Deep eutectic solvents-based three-phase partitioning for tomato peroxidase purification: A promising method for substituting t-butanol
Introduction
Peroxidase (POD) is an oxidoreductase with iron porphyrin as an auxiliary group that is widely present in animals, plants, and microorganisms. POD is widely used in sewage treatment (Klanovicz et al., 2020), the food industry (de Oliveira, Santos, & Buffon, 2021), and analysis and detection (Wei, Lin, & Chang, 2020) because of its high activity, good heat resistance, and good acid-base stability. Horseradish peroxidase (HRP), as a known commercial source of peroxidase, has been widely studied. Numerous studies have reported the extraction and purification of HRP. However, the availability and cost of commercially available HRP limit its application. Many researchers have explored alternative sources of POD, such as soybean (Camiscia, Silva, Pico, & Valetti, 2020), radish (Lucena et al., 2017), and orange peel (Vetal & Rathod, 2015). Tomato is a cheap crop widely cultivated worldwide and can be eaten fresh or processed. Studies have shown that tomato POD is similar to horseradish peroxidase (HRP), so tomatoes can be used as a good source of POD (Kokkinakis & Brooks, 1979). At present, acetone precipitation and (NH4)2SO4 precipitation are widely used to obtain POD. Acetone precipitation is easily performed without desalting, but it is easy to inactivate the enzyme and is unfriendly to the environment. (NH4)2SO4 precipitation does not easily inactivate the enzyme, but the purity of the obtained enzyme is low. Therefore, it is necessary to develop a greener and more efficient method for obtaining POD.
Three-phase partitioning (TPP) is a versatile technology combining the processes of extraction, separation, and purification. In TPP, three phases are usually formed by adding a certain amount of inorganic salt (typically (NH4)2SO4)) and an organic solvent (typically t-butanol) to the crude extract. Proteins (or enzymes) gather in the middle phase, low molecular pigments and lipids are enriched in the top phase, and polar compounds (e.g., sugars) are enriched in the bottom phase (Chew et al., 2019, Yan et al., 2018). To date, TPP has been widely used in the extraction and purification of various bioactive substances, including proteins and enzymes (Chew et al., 2019, Chia et al., 2019, Vetal and Rathod, 2015), polysaccharides (Wang et al., 2019, Yan et al., 2018), and plant oils (Dutta et al., 2015, Panadare et al., 2020). Although the TPP system has many advantages over traditional extraction and separation technologies, many aspects of it should be further studied and improved. First, a more “green” and efficient method to extract bioactive molecules should be developed based on the traditional TPP system. Organic solvents (typically t-butanol) are widely used in TPP systems. T-butanol is volatile and flammable, harmful to the environment, and possesses a potential risk to operators. Therefore, it is important to explore “green” solvents as substitutes for t-butanol in the TPP system. Second, more attention should be paid to the effect of the TPP system on the bioactivity of target bioactive molecules. Third, through the study of recycling mechanisms, the amount of waste produced by the TPP system should be reduced, to obtain a more economical TPP system.
Deep eutectic solvents (DESs) are regarded as one kind of eco-friendly solvents in recent years, which were first put forward by Abbott et al. (Abbott, Boothby, Capper, Davies, & Rasheed, 2004). DESs are a class of eutectic mixtures that commonly consist of hydrogen-bond acceptors (HBAs) and hydrogen-bond donors (HBDs) (Nakhle, Kfoury, Mallard, Landy, & Greige-Gerges, 2021). Hydrogen bond interactions can be the most important factor influencing DESs formation (El Achkar, Greige-Gerges, & Fourmentin, 2021). DESs have numerous advantages, including nonvolatility, low cost, easy preparation, and good biodegradability; thus, DESs can be used as ideal alternatives to conventional organic solvents and ionic liquids (Cheng and Qi, 2021, Tang et al., 2021, Wojeicchowski et al., 2020).To date, DESs have been widely used to extract bioactive compounds from plant resources, including proteins (Chen et al., 2021), enzymes (Álvarez, Rivas, Longo, Deive, & Rodríguez, 2021), phenolic compounds (Ozturk, Parkinson, & Gonzalez-Miquel, 2018), flavonoids (Mansur, Song, Jang, Lim, Yoo, & Nam, 2019), and polysaccharides (Zhang & Wang, 2017).
This study developed a DES-based TPP to extract and purify tomato POD for substituting the conventional t-butanol. Several DESs were prepared and the optimal DES was screened. The influencing factors of (NH4)2SO4 concentration, volume ratio of DES to crude extract, pH, and extraction temperature on the recovery and purification fold of tomato POD were investigated in single-factor experiments. Response surface methodology (RSM) was used to optimize the major factors influencing the extraction process. DES was recycled and reused after extraction. Finally, the obtained tomato POD was analyzed using SDS-PAGE. The results showed that this system could efficiently extract POD and minimize environmental pollution. This study provides a green and efficient strategy to extract and separate bioactive substances using TPP, and provides a reference for the large-scale application of TPP technology to extract bioactive substances.
Section snippets
Materials and reagents
Freshly harvested tomatoes were purchased from the vegetable market in Changsha, Hunan Province, China. The materials for preparing DESs are as follows: hexanoic acid (analytically pure, 99.0%), decanoic acid (analytically pure, 99.0%), octanoic acid (analytically pure, 99.0%), dodecanoic acid (analytically pure, >99.0%), and lidocaine (analytically pure, >98.0%) were provided by Adamas-Beta Reagent Co., Ltd. (Shanghai, China); thymol (analytically pure, ≥99.0%) was purchased from
Screening the optimal DES
T-butanol has been widely used in TPP. Scholars generally believe that t-butanol does not easily penetrate the interior of folded protein molecules due to its size and branching structure, so it generally can’t cause protein denaturation (Dennison, Moolman, Pillay, & Meinesz, 2000). However, t-butanol has many disadvantages, such as toxicity, volatility, and being environmentally unfriendly, which significantly restrict the large-scale application of TPP. In this study, DESs were used to
Conclusion
In this study, a DES-based TPP was used for tomato POD purification as a substitute for t-butanol. DES-5 (menthol and octanoic acid) was screened as the optimal solvent to construct the TPP. The influencing factors of the TPP process were studied in the single-factor experiments, and the three main factors were optimized by RSM. The recovery and purification fold reached the maximum of 104.71% and 9.76, respectively under the optimized conditions: (NH4)2SO4 concentration of 42%, DES: crude
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (21878326), the Huxiang Young Talent Program from Hunan Province (2021RC3116), the Training Program for Excellent Young Innovators of Changsha (kq2106073), the Agricultural Science and Technology Innovation Program (ASTIP-IBFC08), and the earmarked fund for the China Agriculture Research System (CARS-16-E24).
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