Kinetic studies on recombinant stem bromelain
Affiliation(s)
Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia. International Institute for Halal Research & Training (INHART) International Islamic University Malaysia, Kuala Lumpur, Malaysia.
Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia. International Institute for Halal Research & Training (INHART) International Islamic University Malaysia, Kuala Lumpur, Malaysia.
ABSTRACT
Stem bromelain is a plant thiol protease with several industrial and therapeutic applications. This current work presents kinetic studies of recombinant bromelain (recBM) expressed in Escherichia coli BL21-AI on foursynthetic substrates, N-α-carbobenzoxy-L-alanyl-p-nitrophenylester (ZANPE), N-α-carbobenzoxy-L-arginyl-L-ar-ginine-p-nitroanilide (ZAANA), N-α-carbobenzo-xy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanili-de (ZPVANA) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA). Hydrolytic activities of recBM at various pH and temperature conditions were compared to that of commercial bromelain (cBM). Both enzymes demonstrated high activities at 45o C and pH 5 - 8 for recBM and pH 6 - 8 for cBM. recBM showed marginally lower Kmand slightly higher kcat/Kmfor ZAANA, ZANPE and ZPVANA in comparison to cBM.trans Epoxysuccinyl-L-leucylamido {4- guanidino}butane (E-64) severely affected recBM and cBM hydrolysis of the synthetic substrates by competitive inhibition with Kivalues of 3.6 - 5.1 μM and 5.5 - 6.9 μM for recBM and cBM, respectively. The evaluated properties of recBM including temperature and pH optima, substrate specificity and sensitivity to inhibitors or activators, satisfy the requisites required for food industries.
Stem bromelain is a plant thiol protease with several industrial and therapeutic applications. This current work presents kinetic studies of recombinant bromelain (recBM) expressed in Escherichia coli BL21-AI on foursynthetic substrates, N-α-carbobenzoxy-L-alanyl-p-nitrophenylester (ZANPE), N-α-carbobenzoxy-L-arginyl-L-ar-ginine-p-nitroanilide (ZAANA), N-α-carbobenzo-xy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanili-de (ZPVANA) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA). Hydrolytic activities of recBM at various pH and temperature conditions were compared to that of commercial bromelain (cBM). Both enzymes demonstrated high activities at 45o C and pH 5 - 8 for recBM and pH 6 - 8 for cBM. recBM showed marginally lower Kmand slightly higher kcat/Kmfor ZAANA, ZANPE and ZPVANA in comparison to cBM.trans Epoxysuccinyl-L-leucylamido {4- guanidino}butane (E-64) severely affected recBM and cBM hydrolysis of the synthetic substrates by competitive inhibition with Kivalues of 3.6 - 5.1 μM and 5.5 - 6.9 μM for recBM and cBM, respectively. The evaluated properties of recBM including temperature and pH optima, substrate specificity and sensitivity to inhibitors or activators, satisfy the requisites required for food industries.
Cite this paper
Bala, M. , Mel, M. , Saedi Jami, M. , Amid, A. and Mohd Salleh, H. (2013) Kinetic studies on recombinant stem bromelain. Advances in Enzyme Research, 1, 52-60. doi: 10.4236/aer.2013.13006.
Bala, M. , Mel, M. , Saedi Jami, M. , Amid, A. and Mohd Salleh, H. (2013) Kinetic studies on recombinant stem bromelain. Advances in Enzyme Research, 1, 52-60. doi: 10.4236/aer.2013.13006.
References
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John Wiley and Sons, New York, 312. [8] Koh, J., Kang, S.M., Kim, S.J., Cha, M.K. and Kwon, Y.J. (2006) Effect of pineapple protease on the characteristics of protein fibers. Fibers and Polymers, 7, 180-185. Hdoi:10.1007/BF02908264 [9] Ketnawa, S., Rawdkuen, S. and Chaiwut, P. (2010) Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar. Biochemical Engineering Journal, 52, 205-211. Hdoi:10.1016/j.bej.2010.08.012 [10] Yuan, G., Wahlqvist, M.L., He, G., Yang, M. and Li, D. (2006) Natural products and anti-inflammatory activity. Asia Pacific Journal of Clinical Nutrition, 15,143-152. [11] Illanes A. (2008) Enzyme Biocatalysis. Springer Science and Business Media V.B., Brazil, 69. Hdoi:10.1007/978-1-4020-8361-7 [12] Amid, A., Ismail, N., Yusof, F. and Salleh, H.M. (2011) Expression, purification and characterization of a recombinant stem bromelain from Ananas comosus. Process Biochemistry, 46, 2232-2239. Hdoi:10.1016/j.procbio.2011.08.018 [13] Bala, M., Amid, A., Mel, M., Jami, S. and Salleh, H.M. (2011) Recovery of recombinant bromelain from Escherichia coli BL21-AI. African Journal of Biotechnology, 10, 8829-18832. Hdoi:10.5897/AJB11.2761 [14] Muntari, B., Salleh, H.M., Amid, A., Mel, M. and Jami, M.S. (2012) Recombinant bromelain production in Escherichia coli: Process optimization in shake flask culture by Response Surface Methodology. AMB Express, 2, 1-9. Hdoi:10.1186/2191-0855-2-12 [15] Ismail, N. and Amid, A. (2012). Differential scanning calorimetry as tool in observing thermal and storage stability of recombinant bromelain. Food Research International, 19, 727-731. [16] Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 237, 680-685. Hdoi:10.1038/227680a0 [17] Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry, 72, 248-254. Hdoi:10.1016/0003-2697(76)90527-3 [18] Silverstein, R.M. (1974) The assay of the bromelains using N-CBZ-L-lysine-p-nitrophenylester and N-CBZ-L-gly-cine-p-nitrophenyl ester as substrates. Analytical Biochemistry, 62, 478-484. Hdoi:10.1016/0003-2697(74)90180-8 [19] Filippova, I., Lyso-gorskaya, E.N., Oksenoit, E.S., Ruden- skaya, G.N., Stepanov, V.M. (1984) L-Pyroglutamyl-l-phenylala-nyl-l-leucine-p-nitroanilide—A chromogenic substrate for thiol proteinase assay. Analytical Biochemistry, 143, 293-297. Hdoi:10.1016/0003-2697(84)90665-1 [20] Rowan, A.D. and Buttle, D.J. (1994) Pineapple cysteine endopeptidases. Methods in Enzymology, 244, 555-568. Hdoi:10.1016/0076-6879(94)44040-9 [21] Valle, D.M., Bruno, M., Laura, L.M.I., Caffini, N.O., Cantera, A.M.B. (2008) Granulosain I, a Cysteine Protease isolated from ripe fruits of solanum granuloso-leprosum (Solanaceae). The Protein Journal, 27, 267-275. Hdoi:10.1007/s10930-008-9133-4 [22] Benucci, I., Liburdi, K., Garzillo, A.M.V. and Esti, M. (2011) Bromelain from pineapple stem in alcoholic-acidic buffers for wine application. Food Chemistry, 124, 1349- 1353. Hdoi:10.1016/j.foodchem.2010.07.087 [23] Morcelle, S.R., Trejo, A.S., Canals, F., Avilés, F.X., Priolo, N.S. (2004) Funastrain c II: A cysteine endopeptidase purified from the latex of Funastrum clause. The Protein Journal, 23, 205-215. Hdoi:10.1023/B:JOPC.0000026416.90134.7b [24] Napper, A.D., Bennett, P., Borowski, M., Holdridge, M.B., Leonard, M.J.C., Rogers, E.E., Duan, Y., Laursen, R.A., Reinhold, B.S. and Hames, S.L. (1994) Purification and characterization of multiple forms of the pineapplestem-derived cysteine proteinases ananain and comosain. Biochemical Journal, 301, 727-735. [25] Liggieri, M., Arribe, C., Trejo, S.A., Canals, F., Avile, F.X. and Priolo, N.S. (2004) Purification and biochemical characterization of Asclepain c I from the latex of Asclepias curassavica L. constanza. The Protein Journal, 23, 403-411. Hdoi:10.1023/B:JOPC.0000039554.18157.69 [26] M Bruno, M.A., Trejo, S.A., Aviles, X.F., Caffini, N.O. and Lopez, L.M. (2006). Isolation and characterization of Hieronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae). The Protein Journal, 25, 224-223. Hdoi:10.1007/s10930-006-9005-8 [27] Perez, A., Carvajal, C., Trejo, S., Torres, M.J., Martin, M.I., Lorenzo, J.C., Natalucci, C.L. and Hernandez, M. (2010) Penduliflorain I: A cysteine protease isolated from Hohenbergia penduliflora (A.Rich.) Mez (Bromeliaceae). The Protein Journal, 29, 225-233. Hdoi:10.1007/s10930-010-9243-7 [28] Novillo, C., Casta, P. and Ortego, F. (1997) Inhibition of digestive trypsin-like proteases from larvae of several lepidopteran species by the diagnostic cysteine protease inhibitor E-64. Insect Biochemistry and Molecular Biology, 27, 247-254. Hdoi:10.1016/S0965-1748(96)00092-6 [29] Sreedharan, S.K., Patel, H., Smith, S., Gharbia, S.E., Shah, H.N. and Brocklehurst, K. (1993) Additional evidence for the cysteine proteinase nature of gingivain the extracellular proteinase of Porphyromonas gingivalis. Biochemical Society Transactions, 21, 218S-218S. [30] Sreedharan, S.K., Verma, C., Caves, L.S.D., Brocklehurst, S.M., Gharbias, S.E., Shah, H.N. and Brocklehurst, K. (1996) Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis, Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-13-trypsin complex. Biochemical Journal, 316, 777- 786.
[1] Lucia, F. and Tomas, G.V. (2010) Native and biotechnologically engineered plant proteases with industrial applications. Food and Bioprocess Technology, 4, 1066- 1088. [2] Dubey, V.K. and Jagannadham, M.V. (2003) Procerain, a stable cysteine protease from the latex of Calotropis prcera. Phytochemisty, 62, 1057-1071. Hdoi:10.1016/S0031-9422(02)00676-3 [3] Kelly, G.S. (1996) Bromelain: A literature review and discussion of its therapeutic applications. Alternative Medicine Review, 1, 243-257. [4] Maurer, H.R. (2001) Bromelain: Biochemistry, pharmacology and medical uses. Cellular and Molecular Life Sciences, 58, 1234-1245. Hdoi:10.1007/PL00000936H [5] Tochi, B.N., Wang, Z., Xu, S.Y. and Zhang, W. (2009) Effect of stem bromelain on the browning of apple juice, American Journal of Food Technology, 10, 1-8. [6] Aehle, W. (2007) Enzymes in Industry: Production and Applications. Wiley-VCH/Verlag GmbH and Co., Weinheim, 6. [7] Walsh, G. (2002) Protein Biochemistry and Biotechnology. John Wiley and Sons, New York, 312. [8] Koh, J., Kang, S.M., Kim, S.J., Cha, M.K. and Kwon, Y.J. (2006) Effect of pineapple protease on the characteristics of protein fibers. Fibers and Polymers, 7, 180-185. Hdoi:10.1007/BF02908264 [9] Ketnawa, S., Rawdkuen, S. and Chaiwut, P. (2010) Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar. Biochemical Engineering Journal, 52, 205-211. Hdoi:10.1016/j.bej.2010.08.012 [10] Yuan, G., Wahlqvist, M.L., He, G., Yang, M. and Li, D. (2006) Natural products and anti-inflammatory activity. Asia Pacific Journal of Clinical Nutrition, 15,143-152. [11] Illanes A. (2008) Enzyme Biocatalysis. Springer Science and Business Media V.B., Brazil, 69. Hdoi:10.1007/978-1-4020-8361-7 [12] Amid, A., Ismail, N., Yusof, F. and Salleh, H.M. (2011) Expression, purification and characterization of a recombinant stem bromelain from Ananas comosus. Process Biochemistry, 46, 2232-2239. Hdoi:10.1016/j.procbio.2011.08.018 [13] Bala, M., Amid, A., Mel, M., Jami, S. and Salleh, H.M. (2011) Recovery of recombinant bromelain from Escherichia coli BL21-AI. African Journal of Biotechnology, 10, 8829-18832. Hdoi:10.5897/AJB11.2761 [14] Muntari, B., Salleh, H.M., Amid, A., Mel, M. and Jami, M.S. (2012) Recombinant bromelain production in Escherichia coli: Process optimization in shake flask culture by Response Surface Methodology. AMB Express, 2, 1-9. Hdoi:10.1186/2191-0855-2-12 [15] Ismail, N. and Amid, A. (2012). Differential scanning calorimetry as tool in observing thermal and storage stability of recombinant bromelain. Food Research International, 19, 727-731. [16] Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 237, 680-685. Hdoi:10.1038/227680a0 [17] Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry, 72, 248-254. Hdoi:10.1016/0003-2697(76)90527-3 [18] Silverstein, R.M. (1974) The assay of the bromelains using N-CBZ-L-lysine-p-nitrophenylester and N-CBZ-L-gly-cine-p-nitrophenyl ester as substrates. Analytical Biochemistry, 62, 478-484. Hdoi:10.1016/0003-2697(74)90180-8 [19] Filippova, I., Lyso-gorskaya, E.N., Oksenoit, E.S., Ruden- skaya, G.N., Stepanov, V.M. (1984) L-Pyroglutamyl-l-phenylala-nyl-l-leucine-p-nitroanilide—A chromogenic substrate for thiol proteinase assay. Analytical Biochemistry, 143, 293-297. Hdoi:10.1016/0003-2697(84)90665-1 [20] Rowan, A.D. and Buttle, D.J. (1994) Pineapple cysteine endopeptidases. Methods in Enzymology, 244, 555-568. Hdoi:10.1016/0076-6879(94)44040-9 [21] Valle, D.M., Bruno, M., Laura, L.M.I., Caffini, N.O., Cantera, A.M.B. (2008) Granulosain I, a Cysteine Protease isolated from ripe fruits of solanum granuloso-leprosum (Solanaceae). The Protein Journal, 27, 267-275. Hdoi:10.1007/s10930-008-9133-4 [22] Benucci, I., Liburdi, K., Garzillo, A.M.V. and Esti, M. (2011) Bromelain from pineapple stem in alcoholic-acidic buffers for wine application. Food Chemistry, 124, 1349- 1353. Hdoi:10.1016/j.foodchem.2010.07.087 [23] Morcelle, S.R., Trejo, A.S., Canals, F., Avilés, F.X., Priolo, N.S. (2004) Funastrain c II: A cysteine endopeptidase purified from the latex of Funastrum clause. The Protein Journal, 23, 205-215. Hdoi:10.1023/B:JOPC.0000026416.90134.7b [24] Napper, A.D., Bennett, P., Borowski, M., Holdridge, M.B., Leonard, M.J.C., Rogers, E.E., Duan, Y., Laursen, R.A., Reinhold, B.S. and Hames, S.L. (1994) Purification and characterization of multiple forms of the pineapplestem-derived cysteine proteinases ananain and comosain. Biochemical Journal, 301, 727-735. [25] Liggieri, M., Arribe, C., Trejo, S.A., Canals, F., Avile, F.X. and Priolo, N.S. (2004) Purification and biochemical characterization of Asclepain c I from the latex of Asclepias curassavica L. constanza. The Protein Journal, 23, 403-411. Hdoi:10.1023/B:JOPC.0000039554.18157.69 [26] M Bruno, M.A., Trejo, S.A., Aviles, X.F., Caffini, N.O. and Lopez, L.M. (2006). Isolation and characterization of Hieronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae). The Protein Journal, 25, 224-223. Hdoi:10.1007/s10930-006-9005-8 [27] Perez, A., Carvajal, C., Trejo, S., Torres, M.J., Martin, M.I., Lorenzo, J.C., Natalucci, C.L. and Hernandez, M. (2010) Penduliflorain I: A cysteine protease isolated from Hohenbergia penduliflora (A.Rich.) Mez (Bromeliaceae). The Protein Journal, 29, 225-233. Hdoi:10.1007/s10930-010-9243-7 [28] Novillo, C., Casta, P. and Ortego, F. (1997) Inhibition of digestive trypsin-like proteases from larvae of several lepidopteran species by the diagnostic cysteine protease inhibitor E-64. Insect Biochemistry and Molecular Biology, 27, 247-254. Hdoi:10.1016/S0965-1748(96)00092-6 [29] Sreedharan, S.K., Patel, H., Smith, S., Gharbia, S.E., Shah, H.N. and Brocklehurst, K. (1993) Additional evidence for the cysteine proteinase nature of gingivain the extracellular proteinase of Porphyromonas gingivalis. Biochemical Society Transactions, 21, 218S-218S. [30] Sreedharan, S.K., Verma, C., Caves, L.S.D., Brocklehurst, S.M., Gharbias, S.E., Shah, H.N. and Brocklehurst, K. (1996) Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis, Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-13-trypsin complex. Biochemical Journal, 316, 777- 786.