PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

 

e-ISSN 2231-8542
ISSN 1511-3701

Home / Regular Issue / JTAS Vol. 46 (2) May. 2023 / JTAS-2557-2022

 

Isolation of Proteolytic Enzyme from Pineapple Crown

Noor Khaleeda Arsad, Azman Abd Samad, Haryati Jamaluddin and Wan Rosmiza Zana Wan Dagang

Pertanika Journal of Tropical Agricultural Science, Volume 46, Issue 2, May 2023

DOI: https://doi.org/10.47836/pjtas.46.2.14

Keywords: Ananain, bromelain, crown, MD2, proteolytic activity

Published on: 16 May 2023

The pineapple waste from the pineapple industry has contributed to an increase in waste in Malaysia and worldwide every year. A major type of endopeptidase enzymes found in pineapple is fruit bromelain, stem bromelain, ananain, and comasain. This study aims to extract and purify protease from the crown of MD2 pineapple. Protease was extracted and purified using anion exchange chromatography, gel filtration, and desalting before being identified using liquid chromatography-mass spectrometry (LC-MS). Proteolytic activity was determined using the well diffusion method and Casein Digestion Unit. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/ml, a specific activity of 3937.50 U/mg. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/mL, a specific activity of 3937.50 U/mg and the total activity of 3.94 × 109 U. The molecular weight of the purified enzyme was in the range of 25 to 35 kDa under the optimum condition of pH 7 and 37°C. Purification of the extract yielded a band at the molecular weight of 20–25 kDa at the optimum pH of 3 and 9 at 60°C. From LC-MS analysis, the purified enzyme from the crown extract was similar to ananain under accession number A0A199VSS3 (according to Uniprot). It had five unique peptides and covered 97/356 amino acids (44.9% coverage). The ananain (EC 3.4.22.31) is classified in the subfamilies of cysteine protease C1A (clan CA, family C1), a peptidase family related to papain. In conclusion, protease was extracted and identified as an ananain-like protease from the crown.

  • Abreu, D. C. A., & Figueiredo, K. C. D. (2019). Bromelain separation and purification processes from pineapple extract. Brazilian Journal of Chemical Engineering, 36(2), 1029-1039. https://doi.org/10.1590/0104-6632.20190362s20180417

  • Arshad, M. S., Kwon, J., Imran, M., & Sohaib, M. (2017). Plant and bacterial proteases: A key towards improving meat tenderization, a mini review. Cogent Food and Agriculture, 2(1), 1261780. https://doi.org/10.1080/23311932.2016.1261780

  • Arshad, Z. I. M., Amid, A., Yusof, F., Jaswir, I., Ahmad, K., & Loke, S. P. (2014). Bromelain: An overview of industrial application and purification strategies. Applied Microbiology and Biotechnology, 98(17), 7283–7297. https://doi.org/10.1007/s00253-014-5889-y

  • Ataide, J. A., Gerios, E. F., Mazzola, P. G., & Souto, E. B. (2018). Bromelain-loaded nanoparticles: A comprehensive review of the state of the art. Advances in Colloid and Interface Science, 254, 48-55. https://doi.org/10.1016/j.cis.2018.03.006

  • Bala, M., Ismail, N. A., Mel, M., Jami, M. S., Salleh, H. M.., & Amid, A. (2012). Bromelain production: Current trends and perspective. Archives Des Sciences, 65(11), 369–399.

  • Barrera-Núñez, M. C., Yáñez-Vico, R. M., Batista-cruzado, A., Heurtebise-Saavedra, J. M., Castillo-de Oyagüe, R., & Torres-Lagares, D. (2014). Prospective double-blind clinical trial evaluating the effectiveness of bromelain in the third molar extraction postoperative period. Medicina Oral Patologia Oral y Cirugia Bucal, 19(2), e157–e162. https://doi.org/10.4317/medoral.19105

  • Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248–254.https://doi.org/10.1016/0003-2697(76)90527-3

  • Bresolin, I. R. A. P., Bresolin, I. T. L., Silveira, E., Tambourgi, E. B., & Mazzola, P. G. (2013). Isolation and purification of bromelain from waste peel of pineapple for therapeutic application. Brazilian Archives of Biology and Technology, 56(6), 971–979. https://doi.org/10.1590/S1516-89132013000600012

  • Chaurasiya, R. S., & Hebbar, H. U. (2013). Extraction of bromelain from pineapple core and purification by RME and precipitation methods. Separation and Purification Technology, 111, 90-97. https://doi.org/10.1016/j.seppur.2013.03.029

  • Costa, H. B., Fernandes, P. M. B., Romão, W., & Ventura, J. A. (2014). A new procedure based on column chromatography to purify bromelain by ion exchange plus gel filtration chromatographies, Industrial Crops and Products, 59, 163–168. https://doi.org/10.1016/j.indcrop.2014.04.042

  • Falah, M. A. F., Nadine, M. D., & Suryandono, A. (2015). Effects of storage conditions on quality and shelf-life of fresh-cut melon (Cucumis melo L.) and Papaya (Carica papaya L.). Procedia Food Science, 3, 313–322. https://doi.org/10.1016/j.profoo.2015.01.034

  • Ferreira, J. F., Santana, J. C. C., & Tambourgi, E. B. (2011). The effect of pH on bromelain partition from Ananas comosus by PEG4000/phosphate ATPS. Brazilian Archives of Biology and Technology, 54(1), 125–132. https://doi.org/10.1590/s1516-89132011000100017

  • Hale, L. P., Greer, P. K., Trinh, C. T., & James, C. L. (2005). Proteinase activity and stability of natural bromelain preparations. International Immunopharmacology, 5(4), 783–793. https://doi.org/10.1016/j.intimp.2004.12.007

  • Han, J., Cai, Y. F, Xie, X. Q, Wang, Y., Wang, L., Li, C., Rao, W. W., & Ni, L. (2018). A simple method for purification of bromelain in a thermosensitive triblock copolymer-based protection system and recycling of phase components. Separation Science and Technology, 53(4), 636–644. https://doi.org/10.1080/01496395.2017.1398757

  • Hebbar, H. U., Hemavathi, A. B., Sumana, B., & Raghavarao, K. S. M. S. (2011). Reverse micellar extraction of bromelain from pineapple (Ananas comosus L. Merryl) waste: Scale-up, reverse micelles characterization and mass transfer studies. Separation Science and Technology, 46(10), 1656–1664. https://doi.org/10.1080/01496395.2011.572110

  • Hebbar, H. U., Sumana, B., & Raghavarao, K. S. M. S. (2008). Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes. Bioresource Technology, 99(11), 4896–4902. https://doi.org/10.1016/j.biortech.2007.09.038

  • Hebbar, H. U., Sumana, B., Hemavathi, A. B., & Raghavarao, S. M. S. (2012). Separation and purification of bromelain by reverse micellar extraction coupled ultrafiltration and comparative studies with other methods. Food and Bioprocess Technology, 5(3), 1010–1018. https://doi.org/10.1007/s11947-010-0395-4

  • Hidayat, T, Abdullah, F. I., Kuppusamy, C., Samad, A. A., & Wagiran, A. (2012). Molecular identification of Malaysian pineapple cultivar based on internal transcribed spacer region. APCBEE Procedia, 4, 146–151. https://doi.org/10.1016/j.apcbee.2012.11.025

  • Indrajeet, S. O., Singh, S., Chakravarty, I., & Kundu, S. (2017). Extraction and purification of bromelain from pineapple fruit pulp and peel and comparative study of enzymatic activities. International Journal of Basic and Applied Biology, 4(1), 4–7.

  • Kahiro, S. K., Kagira, J. M., Maina, N., Karanja, S. M., & Njonge, F. N. (2017). Enzymatic activity of bromelain from crude extracts of crown, peels and stem of pineapples from different agro-ecological zones of Thika region, Kenya. Asian Journal of Biotechnology and Bioresource Technology, 1(2), 1–6. https://doi.org/10.9734/AJB2T/2017/34314

  • Ketnawa, S, Chaiwut, P., & Rawdkuen, S. (2012). Pineapple wastes: A potential source for bromelain extraction. Food and Bioproducts Processing, 90(3), 385–391. https://doi.org/10.1016/j.fbp.2011.12.006

  • Krishnan, V. A., & Gokulakrishnan, M. (2015). Extraction and purification of bromelain from pineapple and determination of its effect on bacteria causing peridontitis. International Journal of Pharmaceutical Sciences and Research, 6(12), 5284–5294. https://doi.org/10.13040/IJPSR.0975-8232.

  • Kwatra, B. (2019). A review on potential properties and therapeutic applications of bromelain. World Journal of Pharmacy and Pharmaceutical Sciences, 8(11), 488-500. https://doi.org/10.20959/wjpps201911-14941.

  • Lembaga Perindustrian Nanas Malaysia. (2018). Maklumat statistik 2018 [Statistical information of 2018]. LPNM. https://www.mpib.gov.my/en/publication/

  • Mamo, J., & Assefa, F. (2019). Antibacterial and anticancer property of bromelain: A plant protease enzyme from pineapples (Ananas comosus). Current Trends Biomedical Engineering & Bioscience, 19(2), 556009. https://doi.org/10.19080/CTBEB.2019.19.556009

  • Martins, B. C., Rescolino, R., Coelho, D. C., Zanchetta, B., Tambourgi, E. B., & Silveira, E. (2014). Characterization of bromelain from Ananas comosus agroindustrial residues purified by ethanol factional precipitation. Chemical Engineering Transactions, 37, 781–786. https://doi.org/10.3303/CET1437131

  • Mirabella, N., Castellani, V., & Sala, S. (2014). Current options for the valorization of food manufacturing waste: A review. Journal of Cleaner Production, 65, 28–41. https://doi.org/10.1016/j.jclepro.2013.10.051

  • Mohan, R., & Sivakumar, V. (2016). Optimisation of bromelain enzyme extraction from pineapple (Ananas comosus) and application in process industry. American Journal of Biochemistry and Biotechnology, 12(3), 188–195. https://doi.org/10.3844/ajbbsp.2016.188.195

  • Muhammad, Z. A., & Ahmad, T. (2017). Therapeutic uses of pineapple-extracted bromelain in surgical care - A review. Journal of the Pakistan Medical Association, 67(1), 121–125.

  • Munchow, E. A., Hamann, H. J., Carvajal, M. T., Pinal, R., & Bottino, M. C. (2016). Stain removal effect of novel papain and bromelain-containing gels applied to enamel. Clinical Oral Investigations, 20(8), 2315–2320. https://doi.org/10.1007/s00784-016-1840-1

  • Murachi, T. (1976). Bromelain enzymes. In Methods in enzymology (Vol. 45, pp. 475–485). Academic Press.

  • Napper, A. D., Bennett, S. P., Borowski, W., Holdridge, M. B., Leonard, M. J., Rogers, E. E., Duan, Y., Laursen, R. A., Reinhold, B., & Shames, S. L. (1994). Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain. Biochemical Journal, 301(3), 727–735. https://doi.org/10.1042/bj3010727

  • Neh, A., & Ali, N. E. (2020). Agricultural waste management system [AWMS] in Malaysian. Open Access Journal of Waste Management & Xenobiotics, 3(2), 000140. https://doi.org/10.23880/oajwx-16000140

  • Neta, J. L. V., Lédo, A. D., Lima, A. A. B., Santana, J. C. C., Leite, N. S., Ruzene, D. S., Silva, D. P., & de Souza, R. R. (2012). Bromelain enzyme from pineapple: In vitro activity study under different micropropagation conditions. Applied Biochemistry and Biotechnology, 168(2), 234–246. https://doi.org/10.1007/s12010-012-9753-1

  • Ngoc, U. N., & Schnitzer, H. (2009). Sustainable solutions for solid waste management in Southeast Asian countries. Waste Management, 29(6), 1982–1995. https://doi.org/10.1016/j.wasman.2008.08.031

  • Nor, M. Z. M., Ramchandran, L., Duke, M., & Vasiljevic, T. (2015). Characteristic properties of crude pineapple waste extract for bromelain purification by membrane processing. Journal of Food Science and Technology, 52(11), 7103–7112. https://doi.org/10.1007/s13197-015-1812-5

  • Novaes, L. C. D, Jozala, A. F., Lopes, A. M., Santos-Ebinuma, V. D, Mazzola, P. G., & Pessoa, A. (2016). Stability, purification, and applications of bromelain: Review. Biotechnology Progress, 32(1), 5–13. https://doi.org/10.1002/btpr.2190

  • Omotoyinbo, O. V., & Sanni, D. M. (2017). Characterization of bromelain from parts of three different pineapple varieties in Nigeria. American Journal of Bioscience, 5(3), 35–41. https://doi.org/10.11648/j.ajbio.20170503.11

  • Piper, D. W., & Fento, B. H. (1965). pH stability and activity curves of pepsin with special reference to their clinical importance. Gut, 6(5), 506–508. https://doi.org/10.113/gut.6.5.506

  • Ramalingam, C., Srinath, R., & Islam, N. N. (2012). Isolation and characterization of bromelain from pineapple (Ananas comosus) and comparing its anti-browning activity on apple juice with commercial anti-browning agents. Elixir Food Science, 45, 7822–7826.

  • Ramli, A. N. M., Aznan, T. N. T., & Illias, R. M. (2017). Bromelain: From production to commercialisation. Journal of the Science of Food and Agriculture, 97(5), 1386–1395. https://doi.org/10.1002/jsfa.8122

  • Ramli, A. N. M., Manas, N. H. A., Hamid, A. A., Hamid, H., & Illias, R. (2018). Comparative structural analysis of fruit and stem bromelain from Ananas comosus. Food Chemistry, 266, 183-191. https://doi.org/10.1016/j.foodchem.2018.05.125

  • Rathnavelu, V., Alitheen, N. B., Sohila, S., Kanagesan, S., & Ramesh, R. (2016). Potential role of bromelain in clinical and therapeutic applications (Review). Biomedical Reports, 5(3), 283–288. https://doi.org/10.3892/br.2016.720

  • Sahoo, R., & Das, P. K (2017). Bromelain: Applications and purification strategies. PharmaTutor, 5(11), 40–48.

  • Soares, P. A. G., Vaz, A. F. M., Correia, M. T. S., Pessoa Jr., A., Carneiro-da-Cunha, M. G. (2012). Purification of bromelain from pineapple wastes by ethanol precipitation. Separation and Purification Technology, 98, 389–395. https://doi.org/10.1016/j.seppur.2012.06.042

  • Thalip, A. A., Tong, P. S., & Ng, C. (2015). The MD2 ‘super sweet’ pineapple (Ananas comosus). UTAR Agriculture Science, 1(4), 14–17.

  • Vicente, F. A., Lario, L. D., Pessoa, A., & Ventura, S. P. M. (2016). Recovery of bromelain from pineapple stem residues using aqueous micellar two-phase systems with ionic liquids as co-surfactants. Process Biochemistry, 51(4), 528–534. https://doi.org/10.1016/j.procbio.2016.01.004

  • Vijayaraghavan, P., & Vincent, S. G. P. (2013). A simple method for the detection of protease activity on agar plate using bromocresolgreen dye. Journal of Biochemical Technology, 4(3), 628–630. https://doi.org/10.101/j.bej.2007.08.016

  • Wan, J., Guo, J. J., Miao, Z. T., & Guo, X. (2016). Reverse micellar extraction of bromelain from pineapple peel – Effect of surfactant structure. Food Chemistry, 197(Part A), 450–456. https://doi.org/10.1016/j.foodchem.2015.10.145

  • Youryon, P., Supapvanich, S., Kongtrakool, P., & Wongs-Aree, C. (2018). Calcium chloride and calcium gluconate peduncle infiltrations alleviate the internal browning of queen pineapple in refrigerated storage. Horticulture Environment and, Biotechnology, 59(2), 205–213. https://doi.org/10.1007/s13580-018-0028-9