One-step purification and freeze stability of papain at acidic pH values

Authors

DOI:

https://doi.org/10.2298/ABS201217001M

Keywords:

papain, papaya leaves, precipitation techniques, structural stability, activity recovery

Abstract

  • Papain is a protease of great commercial value. The aim of this work was to provide a scalable, inexpensive, and simple papain purification procedure from papaya leaves that would enable the utilization of this by-product.
  • Papain was purified by an optimized combination of salt precipitation at acidic pH. Its freeze stability was determined by activity and secondary structure recovery.
  • Purification of papain at acidic pH values suppressed autoproteolysis and provided a high yield. The obtained protein was stored at slightly alkaline pH to avoid cold denaturation.
  • The purification procedure may be of major importance for industrial, large-scale papain purification from papaya leaves.

Abstract: Papain is a proteolytic enzyme of great commercial value. It is a cysteine protease highly expressed in Carica papaya fruit latex, but also present in papaya leaves. Purification procedures mostly deal with the latex and include a combination of precipitation and/or chromatographic techniques. Due to its solubility, structure and activity characteristics, the pH and salt content play significant roles in handling papain extracts. Here we report a simple, rapid and easily scalable procedure for papain purification from papaya leaves, which contain different contaminants as compared to papaya latex. Sodium chloride precipitation of contaminants at pH 5 followed by ammonium sulphate precipitation resulted in the removal of other leaf proteins and protein fragments from papain solution and about a 3-fold purification. The procedure also benefits from the suppression of autoproteolysis and preservation of the native structure, as confirmed by FTIR analysis, and the high recovery of activity of over 80%.

 

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References

Vatić S, Mirković N, Milošević JR, Jovčić B, Polović NĐ. Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes. Int J Food Microbiol. 2020;334:108851.

Milošević J, Vrhovac L, Đurković F, Janković B, Malkov S, Lah J, et al. Isolation, identification, and stability of Ficin 1c isoform from fig latex. New J Chem. 2020;44(36):15716–23.

Liggieri C, Obregón W, Trejo S, Priolo N. Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L. Acta Biochim Biophys Sin (Shanghai). 2009;41(2):154–62.

González-Rábade N, Badillo-Corona JA, Aranda-Barradas JS, Oliver-Salvador M del C. Production of plant proteases in vivo and in vitro - A review. Biotechnol Adv. 2011;29(6):983–96.

Sharma A, Kumari M, Jagannadham M V. Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis. J Agric Food Chem. 2009;57(23):11120–6.

Weiss J, Gibis M, Schuh V, Salminen H. Advances in ingredient and processing systems for meat and meat products [Internet]. Vol. 86, Meat Science. Meat Sci; 2010. p. 196–213.

Lopes MC, Mascarini RC, Da Silva BMCG, Flório FM, Basting RT. Effect of a papain-based gel for chemomechanical caries removal on dentin shear bond strength. J Dent Child. 2007;74(2):93–7.

Aibara N, Aizawa R, Nakashima M, Ohyama K. Optimization of pH Elution Conditions in Immune Complexome Analysis for Comprehensive Identification of Immune Complex Antigens. Anal Sci. 2020;36(11):1423–6.

Sim YC, Lee SG, Lee DC, Kang BY, Park KM, Lee JY, et al. Stabilization of papain and lysozyme for application to cosmetic products. Biotechnol Lett. 2000;22(2):137–40.

Khaparde SS, Singhal RS. Chemically modified papain for applications in detergent formulations. Bioresour Technol. 2001;78(1):1–4.

Baines BS, Brocklehurst K. A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods. Biochem J. 1979;177(2):541–8.

Nitsawang S, Hatti-Kaul R, Kanasawud P. Purification of papain from Carica papaya latex: Aqueous two-phase extraction versus two-step salt precipitation. Enzyme Microb Technol. 2006;39(5):1103–7.

Rašković B, Popović M, Ostojić S, Anđelković B, Tešević V, Polović N. Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage. Spectrochim Acta Part A Mol Biomol Spectrosc. 2015;150:238–46.

Azarkan M, El Moussaoui A, Van Wuytswinkel D, Dehon G, Looze Y. Fractionation and purification of the enzymes stored in the latex of Carica papaya. J Chromatogr B Anal Technol Biomed Life Sci. 2003;790(1–2):229–38.

Thomás GE, Rodolfo HG, Juan MD, Georgina SF, Luis CG, Ingrid RB, et al. Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products. Process Biochem. 2009;44(1):77–82.

Bradford M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem. 1976;72(1–2):248–54.

Milošević J, Janković B, Prodanović R, Polović N. Comparative stability of ficin and papain in acidic conditions and the presence of ethanol. Amino Acids. 2019;51(5):829–38.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5.

Rašković B, Vatić S, Anđelković B, Blagojević V, Polović N. Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life. Biochem Eng J. 2016;105:168–76.

Rodrigues SP, Ventura JA, Zingali RB, Fernandes PMB. Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis. Phytochem Anal. 2009;20(6):456–64.

Shaw KL, Grimsley GR, Yakovlev GI, Makarov AA, Pace CN. The effect of net charge on the solubility, activity, and stability of ribonuclease Sa. Protein Sci. 2001;10(6):1206–15.

Arakawa T, Timasheff SN. [3]Theory of Protein Solubility. Methods Enzymol. 1985;114(C):49–77.

Devaraj KB, Kumar PR, Prakash V. Purification, Characterization, and Solvent-Induced Thermal Stabilization of Ficin from Ficus carica. J Agric Food Chem. 2008;56(23):11417–23.

Naeem A, Fatima S, Khan RH. Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH. Biopolymers. 2006;83(1):1–10.

Homaei A. Enhanced activity and stability of papain immobilized on CNBr-activated sepharose. Int J Biol Macromol. 2015;75:373–7.

Deulgaonkar SU, Thorat BN. The purification, formulation and drying of papain. Int J Food Eng. 2008;4(8).

Li M, Su E, You P, Gong X, Sun M, Xu D, et al. Purification and in situ immobilization of papain with aqueous two-phase system. PLoS One. 2010;5(12).

Yu L, Zhang H, Yang L, Tian K. Optimization of purification conditions for papain in a polyethylene glycol-phosphate aqueous two-phase system using quaternary ammonium ionic liquids as adjuvants by BBD-RSM. Protein Expr Purif. 2019;156:8–16.

Jiang ZG, Zhang H De, Wang WT. Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems. J Sep Sci. 2015;38(8):1426–32.

Edwin F, Jagannadham M V. Single disulfide bond reduced papain exists in a compact intermediate state. Biochim Biophys Acta - Protein Struct Mol Enzymol. 2000;1479(1–2):69–82.

Shivu B, Seshadri S, Li J, Oberg KA, Uversky VN, Fink AL. Distinct β-sheet structure in protein aggregates determined by ATR-FTIR spectroscopy. Biochemistry. 2013;52(31):5176–83.

Meersman F, Smeller L, Heremans K. Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin. Biophys J. 2002;82(5):2635–44.

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Published

2021-03-19

How to Cite

1.
Marković S, Milošević J, Đurić M, Lolić A, Polović N. One-step purification and freeze stability of papain at acidic pH values. Arch Biol Sci [Internet]. 2021Mar.19 [cited 2022Aug.7];73(1):57-64. Available from: https://www.serbiosoc.org.rs/arch/index.php/abs/article/view/6067

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