Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer

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1 Indian Journal of Experimental Biology Vol. 45, November 2007, pp Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer A Josephrajkumar 1, R Chakrabarty 2 & G Thomas 3 1 Cardamom Research Station, Kerala Agricultural University, Pampadumpara , Dist. Idukki, India 2 Department of Plant Biology and Forest Genetics, Genetics Centre, P.B. No. 7080, Swedish University of Agricultural Sciences, SE 75007, Uppsala, Sweden 3 Interfield Laboratories, Kochi , India Received 19 October 2006; revised 3 July 2007 An elastase-like chymotrypsin was purified by aprotinin-agarose affinity chromatography from the midgut extract of cardamom shoot and capsule borer, Conogethes punctiferalis. The purified enzyme had a V max of ± 22.1 nmole pna released/min/mg protein, K m of ± mm with SAAPLpNA as substrate and gave a single band on SDS-PAGE with a molecular mass of 72.1 kda. Casein zymogram revealed one clear zone of proteolytic activity, which corresponded to the band obtained with SDS-PAGE indicating that this could be a single-polypeptide enzyme. Keywords: Aprotinin, Cardamom, Chymotrypsin, Conogethes punctiferalis, Trypsin Small cardamom (Elettaria cardamomum Maton) popularly known as Queen of Spices is an important spice crop extensively cultivated in Cardamom Hill Reserves of Idukki district, India. Shoot and capsule borer (Conogethes punctiferalis Guenée Lepidoptera : Pyralidae) has become a major production constraint infesting shoots, succulent panicles, racemes, immature capsules and stem causing typical dead heart. Moreover, the late instar larvae thriving inside the stem is inaccessible to the insecticidal spray. In severely infested plantations a yield loss up to 70-80% has been recorded 1. It is well established that proteolytic enzymes in insect guts are primarily responsible for breakdown of plant proteins. Proteins are digested in the insect gut by enzymes that are active in slightly alkaline (Lepidoptera) to slightly acidic ph (Coleoptera) 2. Protein breakdown in lepidopteran guts is mediated by the concerted action of digestive enzymes, particularly trypsin and chymotrypsin, which are the primary digestive proteases in this insect order 3. The digestive enzymes of lepidopteran larvae are of interest first, as a target for insect pest management and second, due to their unusual ability to function in alkaline lepidopteran guts, (ph 10-12) 4 in which serine proteases and metallo-exopeptidases are most active. Serine proteases have been identified from the Phone: entojoe2003@yahoo.co.in digestive tracts of insects from many families. Trypsins have been well characterized while chymotrypsins have been less thoroughly studied. A study of midgut proteases in C. punctiferalis reveals trypsin and elastase-like chymotrypsin as prominent digestive proteases and age-related modulation of midgut proteases exists for trypsin, chymotrypsin, elastase-like chymotrypsin and leucine aminopeptidase 5. Protein digestion in C. punctiferalis has been found to be primarily due to serine proteinases that are sensitive to aprotinin. Specific activities of elastase and trypsin are drastically inhibited by aprotinin in feeding bioassays 6. Proteolysis is therefore, an essential part of food digestion in insects and studies on insect proteases are important. This requires a thorough understanding of biochemical properties of proteases and purification of specific proteases from the midgut homogenate so as to evolve strategies for effective insect pest management programme including development of transgenic cardamom using protease inhibitors. In this paper, we report for the first time the biochemical and kinetic parameters of the purified succinyl-ala-alapro-leu-p-nitroanilide (SAAPLpNA) specific elastaselike chymotrypsin from C. punctiferalis. Materials and Methods Chemicals and equipment All substrates, protease inhibitors, aprotinin-agarose affinity column and electrophoretic reagents were obtained from Sigma-

2 JOSEPHRAJKUMAR et al.: PURIFICATION OF ELASTASE-LIKE CHYMOTRYPSIN 999 Aldrich (St. Louis, USA). Rainbow markers were obtained from Amersham Biosciences, Sweden. Spectrophotometric measurements were recorded using a Shimadzu UV-VIS 1601 double beam spectrophotometer. Insect source and rearing condition Field strains of C. punctiferalis larvae (100 numbers) were collected from the research farm of Cardamom Research Station, Kerala Agricultural University, Pampadumpara, Kerala, India, situated at an elevation of 1100m bove mean sea level. This population has been maintained on cardamom shoots in optimum rearing conditions of 26 C, RH 60-70% and photoperiod of 14h light and 10h dark regime 7. One day old fifth-instar larvae weighing mg each were used in the investigation. Preparation of gut extracts One day old fifth-instar C. punctiferalis larvae were selected for gut extraction. Individual guts of cold-anesthetized larvae were dissected out, cleaned with tissue paper to remove foodstuff, weighed in the Eppendorf tubes and stored at -20 C. Guts were homogenized in a plastic homogenizer in 150 μl of ice-cold 20mM Tris-HCl buffer (ph 8.0) and then spun at 12,000 g for 15 min at 4 C. Supernatant was transferred to clean tubes, spun again as above and stored at -20 C until use. Purification of elastase-like chymotrypsin by affinity chromatography Supernatants from 10 midgut homogenates in 10 mm of Tris-HCl buffer (ph 8.0) from early fifth-instar larvae were applied to an aprotinin agarose column equilibrated with 10 mm of Tris-HCl buffer (ph 8.0). The column was washed with 10 mm of Tris-HCl buffer (ph 8.0; 30ml) to free it from unbound proteins. After the washing step, enzyme was eluted in 500 μl fraction using sodium chloride 0.5 M in10 mm Tris-HCl buffer (ph 8.0). A total of 15 fractions were collected and monitored for protein content according to the method of Bradford 8 and also assayed for enzyme activity using the following substrates viz. benzoyl-arg-p-nitroanilide (BApNA), benzoyl-tyr-p-nitroanilide (BTpNA), succinyl-ala-ala-pro-leu-p-nitroanilide (SAAPLpNA) and leu-p-nitroanilide (LpNA) at 1mM to deduce the enzyme specificity 5. The enzyme activity and protein concentration were expressed as nmole of pna released/min/ml and μg/ml of fraction, respectively. Enzyme kinetics (gut enzyme) The K m for SAAPLpNA and V max was determined for the purified enzyme in 50 mm of Tris-HCl buffer (ph 8) with 20 mm of CaCl 2 at 37 C. The enzyme activities were measured using six different concentrations of substrate ranging from mm and each assay repeated thrice. The results were analyzed by Hanes- Woolf plot and values estimated by plotting [s] verses [s]/v. SDS-PAGE of midgut proteases Proteins were separated under reducing condition using vertical slab sodium dodecyl sulfate-polyacrylamide gel (12%) electrophoresis 9. Proteins were detected staining with Coomassie Brilliant Blue (CBB) R-250 (0.5%) and molecular weights were determined using the full range ( kda) rainbow markers. The molecular weights were calculated by comparing the relative mobility and log molecular weight of protein using a linear regression equation. Reverse zymogram Crude gut protease as well as the purified enzyme were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% denaturing gels for zymogram analysis 10. After the run, the strips were incubated in 100 mm glycine-sodium hydroxide buffer (ph 10) containing 2% casein for 90 min at 30 C. Finally, the gel strips were stained with 0.5% CBB R-250 and destained briefly with methanol-acetic acid-water (40:10:50 v/v). Proteolytic activity was revealed as a zone of white clearing in a dark blue background 11. Results and Discussion Purification of elastase-like chymotrypsin The elution profile of C. punctiferalis midgut protease with 10 mm of Tris-HCl buffer (ph 8) containing 0.5 M of NaCl using an aprotinin-agarose affinity column chromatography has been shown in (Fig. 1). Fig. 1 Profile of affinity chromatography on aprotinin-agarose of midgut extract of C. punctiferalis.

3 1000 INDIAN J EXP BIOL, NOVEMBER 2007 Activities of the 0.5 M of NaCl eluates were examined with BApNA, BTpNA, SAAPLpNA and LpNA (1 mm), and the eluates were found positive only for SAAPLpNA hydrolyzing activity. The single elastase-like chymotrypsin activity peak coincided with the broad peak of protein. The enzyme preparation showed a single protein and elastase-like chymotrypsin band on SDS-PAGE (Fig. 2) and SDS- PAGE-zymogram (casein) (Fig. 3). The molecular mass of the protease corresponded to approximately 72.1 kda. A total of six activity bands were detected with the casein zymogram following electrophoresis of crude midgut preparations from C. punctiferalis. Three bands with molecular masses above 72.1 kda and two below 72.1 kda were resolved in the gel. It is well documented that gut proteolytic profiles of most lepidopteran species including C. punctiferalis comprise trypsin, chymotrypsin, and elastase-like proteinases, amino-peptidases and carboxy-peptidases 5,12. Several proteinases have been purified and characterized from many insect species It has also been reported earlier that protein digestion in C. punctiferalis is primarily due to serine proteases that are sensitive to the serine protease inhibitors such as aprotinin and soybean trypsin inhibitor 5. Purification of elastase-like chymotrypsin was performed using aprotinin affinity chromatography since the major proteolytic activity was affected by aprotinin 5,6. Enzyme kinetics A linear plot (Hanes-Woolf) between [s] verses [s]/v was obtained with SAAPLpNA as a substrate (Fig. 4). The K m and V max values for C. punctiferalis elastase-like chymotrypsin were found to be ± mm and ± 22.1 nmole pna released/min/mg protein, respectively. Several investigators purified more than one protease from the larval guts, and a unique 72.1 kda SAAPLpNA specific elastase-like chymotrypsin from C. punctiferalis (CpELC) was reported from this investigation. The K m value for M. sexta chymotrypsin using SAAPLpNA as a substrate at Fig. 2 SDS-PAGE analysis of midgut extract and purified elastase-like chymotrypsin from C. punctiferalis [Lane A- Molecular weight markers (Rainbow); Lane B- Midgut extract from C. punctiferalis; and Lane C- Purified elastase-like chymotrypsin from C. punctiferalis]. Fig. 3 Zymogram of midgut extract and purified elastase-like chymotrypsin from C. punctiferalis [Lane A- Molecular weight markers (Rainbow); Lane B- Zymogram of midgut extract from C. punctiferalis on denaturing SDS-PAGE; Lane C- Zymogram of purified elastase-like chymotrypsin from C. punctiferalis]. Arrow ( ) indicates one clear zone of proteolytic activity.

4 JOSEPHRAJKUMAR et al.: PURIFICATION OF ELASTASE-LIKE CHYMOTRYPSIN 1001 their role in insecticide resistance mechanisms. Any alterations in the chymotrypsin profile of C. punctiferalis would invariably affect its growth and development due to modulation in protein metabolism. Acknowledgement The author (AJ) is grateful to Department of Biotechnology, New Delhi, India for the award of DBT-post doctoral fellowship for undertaking the project entitled Development of insect resistant transgenic cardamom as well as SPIC Science Foundation, Chennai, India for extending laboratory facilities. Fig. 4 Hanes-Woolf Plot of elastase-like chymotrypsin from C. punctiferalis with SAAPLpNA as a substrate (n=3). ph 8 was reported to be 0.68 mm 18. The reduced K m value for CpELC suggested higher affinity towards SAAPLpNA. At least seven activity bands comprising four minor bands with molecular masses above 50 kda and three major bands with molecular masses estimated between 23 and 32 kda were detected from midgut preparations of Rhyzopertha dominica 14. Two novel chymotrypsins (CTR 1 and CTR 2) that are hydrolyzed by N-terminally extended substrate succinyl-ala-ala-pro-phe-p-nitroanilide (SAAPPpNA) have been reported from locust midguts 13. Chromatography on DEAE columns further indicated that CTR 1 and CTR 2 represent cationic and anionic isoforms of chymotrypsin, respectively, and produced bands on silver-stained SDS-PAGE corresponding to M r 24,000 and was judged to be homogenous. Targeting these enzymes may be a good strategy for the development of effective bio-pesticides and developing transgenics. Transgenic tobacco with aprotinin gene effectively reduced growth and development of Helicoverpa armigera 19. Purification and characterization of midgut proteinases would unravel unique properties that could be selectively exploited in successful pest management strategy or deciphering the physiological mechanism in adaptation of some insect species or resistant strains to the effective protease inhibitors. It will be of paramount importance to use this information about the purified enzymes to improve effective inhibitors through methods of rational design and also proving References 1 Varadarasan S, Insect Pest Management in cardamom-key to reduce cost of production, Spice India, 14 (2001)19. 2 Applebaum S W, Biochemistry of digestion, in Comprehensive insect physiology, biochemistry and pharmacology, vol IV, edited by G A Kerkut & L I Gilbert (Pergamon press, Toronto) 1985, Terra W R, Ferreira C, Jordao B P & Dillon R J, Digestive enzymes, in Biology of the insect midgut, edited by M J Lehane & R F Billingsley (Chapman and Hall, London) 1996, Christeller J T, Liang W A, Markwick N P & Burgess E P J, Midgut protease activities in 12 phytophagous lepidopteran larvae: Dietary and proteases inhibitory interactions, Insect Biochem Mol Biol, 22 (1992) Josephrajkumar A, Chakrabarty R & Thomas G, Midgut proteases of the cardamom shoot and capsule borer, Conogethes punctiferalis (Lepidoptera : Pyralidae) and their interaction with aprotinin, Bull Entomol Res, 96 (2006) Josephrajkumar A, Chakrabarty R & Thomas G, Aprotinin induced modulation of digestive proteinases and growth suppression in cardamom shoot and capsule borer, Conogethes punctiferalis Guenée, Entomon, 30 (2005) Kumaresan D, Regupathy A & Baskaran P, Pests of spices (Rajalakshmi publications, Nagercoil) 1988, Bradford M M, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal Biochem, 72 (1976) Laemmli U K, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) Garcia-Carreno F, Dimes L & Haard N, Substrate gelelectrophoresis for composition and molecular weight of proteases or protease inhibitors, Anal Biochem, 214 (1993) Mohan M & Gujar G T, Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera), J Invertebr Pathol, 82 (2003) 1.

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