Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Asghar Ali Shaikh; Muhammad Umer Dahot; Abdul Sajid; Syed Habib Ahmed Naqvi

doi:10.38211/joarps.2024.05.201

Authors

Asghar Ali Shaikh Department of Chemistry, Government College University, Hyderabad, Pakistan
Muhammad Umer Dahot Institute of Biotechnology & Genetic Engineering, University of Sindh, Jamshoro, Pakistan
Abdul Sajid Department of Chemistry, Government College University, Hyderabad, Pakistan
Syed Habib Ahmed Naqvi Institute of Biotechnology & Genetic Engineering, University of Sindh, Jamshoro, Pakistan

DOI:

https://doi.org/10.38211/joarps.2024.05.201

Keywords:

Cotton seed, Protease, Purification, Alkaline, Cysteine

Abstract

Proteases are widely utilized both in physiological and commercial fields such as medicine, food, detergent, and leather. Plant-originated proteases play a significant role in several biomedical fields due to their easy accessibility and activity. Pakistan is an agro-based country and can be an ideal place for the isolation of industrially important proteases from plant sources such as cotton, which is the main crop and frequently available and low cost. Purification of protease was carried out by fractionation with two-fold acetone, ethanol, methanol and various concentrations (40-80%) of ammonium sulphate. The precipitates formed were collected after centrifugation and dialyzed for 24 hours against universal buffer pH 7.0 and was centrifuged in a cooled refrigerated. The dialyzed sample was loaded on Sephadex G–100 gel column. The fractions of the samples were collected and their absorbance of protein was monitored at 280 nm. The homogeneity of the purified enzyme was checked by SDS gel electrophoresis The purified protease enzyme has optimum activity at 30°C and pH 8.0 when casein was used as substrate. The Km and Vmax values of purified cotton seed's alkaline protease activity was recorded as 0.03M and 17 μmol/minute respectively. Protease activity was increased by the addition of cysteine but inhibited by Iodoacetic acid and β-Mercaptoethanol and decreased with some metal ions. These characteristics of the purified enzyme allowed classifying it as a cysteine protease. In conclusion, this study suggests that the alkaline protease enzyme is the best choice for commercial use

Downloads

Download data is not yet available.

References

Abbas, F., & Abdelrahman, A. (2021). Isolation, Purification and Partial Characterization of a Serine-Like Protease from Solanum dubium Seeds. Biochem Mol Biol, 7(3), 12.

Abd-ElKhalek, A. M., Seoudi, D. M., Ibrahim, O. A., Abd-Rabou, N. S., & Abd ElAzeem, E. M. (2020). Extraction, partial purification, characteristics, and antimicrobial activity of plant protease from Moringa Oleifera leaves. Journal of Applied Biotechnology Reports, 7(4), 243-250.

Abdulaal, W. H. J. B. b. (2018). Purification and characterization of cysteine protease from miswak Salvadora persica. 19(1), 1-6. DOI: https://doi.org/10.1186/s12858-018-0100-1

Akcan, N., & Uyar, F. (2011). Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation. EurAsian Journal of BioSciences, 5. DOI: https://doi.org/10.5053/ejobios.2011.5.0.8

Alhashem, Y. N., Farid, A., Al Mohaini, M., Muzammal, M., Khan, M. H., Dadrasnia, A., . . . Almusalami, E. M. (2022). Protein Isolation and Separation Techniques of Pasteurella multocidavia One-and Two-Dimensional Gel Electrophoresis. Int. J. Cur. Res. Rev, 14(12), 1-8. DOI: https://doi.org/10.31782/IJCRR.2022.141208

Ali, A., & Dahot, M. (2009). Characterization of crude alkaline protease of soybean (Glycine max) seeds. Sindh University Research Journal-SURJ (Science Series), 41(2).

Ali, A. J. P. J. o. A., & Chemistry, E. (2022). Evaluation of Cotton Seeds as Environmentally Liable Source for Neutral Protease. 23(2), 215-224. DOI: https://doi.org/10.21743/pjaec/2022.12.04

Antao, C. M., & Malcata, F. X. (2005). Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol Biochem, 43(7), 637-650. DOI: https://doi.org/10.1016/j.plaphy.2005.05.001

Anupama, Marimuthu, M., Sundaram, U., & Gurumoorthi, P. (2013). Optimization, Isolation and Partial Characterization of Proteases from Underutilized and Common Food Legumes. International research journal of pharmacy, 4, 99-106. DOI: https://doi.org/10.7897/2230-8407.04722

Ashouri, S., Abad, R. F. P., Zihnioglu, F., & Kocadag, E. (2017). Extraction and purification of protease inhibitor (s) from seeds of Helianthus annuus with effects on Leptinotarsa decemlineata digestive cysteine protease. Biocatalysis and Agricultural Biotechnology, 9, 113-119. DOI: https://doi.org/10.1016/j.bcab.2016.12.005

Atrooz, O. M., & Alomari, F. N. (2020). Determination of the activity and kinetics parameters of proteases in the crude plant extracts of Mentha piperita L. and Thymus capitatus L. Journal of Applied Biology and Biotechnology, 8(6), 3-7.

Badgujar, S. B. (2014). Evaluation of hemostatic activity of latex from three Euphorbiaceae species. Journal of ethnopharmacology, 151 1, 733-739. DOI: https://doi.org/10.1016/j.jep.2013.11.044

Baidamshina, D. R., Koroleva, V. A., Olshannikova, S. S., Trizna, E. Y., Bogachev, M. I., Artyukhov, V. G., . . . Kayumov, A. R. (2021). Biochemical properties and anti-biofilm activity of chitosan-immobilized papain. Marine Drugs, 19(4), 197.

Baidamshina, D. R., Koroleva, V. A., Olshannikova, S. S., Trizna, E. Y., Bogachev, M. I., Artyukhov, V. G., . . . Kayumov, A. R. (2021). Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain. Marine Drugs, 19. DOI: https://doi.org/10.3390/md19040197

Banerjee, R., & Bhattacharyya, B. C. (1992). Purification and characterization of protease from a newly isolated Rhizopus oryzae. Bioprocess Engineering, 7(8), 369-374. doi:10.1007/BF00369493 DOI: https://doi.org/10.1007/BF00369493

Banik, S., Biswas, S., & Karmakar, S. (2018). Extraction, purification, and activity of protease from the leaves of Moringa oleifera. F1000Research, 7. DOI: https://doi.org/10.12688/f1000research.15642.1

Chanda, I., Basu, S. K., Dutta, S. K., & Das, S. R. C. (2011). A Protease Isolated from the Latex of Plumeria rubra Linn (Apocynaceae) 1: Purification and Characterization. Tropical journal of pharmaceutical research, 10, 705-711. DOI: https://doi.org/10.4314/tjpr.v10i6.2

da Silva, A. V., do Nascimento, J. M., Rodrigues, C. H., Nascimento, D. C. S., Costa, R. M. P. B., Marques, D. d. A. V., . . . Converti, A. (2020). Partial purification of fibrinolytic and fibrinogenolytic protease from Gliricidia sepium seeds by aqueous two-phase system. Biocatalysis and Agricultural Biotechnology, 27, 101669.

Davis, B. (1964). Method and application tohuman serum protein. Ann. NY Acad. Sci., 121, 404-427. DOI: https://doi.org/10.1111/j.1749-6632.1964.tb14213.x

Devi, B. G., & HemaLatha, K. (2014). Isolation, partial purification and characterization of alkaline serine protease from seeds of Cucumis melo var agrestis. International Journal of Research in Engineering and Technology, 3(6), 88-95. DOI: https://doi.org/10.15623/ijret.2014.0306016

Doi, O., & Nojima, S. (1975). Lysophospholipase of Escherichia coli. J Biol Chem, 250(13), 5208-5214. DOI: https://doi.org/10.1016/S0021-9258(19)41297-0

Gangaraju, S., Manjappa, B., Subbaiah, G. K., Kempaiah, K., Shashidharamurthy, R., Plow, J. H., . . . Sannaningaiah, D. (2015). Jackfruit (Artocarpus heterophyllus) seed extract exhibits fibrino(geno)lytic activity. Pharmacognosy Journal, 7, 171-177. DOI: https://doi.org/10.5530/pj.2015.3.5

Gonçalves, R. N., Gozzini Barbosa, S. D., & Silva-López, R. E. d. (2016). Proteases from Canavalia ensiformis: active and thermostable enzymes with potential of application in biotechnology. Biotechnology Research International, 2016. DOI: https://doi.org/10.1155/2016/3427098

Holme, D., & Peck, H. In Analytical Biochemistry (1983): Published by Longman Group Ltd., England.

Holme, D. J., & Peck, H. (1983). Analytical Biochemistry: Longman.

Iqbal, A., Hakim, A., Hossain, M. S., Rahman, M. R., Islam, K., Azim, M. F., . . . Azad, A. K. (2018). Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2. Journal of Genetic Engineering & Biotechnology, 16, 29 - 37. DOI: https://doi.org/10.1016/j.jgeb.2017.10.011

Jinka, R., Ramakrishna, V., Rao, S. K., & Rao, R. P. (2009). Purification and characterization of cysteine protease from germinating cotyledons of horse gram. BMC Biochem, 10, 28. doi:10.1186/1471-2091-10-28 DOI: https://doi.org/10.1186/1471-2091-10-28

Kavoosi, G., & Ardestani, S. K. (2012). Gel electrophoresis of protein–from basic science to practical approach. Gel Electrophoresis–Principles and Basics, 69. DOI: https://doi.org/10.5772/38062

Khan, M. B., Khan, H.-u., Shah, M. U., & Khan, S. (2016). Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus colocynthis. Natural Product Research, 30, 935 - 940. DOI: https://doi.org/10.1080/14786419.2015.1079909

Kim, M., Si, J.-B., Reddy, L. V., & Wee, Y.-J. (2016). Enhanced production of extracellular proteolytic enzyme excreted by a newly isolated Bacillus subtilis FBL-1 through combined utilization of statistical designs and response surface methodology. Rsc Advances, 6(56), 51270-51278. DOI: https://doi.org/10.1039/C6RA07724B

Kumar, V., Anjana, P., & Sharma, S. (2019). Latest Overview of Proteases: A Review. Asian J. Adv. Basic Sci, 7(2), 20-28. DOI: https://doi.org/10.33980/ajabs.2019.v07i02.004

Lowry, O., Rosebrough, N., Farr, A., & Randall, R. J. B. C. (1951). of these enzymes than MQ. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA. 193, 265-275.

Maqtari, M. A. A., Naji, K. M., & Ali, L. K. Extraction and Immobilization of Proteolytic Enzymes from Local Yemeni Bean Seeds (Dolichos Lablab L.).

Matagne, A., Bolle, L., El Mahyaoui, R., Baeyens-Volant, D., & Azarkan, M. (2017). The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms. Phytochemistry, 138, 29-51. DOI: https://doi.org/10.1016/j.phytochem.2017.02.019

Matkawala, F., Nighojkar, S., Kumar, A., & Nighojkar, A. (2019). A novel thiol-dependent serine protease from Neocosmospora sp. N1. Heliyon, 5. DOI: https://doi.org/10.1016/j.heliyon.2019.e02246

Miyazaki-Katamura, S., Yoneta-Wada, M., Kozuka, M., Sakaue, T., Yamane, T., Suzuki, J., . . . Ohkubo, I. (2019). Purification and Biochemical Characterization of Cysteine Protease from Baby Kiwi (Actinidia arguta). The Open Biochemistry Journal. DOI: https://doi.org/10.2174/1874091X01913010054

Mnif, I. H., Siala, R., Nasri, R., Mhamdi, S., Nasri, M., & Kamoun, A. S. (2014). A Cysteine Protease Isolated from the Latex of Ficus microcarpa: Purification and Biochemical Characterization. Applied biochemistry and biotechnology, 175, 1732-1744. DOI: https://doi.org/10.1007/s12010-014-1376-2

Moo-Young, M. (2011). Comprehensive Biotechnology: Elsevier Science.

Ogbonna, A., & Okolo, B. (2005). Purification and some properties of a metalloprotease from sorghum malt variety KSV8-1. World Journal of Microbiology and Biotechnology, 21(6), 1051-1056. DOI: https://doi.org/10.1007/s11274-004-8026-8

Paul, B., & Gowda, L. R. (2000). Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab). J Agric Food Chem, 48(9), 3839-3846. doi:10.1021/jf000296s DOI: https://doi.org/10.1021/jf000296s

Rawski, R. I., Sanecki, P. T., Dżugan, M., & Kijowska, K. (2018). The evidence of proteases in sprouted seeds and their application for animal protein digestion. Chemical Papers, 72(5), 1213-1221. DOI: https://doi.org/10.1007/s11696-017-0341-2

Reagent, F. P. (1951). PROTEIN MEASUREMENT WITH THE. J. biol. Chem, 193, 265-275. DOI: https://doi.org/10.1016/S0021-9258(19)52451-6

Sarkar, G., & Suthindhiran, K. (2020). Extraction and characterization of alkaline protease from Streptomyces sp. GS-1 and its application as dehairing agent. Biocatalysis and Agricultural Biotechnology, 25, 101590. DOI: https://doi.org/10.1016/j.bcab.2020.101590

Sathya Prabhu, D., Apoorva, S., Nandita, J., Chamy, P., & Devi Rajeswari, V. (2018). Purification of protease enzyme from the leaf, seed and pod samples of Vicia faba L. International Food Research Journal, 25(5).

Segel, I. (1976). Biochem. calculations, 273-277: New York, John Wiley and Sons.

Sharma, M., Gat, Y., Arya, S., Kumar, V., Panghal, A., & Kumar, A. (2019). A review on microbial alkaline protease: an essential tool for various industrial approaches. Industrial Biotechnology, 15(2), 69-78. DOI: https://doi.org/10.1089/ind.2018.0032

Silva, A. V. d., Nascimento, J. M., Rodrigues, C. H., Nascimento, D. C. S., Costa, R. M. P. B., Marques, D. A. V., . . . Porto, A. L. F. (2020). Partial purification of fibrinolytic and fibrinogenolytic protease from Gliricidia sepium seeds by aqueous two-phase system. Biocatalysis and Agricultural Biotechnology, 27, 101669. DOI: https://doi.org/10.1016/j.bcab.2020.101669

Turk, B., Turk, V., & Turk, D. J. B. c. (1997). Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. 378(3-4), 141-150.

Uday, P., Maheshwari, M., Sharanappa, P., Nafeesa, Z., Kameshwar, V. H., Priya, B. S., & Nanjunda Swamy, S. (2017). Exploring hemostatic and thrombolytic potential of heynein - A cysteine protease from Ervatamia heyneana latex. Journal of ethnopharmacology, 199, 316-322. DOI: https://doi.org/10.1016/j.jep.2016.12.047

Yang, R., Song, J., Gu, Z., & Li, C. (2011). Partial purification and characterisation of cysteine protease in wheat germ. Journal of the Science of Food and Agriculture, 91(13), 2437-2442. DOI: https://doi.org/10.1002/jsfa.4484

Zanphorlin, L. M., Facchini, F. D. A., Vasconcelos, F. N., Bonugli-Santos, R. C., Rodrigues, A., Sette, L. D., . . . Bonilla-Rodriguez, G. O. (2010). Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp. The Journal of Microbiology, 48, 331-336. DOI: https://doi.org/10.1007/s12275-010-9269-8