accession number AAB02650), BSA (GenBank: accession number X58989) and lysozyme (Swiss-Prot: accession number P00698) gave the same values as reported in database. 3.5. Substrate specificity Neriifolin hydrolyzes denatured natural substrates such as casein and hemoglobin with high efficiency. The enzyme also shows amidolytic activity against synthetic substrates. It hydrolyzes synthetic substrates such as N-succinyl-l-Phe-p-nitroanilide and ␣-leucine-p-nitroanilide with high affinity (Km of 1.39 mM and 1.94 mM, respectively) and specificity constant (Kcat /Km of 539 and 299.7 mM−1 min−1 , respectively). However, neriifolin fails to hydrolyze l-glutamyl-p-nitroanilide, l-alanine-p-nitroanilide and N-succinyl-alanine-alanine-alanine-p-nitroanilide. These results indicate that the enzyme cleaves peptides at the carboxyl side of amino acids containing phenyl rings and hydrolyzes other amide bonds, particularly those with l-donated carboxyl groups which suggest that neriifolin is a protease with a cleaving site similar to chymotrypsin [33,34]. 3.6. Effect of various inhibitors on the protease activity of neriifolin

Fig. 3. (a) Zymogram (in gel activity) of neriifolin. The clear region (indicated by arrow) shows the hydrolysis of casein by the enzyme. (b) Glycostaining of neriifolin by Schiff’s base. The magenta color (indicated by arrow) shows the glycosylation. (c) Isoelectric focusing of neriifolin. Isoelectric focusing was performed using 5% polyacrylamide gels with ampholine ampholytes at pH 5.0–8.0. Isoelectric point of the purified enzyme is indicated by the arrow.

The minimum amounts of inhibitors required for the inhibition of the proteolytic activity of neriifolin is summarized in Table 3. Inhibitors of metalloproteases, cysteine proteases and trypsin-like proteases do not affect the enzymatic activity of neriifolin. However, the enzyme activity of neriifolin is considerably inhibited by chymostatin, DFP and PMSF, suggesting that neriifolin is a serine protease. Moreover, the enzymatic activity of neriifolin is completely inhibited by chymostatin, indicating that neriifolin belongs to the chymotrypsin-like serine protease class. A proteinaceous inhibitor such as soybean trypsin inhibitor (SBTI), which is present in a typical protein-rich food, did not inhibit the activity of the enzyme. This property could, therefore, pave the way for the application of the purified enzyme in the food industries. Generally, soybean trypsin inhibitor successfully inhibits the activity of bacterial or animal serine proteases, while failing to do so in the case of plant serine proteases such as cucumisin and bamboo sprout proteases [34,35]. Some enhancement to the activity of neriifolin, as well as a small decrease in activity, was observed in the presence of ␤-mercaptoethanol and IAA, respectively, indicating the presence of a cysteine residue in close vicinity to the active site. This is further evidenced by the inhibition of activity by mercuric chloride. Similar observations are reported in the case of cucumisin, a serine protease, and dubumin, a chymotrypsin-like serine protease [34,36].

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Table 3 Effect of various inhibitors on the activity of neriifolin. Inhibitor

Name of inhibitor

Concentrationa

Residual activity (%)b

Serine protease

Chymostatin DFP PMSF SBTI

1 mM 1 mM 1 mM 0.25 mM

0.00 6.50 ± 0.05 20.00 ± .0.75 105.00 ± .0.95

Cysteine protease

IAA HgCl2 Iodoacetamide Sodium tetrathionate ␤-Mercaptoethanol

1 mM 0.5 mM 1 mM 5 mM 1 mM

80.00 ± 0.67 60.00 ± 0.88 99.13 ± 0.79 98.34 ± 0.91 110.00 ± 1.75

Metalloprotease

EDTA o-Phenanthroline

1 mM 5 mM

a b

96.00 ± 0.83 98.00 ± 0.89

Minimum amount of inhibitor required for inhibition of proteolytic activity. Residual activities are shown as the mean ± SD (n = 3).

3.7. pH and temperature optima and stability The enzyme is active over broad ranges of pH and temperature. The enzyme retained proteolytic activity in the range of pH 6.0 to 10.5, with optimum activity at pH 8.5 (Fig. 4a). Similarly, the enzyme is active in the temperature range of 20–70 ◦ C, with a maximal activity at 55 ◦ C (Fig. 4b). The stability of enzymes is one of the most important factors that limit their industrial application. Neriifolin retains more than 75% of its proteolytic activity in the range of pH 5.0–10.5 and between 20 ◦ C and 65 ◦ C (Fig. 4a and b). The stability of the enzyme in the presence of different additives commonly used in protein chemistry is summarized in Table 4. The observed high stability of the enzyme under adverse conditions such as extreme pH, extreme temperature, strong denaturants, organic solvents, and various metallic salts could facilitate exploring the utilization of the enzyme in industrial and biotechnological applications. 3.8. Kinetic parameters Neriifolin obeyed Michaelis–Menten kinetics with synthetic substrates specific to chymotrypsin. The effect of increasing substrate concentration on the reaction velocity follows typical Michaelis–Menten equation with N-succinyl-l-Phe-p-nitroanilide and ␣-leucine-p-nitroanilide as a substrate. The value of Km obtained from the more reliable nonlinear regression method were 1.39 ± .079 mM and 1.94 ± 0.17 mM with N-succinyl-lTable 4 Stability of neriifolin under various conditions. Conditions

Concentration

Residual activity (%)a

Urea GuHCl GuSCN Methanol Ethanol Isopropanol Butanol Acetonitrile DMSO Hg2+ Cu2+ Ca2+ Mn2+ Mg2+ SDS Triton X H2 O2 H2 O2

6.0 M 2.0 M 1.0 M 40% 70% 55% 25% 30% 50% 1 mM 1 mM 5 mM 1 mM 1 mM 0.4% 0.8% 1.0% 4.0%

100.00 80.00 80.00 84.00 55.00 55.00 60.00 82.00 80.00 60.00 75.00 110.00 98.00 97.00 62.00 70.00 100.00 120.00

a

Residual activities are shown as the mean ± SD (n = 3).

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.17 0.13 0.15 0.16 0.12 0.12 0.13 0.16 0.17 0.16 0.17 0.21 0.16 0.17 0.13 0.13 0.19 0.23

Fig. 4. (a) Effect of pH and (b) the effect of temperature on stability () and activity (). For pH optima measurement, 15 ␮g of enzyme in 0.5 mL buffer at the required pH was used and the activity was determined using substrates prepared in corresponding buffers, as described. Stability was determined after overnight incubation of 15 ␮g of enzyme at room temperature and activity was measured. Similarly to see effect of temperature on activity, 15 ␮g of enzyme was incubated at required temperature for 15 min and activity was measured at the same temperature. For temperature stability experiments, 15 ␮g of enzyme was incubated at required temperature for 15 min, and the activity was measured at 37 ◦ C and pH 8.0. The maximum activity obtained was taken as 100% and residual activity was calculated.

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Residual activity (%)a

Ariel Tide Rin Surf Excel Henko

65.00 + 0.09 70.00 ± 0.11 64.00 ± 0.09 67.00 ± 0.09 60.00 ± 0.08

Enzyme (1 U) was incubated with detergent (7 mg/mL) at 55 ◦ C pH 8.5 for 60 min, and the residual activity was estimated. a Residual activities are shown as the mean ± SD (n = 3).

in the presence of various commercially used detergents in India (Table 5). The enzyme was found to be promising with regard to pH and temperature stability, detergent compatibility, stability to surfactants and oxidizing agents and for its application in detergent formulations. 3.10. Autodigestion Generally, a protease undergoes autolysis, which is dependent on protein concentration, temperature, time, and activators, if any are required. Therefore, it is important to check the conditions for storage of the enzyme without any loss of proteolytic activity. Autocatalysis, if any, of neriifolin was monitored as a function of increasing protein concentration in the range of 0.05–0.5 mg/mL at room temperature under neutral conditions (data not shown). The magnitude of the loss of proteolytic activity decreases with increases in enzyme concentration from 0.05 to 0.5 mg/mL, and no loss of activity was seen at higher concentrations. The enzyme retains more than 75% of activity even at a very low protein concentration of 0.05 mg/mL, indicating its higher stability and possible utilization in the food, textile and biotechnology industries. Fig. 5. Effect of substrate concentration on the reaction velocity of neriifolin (a) N-succinyl-l-Phe-p-nitroanilide and (b) ␣-leucine-p-nitroanilide. The value of Km and Vmax were calculated by the non linear regression analysis of the data using the software Origin 8.

3.11. Milk-clotting activity

Phe-p-nitroanilide and ␣-leucine-p-nitroanilide as substrate, respectively (Fig. 5a and b). Values of Vmax , Kcat and the specificity constant (Kcat /Km ) with N-succinyl-l-Phe-p-nitroanilide as the substrate were 5.28 × 10−6 ± .046 × 10−8 mol min−1 , 0.75 min−1 and 54 mol−1 min−1 , respectively. The same values with ␣-leucine-p-nitroanilide as a substrate were 4.06 × 10−6 ± 1.25 × 10−8 mol min−1 , 0.58 min−1 and 299.70 mol−1 min−1 , respectively.

Most of the proteolytic enzymes clot milk, but the ratio of the milk-clotting activity and proteolytic activity is a useful indicator of the protease efficiency when used as a coagulant for cheese making. The enzyme neriifolin also has milk-clotting activity, and the ratio of milk-clotting activity to the proteolytic activity of neriifolin was 106 U/OD 660 nm, comparable to cucumisin, ficin and papain [42]. The ability of the enzyme to produce milk curds, indicating a high ratio of milk-clotting to proteolytic activity, could make it useful as a milk coagulants, however, more studies investigating the quality of both the milk curds and the cheese formed should be conducted to confirm neriifolin’s efficacy in the dairy industry.

3.9. Application of the neriifolin as a detergent additive

3.12. Polyclonal antibodies and immunoassays

The enzyme neriifolin retained 62% activity in the presence of 0.4% SDS, an anionic detergent and 70% activity in the presence of 0.8% Triton X-100, a non-ionic detergent. The activity of neriifolin increased with increasing concentrations of H2 O2 . Activity was 100% with 1% H2 O2 and was 120% with 4% H2 O2 after incubation for 60 min. The stability of neriifolin in the presence of surfactants such as SDS, Triton X-100 and oxidizing agent such as H2 O2 (summarized in Table 4) are an important parameters because very few reports are available on SDS and Triton X-100 stable enzymes [37–39] or bleach-stable enzymes [40,41]. The suitability of an enzyme as detergents additive depends on its compatibility with the detergents over wide ranges of pH and temperature. An ideal detergent enzyme should be stable and active in the detergent solution for a long period of time and should have enough temperature stability to be effective over a wide range of washing temperatures. The enzyme neriifolin is fairly stable

Polyclonal antibodies against neriifolin were raised in a male albino rabbit, and cross reactivity was checked using the Ouchterlony double immunodiffusion technique [25]. No precipitin line was observed with the pre-immune serum, indicating that there is no prior antibody against neriifolin in the pre-immune serum. However, with raised polyclonal anti-neriifolin antibodies, precipitin lines begin to appear after 10–12 h of incubation at room temperature and are distinctly visible by 24–30 h (Fig. 6). The precipitin line is formed only against neriifolin as a result of precipitation of antigen-antibody complexes near the equivalence zone. Antisera to neriifolin did not cross-react with any other serine proteases from other plants that were tested. Similarly, the polyclonal antibodies specific to neriifolin did not cross-react with the protein pool of fractions from Peak I of the SP Sepharose column (data not shown). These observations confirm that the proteins present in the two active peaks from the SP Sepharose column (Fig. 1a) are

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Fig. 6. Ouchterlony double immunodiffusion assay. (a) Anti-neriifolin serum was added in the central well and neriifolin was added in the peripheral wells. (b) Anti-neriifolin serum was added in the central well and neriifolin, carnein, milin and indicain were added in the peripheral wells. The appearance of a precipitin band (indicated by arrow) was observed after 24 h of incubation. Table 6 Amino terminal sequence of neriifolin as compared to other serine proteases. Enzyme

Amino terminal sequence (first 14 residues)

Neriifolin LGPa Milinb Cucumisinc Tomato P69Ad Tomato P69Be

D D D T T T

F F V T T T

P P S R H R

P S Y S T S

N D V W S P

T W G D S T

H Y L F F F

I A I L L L

G Y L G G G

I E E F L L

P G T P Q E

N Y D L Q G

G V – T N R

Y I – V M E

Bold letter represent the similar amino acid residues. a Rajesh et al. [43]. b Yadav et al. [26]. c Yagamata et al. [44]. d Tornero et al. [45]. e Tornero et al. [46].

distinct, suggesting that the antigenic determinants of neriifolin are unique. 3.13. N-terminal protein sequence The amino terminal sequence of the first 14 residues of neriifolin is D–F–P–P–N–T–H–I–G–I–P–N–G–Y. This N-terminal sequence of neriifolin was aligned with some other known serine proteases from plants, and the result is presented in Table 6. The N-terminal sequence of neriifolin did not show considerable similarity to the sequences of other known plant serine proteases when aligned using NCBI-BLAST and CLUSTALW [28,29]. The N-terminal amino acid sequence, as well as sequences generated using mass data for MALDI-MS trypsin-digested peptide fragments, were used to search for matches in the SWISS-PROT protein data bank (data not shown). The absence of any positive match confirms the uniqueness of neriifolin. Further additional sequence analysis, catalytic site studies and structural determination may refine the classification of neriifolin.

its various aspects as well as allowing exploration of the possibilities of utilizing the enzyme in industrial and biotechnological application. The enzyme exhibits activity and high stability over broad ranges of pH and temperature, as well as in the presence of various chemical denaturants, oxidizing agents and surfactants. In addition to its stability and activity, the low susceptibility to autodigestion at ambient temperature, milk-clotting activity and compatibility as a detergent additive also offers promises for the usefulness neriifolin in the food, dairy, and textile industries as well as in detergent formulation. Despite the extensive research on several aspects of proteases from ancient times, there are several gaps in our knowledge of these enzymes, and there is tremendous scope for improving their properties to suit projected applications. The future lines of development would include elucidating the tissue localizations of the protease, identifying physiological substrates, analyzing the structure–function relationship and finding genetic approaches to generate recombinant systems for hyperproduction of the enzyme.

4. Conclusion

Acknowledgments

This is the first report of the purification and characterization of a chymotrypsin-like serine protease from the latex of E. neriifolia. A simple and economic purification procedure, combined with the easy availability of the plant latex, could be used for the large-scale production of the enzyme, allowing a broad study of

The financial assistance to RPY from the DBT Government of India and to AKP from the CSIR Government of India in the form of research fellowships is gratefully acknowledged. We thank Mr. Vijay Kumar Singh (Molecular Biology Unit, Banaras Hindu University, India) for the assistance in experiments and for fruitful

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discussions. We are thankful to Dr. AJG Moir of the University of Sheffield, UK, for N-terminal sequencing. References [1] Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 1998;62:597–635. [2] Haarasilta S, Pullinen T. Novel enzyme combinations: a new tool to improve baking results. Agro-Food-Ind Hi-Tech 1992;3:12–3. [3] Sumantha A, Larroche C, Pandey A. Microbiology and industrial biotechnology of food grade proteases: a perspective. Food Technol Biotechnol 2006;44(2):211–20. [4] Kundu S, Sundd M, Jagannadham MV. Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria. J Agric Food Chem 2000;48:171–9. [5] Yadav SC, Jagannadham MV, Kundu S, Jagannadham MV. A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties. Biophys Chem 2009;139:13–23. [6] Singh AN, Shukla AK, Jagannadham MV, Dubey VK. Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain. Process Biochem 2010;45:399–406. [7] Schnölzer M, Jedrzejewski P, Lehmann WD. Protease-catalyzed incorporation of 18 O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 1996;17(5):945–53. [8] Capiralla H, Hiroi T, Hirokawa T, Maeda S. Purification and characterization of hydrophobic amino acid-specific endopeptidase from Halobactrium halobium S9 with potential application in debittering of protein hydrolysates. Process Biochem 2002;38:571–9. [9] Tomar R, Kumar R, Jagannadham MV. A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.): purification and biochemical properties. J Agric Food Chem 2008;56:1479–87. [10] Antao CM, Malcata FX. Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol Biochem 2005;4:637–50. [11] Bigoniya P, Rana AC. A comprehensive phyto-pharmacological review of Euphorbia neriifolia Linn. Pharmacogn Rev 2008;2(4):57–66. [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [13] Arnon R. Papain. Methods Enzymol 1970;19:226–44. [14] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [15] Patel AK, Singh VK, Jagannadham MV. Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory). J Agric Food Chem 2007;55:5809–18. [16] Hounsell EF, Davies MJ, Smith KD. Chemical methods of analysis of glycoproteins. In: Walker JM, editor. The Protein Protocol Hand Book. Totawa, NJ: Humana Press; 1997. p. 633–4. [17] Wilson NL, Karlsson NG, Packer NH. Enrichment and analysis of glycoproteins in the proteome. In: Smejkal GB, Lazarev A, editors. Separation methods in proteomics. Boca Raton, FL: CRC Press; 2006. p. 350–5. [18] Goodwin W, Morton RA. The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem J 1946;40:628–32. [19] Ellman L. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. [20] Aitken A, Learmonth M. Protein determination by UV absorption. In: Walker JM, editor. The protein protocols handbook. Totowa, NJ: Humana Press; 1997. p. 3–6. [21] Erlanger BF, Kokowsky N, Cohen N. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961;95:271–8. [22] Seber GAF, Wild CJ. Nonlinear regression. New York, NY, USA: John Wiley & Sons; 1989. p. 325–367. [23] Banerjee UC, Sani RK, Azmi W, Sani R. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem 1999;35:213–9.

[24] Arima K, Yu J, Iwasaki S. Milk-clotting enzyme from Mucor pusillus var. Lindt. In: Pearlman G, Lorand L, editors. Methods in enzymology. New York: Academic Press; 1970. p. 446–59. [25] Ouchterlony O, Nilsson LA. Immunodiffusion and immunoelectrophoresis. In: Weir DM, Herzerberg LA, Blackwell C, Herzerberg LA, editors. Handbook of experimental immunology. Oxford: Blackwell; 1986. p. 32.1–50. [26] Yadav SC, Pande M, Jagannadham MV. Highly stable glycosylated serine protease from the medicinal plant Euphorbia milli. Phytochemistry 2006;67:1414–26. [27] Singh VK, Patel AK, Moir AJ, Jagannadham MV. Indicain, a dimeric serine protease from Morus indica cv K2. Phytochemistry 2008;69:2110–9. [28] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215(3):403–10. [29] Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 2003;31(13):3497–500. [30] Lynn KR, Clevette-Radford NA. Proteases of Euphorbiaceae. Phytochemistry 1998;27:45–50. [31] Noda K, Koyanagi M, Kamiya C. Purification and characterization of an endoprotease from melon fruit. J Food Sci 1994;59:585–7. [32] Van Teeffelen MMA, Broersen K, De Jongh HJ. Glucosylation of b-lactoglobulin lowers the capacity change of unfolding, a unique way to affect protein thermodynamics. Protein Sci 2005;14:2187–94. [33] Singh KA, Kumar R, Rao GRK, Jagannadham MV. Crinumin, a chymotrypsinlike but glycosylated serine protease from Crinum asiaticum: purification and physicochemical characterization. Food Chem 2010;119:1352–8. [34] Uchikoba T, Yonezawa H, Kaneda M. Cleavage specificity of cucumisin, a plant serine protease. J Biochem 1995;117:1126–30. [35] Arima K, Uchikoba T, Yonezawa H, Shimada M, Kaneda M. Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai. Phytochemistry 2000;54:559–65. [36] Ahmed IAM, Morishima I, Babiker EE, Mori N. Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen. Phytochemistry 2009;70:483–91. [37] Deane SM, Robb FT, Woods DR. Production and activation of SDS resistant alkaline serine exoprotease of Vibrio alginolyticus. J Gen Microbiol 1987;133:272–94. [38] Oberoi R, Beg QK, Puri S, Saxena RK, Gupta R. Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp. World J Microbiol Biotechnol 2001;17:493–7. [39] Seong CN, Jo JS, Choi SK, Kim SW, Kim SJ, Lee OH, et al. Production, purification and characterization of a novel thermo stable serine protease from soil isolate Streptomyces tendae. Biotechnol Lett 2004;26:907–9. [40] Tunga R, Shrivastava B, Banerjee R. Purification and characterization of a protease from solid-state cultures of Aspergillus parasiticus. Process Biochem 2003;38:1553–8. [41] Moriera KA, Albuquerque BF, Teixeira MFS, Porto ALF, Fihlo L. Application of protease from Nocardiopsis spp. as laundry detergent additive. World J Microbiol Biotechnol 2002;18:307–12. [42] Uchikoba T, Kaneda M. Milk-clotting activity of cucumisin, a plant serine protease from melon fruit. Appl Biochem Biotechnol 1996;56:325–30. [43] Rajesh R, Nataraju A, Gowda CDR, Frey BM, Frey FJ, Vishwanath BS. Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot. Biochimie 2006;88:1313–22. [44] Yagamata H, Masuzawa T, Nagaoka Y, Ohnishi T, Iwasaki T. Cucumisin, a serine protease from melon fruit, shares structural homology with subtilisin and is generated from a large precursor. J Biol Chem 1994;260:32725–31. [45] Tornero P, Conezaro V, Vera P. Primary structure and expression of a pathogen induced protease (PR-P69) in tomato plants. Proc Natl Acad Sci USA 1996;93:6332–7. [46] Tornero P, ConezaroV, Vera P. Identification of a new pathogen induced member of the subtilisin-like processing protease family from plants. J Biol Chem 1997;272:14412–9.