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School of Chemical Science and Technology, Key Laboratory of Medicinal Chemistry for Natural Resource-Ministry of Education, Yunnan University, 2 North Cuihu Road, Kunming 650091, People's Republic of China
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, 2 North Cuihu Road, Kunming 650091, People's Republic of China
School of Chemical Science and Technology, Key Laboratory of Medicinal Chemistry for Natural Resource-Ministry of Education, Yunnan University, 2 North Cuihu Road, Kunming 650091, People's Republic of China
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, 2 North Cuihu Road, Kunming 650091, People's Republic of China
Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free electrochemical method for determining chymosin activity based on a peptide-based enzyme substrate. The synthesized substrate peptide for chymosin was assembled onto the surface of the Au-deposited grassy carbon electrode. The current was proportional to chymosin activity, and thus chymosin activity could be determined. The detection ranges of chymosin activity were 2.5 to 25 U mL−1. The detection limit of chymosin activity was 0.8 U mL−1. The sensing platform was used to quantify chymosin activity in commercial rennet with high selectivity, excellent stability, and satisfactory reproducibility. We developed a facile, fast, and effective electrochemical assay for detecting chymosin activity, which has potential applications in cheesemaking.
Cheese is a major dairy food consumed worldwide. Most cheeses are made using rennet as a coagulant. Chymosin is a specific milk-clotting aspartyl proteinase secreted in the abomasum of calves during the initial days after birth. This enzyme specifically cleaves the Phe105-Met106 peptide bond of κ-casein and releases a hydrophilic glycomacropeptide from the C-terminus of the protein (
). This leads to the coagulation of casein micelles and gel formation, which can be easily separated into cheese and whey. Chymosin has higher milk-clotting activity, but lower nonspecific protease activity than other milk-clotting enzymes, which is advantageous for cheesemaking (
). The proportion of chymosin differs by source or animal age (cow, lamb, goat, camel, or nonanimal sources). For example, the enzymatic activity of camel rennet is higher than that of bovine for cleaving the Phe105-Met106 bond of bovine κ-casein (
). In calf rennet, this activity depends on both the age and diet of the animal. Chymosin accounts for ∼90% of the clotting enzymes, and pepsin accounts for only ∼10% in the rennet of milk-fed young calves. In contrast, pepsin predominates in older fodder-eating bovines (
). It is well known that chymosin and pepsin have different specific hydrolysis contributions to κ-casein and significantly affect cheese maturation and its ultimate quality (in flavor or texture). Residual chymosin is considered to cause cheese softening by slowly cleaving caseins other than κ-casein (
). Therefore, detecting chymosin is very important in cheesemaking from the scientific, technological, industrial, and economic perspectives.
Many techniques have been described for quantifying chymosin. The classical method for determining protease activity involves measuring the clotting time of milk after adding milk-clotting protease by visual observation when flocculation of the milk precipitate begins (
standard method for chymosin determination in which chymosin is chromatographically separated from pepsin, and its clotting-milk activity is determined on a standardized milk substrate (standard 110A). Unlike typical assays of milk clotting, several methods for chymosin determination with different analytical principles have been reported. Additionally,
described an assay for chymosin determination with a spectrophotometric method using a synthetic peptide as a substrate. Methods including ELISA (Rolet- Répécaud et al., 2015), HPLC (
) have also been developed for chymosin quantification. Although chymosin can be efficiently determined using these methods, they have some limitations, such as the subjective nature of the observation, costly instruments, inefficiency, or tedious pretreatment, preventing analytical applications in a real sample. Thus, a rapid, stable, and simple quantitative method for chymosin is needed.
Electrochemical sensors are highly sensitive, have a fast response time, use simple instrumentation, and have been widely used in bioanalysis. However, few studies have examined electrochemical determination of chymosin.
reported an electrochemical assay for evaluating the milk-clotting activity of rennet, which was an efficient and rapid analytical method; however, the problem is that the preparation of artificial casein micelles is needed, which is a bureaucratic procedure. A peptide-based sensing platform (
Bovine serum albumin as an effective sensitivity enhancer for peptide-based amperometric biosensor for ultrasensitive detection of prostate specific antigen.
Target protein induced cleavage of a specific peptide for prostate-specific antigen detection with positively charged gold nanoparticles as signal enhancer.
Peptide substrates for chymosin (rennin). Interaction sites in κ-casein-related sequences located outside the (103–108)-hexapeptide region that fits into the enzyme's active-site cleft.
). With previous methods, however, further modification of the chromophoric or luminous group with peptide is necessary. Moreover, labeled substrate peptides must be accurately synthesized, which is complex and costly, and the peptides do not exhibit their natural properties after this process, limiting their further applications (
). Therefore, unmodified peptides should be used as a substrate.
In this study, we developed an electrochemical sensor for determining chymosin activity based on a label-free peptide substrate. The substrate peptide was assembled onto the surface of an Au-deposited grassy carbon electrode (Au-GCE) through Cys at the C-terminus of the peptide. This substrate peptide is positively charged at pH 7.0, inhibiting permeation of [Ru(NH3)5Cl]2+ and resulting in a weak peak current. In the presence of chymosin, the Phe-Met bond of peptide can be cleaved by chymosin, and the positively charged peptide fragment released, leaving the neutral peptide portion that can promote the penetration of [Ru(NH3)5Cl]2+ on the electrode surface, leading to enhanced peak current. The current change is proportional to the chymosin activity. The sensor successfully quantified the chymosin activity in commercial rennets with good selectivity, excellent stability, and satisfactory reproducibility, thus providing a facile, fast, and efficient electrochemical method for chymosin detection.
MATERIALS AND METHODS
Materials and Reagents
Substrate peptide for chymosin (>98%) was synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Chymosin was purchased from GL Biochem., Ltd. (Shanghai, China). Rennets were from Anke Bioengineering Co. Ltd. (Jinan, China). Human thrombin, lipase, papain, lysozyme, trypsin, and chymotrypsin were purchased from Sigma Aldrich (Shanghai, China). Whey was prepared as follows. The pH of skim milk was adjusted to pH 4.6, and then the supernatant was obtained by centrifugation at 5,000 × g for 30 min. Subsequently, the pH of supernatant was adjusted to 7.0 with 1 M HCl to obtain the whey. 1-Hexanethiol (HT), pentaamminechlororuthenium (III) chloride ([Ru(NH3)5Cl]2+), gold chloride hydrate (HAuCl4), and potassium hexacyanoferrate (III) K3Fe(CN)6 were obtained from Aladdin Industrial Corporation (Shanghai, China). Solution for electrochemical impedance spectroscopy (EIS) comprised 1 mM [Fe(CN)6]3−/4− with 0.1 M KCl. Solution for differential pulse voltammetry (DPV) was 10 mM [Ru(NH3)5Cl]2+. All other reagents were analytical grade and used without further purification. All aqueous solutions were prepared with deionized water (DW; 18 MΩ cm−1).
Apparatus
The morphologies of the prepared samples were characterized by scanning electron microscopy (QUNT200, Hillsboro, OR) and Nanoscope III atomic force microscope (AFM). All electrochemical measurements were performed on a CHI 660E Electrochemical Workstation from Shanghai Chenhua Instrument (Shanghai, China).
Preparation of Peptide-Modified Electrode
A GCE (3 mm in diameter) was polished with 0.3- and 0.05-μm Al2O3 powder, respectively, and then sonicated in ethanol and DW to remove the physically adsorbed substance and dried in air. Then, Au nanoparticles were electrodeposited on the cleaned and dried GCE surface in 10 mM HAuCl4 aqueous solution under the potential of −0.2 V for 200 s. The Au-modified electrode was submerged into a solution of 10 μL of peptide solution overnight at 4°C. Subsequently, to block the possible remaining active sites and eliminate the risk of nonspecific binding, the electrode was treated with 10 μL of 10 mM HT at room temperature for 1 h. The modified electrode was cleaned with DW to remove the physically adsorbed species. The electrode was preactivated at 37°C for 5 min. Finally, 10-μL samples of chymosin solution with different chymosin activity were incubated at 37°C for 10 min. The electrode was thoroughly washed with DW to terminate the reaction.
Electrochemical Measurements
The DPV and EIS experiments were performed with a CHI 660E Electrochemical Workstation and conducted using a 3-electrode system, with the modified GCE as the working electrode, a platinum wire as the counter-electrode, and a saturated calomel electrode as the reference electrode. The DPV response was recorded in 10 mM [Ru(NH3)5Cl]2+ with a potential range of 0 to −0.5 V, modulation amplitude of 0.05 V, and pulse width of 0.05 s. In addition, electrochemical characterization of the biosensor was performed by EIS in 0.1 M KCl containing 1 mM [Fe(CN)6]3−/4−. The EIS was tested in the frequency range of 10−1 to 10−5 Hz with an amplitude of 5 mV.
Assembly of Modified Electrodes Characterized by EIS and Selectivity of Constructed Electrochemical Sensor
The EIS was performed in 1.0 mM [Fe(CN)6]3−/4− and 0.1 M KCl with a frequency range of 10−1 to 10−5 Hz with an amplitude of 5 mV. The interferent included chymotrypsin, thrombin, lipase, lysozyme, trypsin, and papain. Concentration of interferent was 300 μg mL−1.
RESULTS AND DISCUSSION
Principle of the Constructed Electrochemical Sensor
The substrate peptide for chymosin was designed based on a previous study (
Peptide substrates for chymosin (rennin). Interaction sites in κ-casein-related sequences located outside the (103–108)-hexapeptide region that fits into the enzyme's active-site cleft.
) with minor modifications. The AA sequence of the peptide is NH2-Cys-Gly-Gly-Gly-His-Pro-His-Pro-His-Leu-Ser-Phe-Met-Ala-Ile-Pro-Pro-Lys-Lys-COOH, where the Cys residue at peptide N terminus was used to combine with Au-GCE, and Gly-Gly-Gly is a flexible domain enabling efficient binding of the peptide to Au-GCE. Chymosin can cleave the peptide bond between underlined Phe and Met AA residues (
). Figure 1A illustrates the sensing principle of the constructed sensor. The substrate peptide showed an isoelectric point (pI) of 9.9 and formed a positively charged (+2.2) interface at pH 7.0, preventing access of the positive electrochemical probe [Ru(NH3)5Cl]2+ to reach the electrode surface and resulting in a low peak current. When the amino bond between Phe and Met in the substrate peptide was cleaved by chymosin, the positively charged peptide portion (MAIPPKK, pI 10.4) was released from the electrode surface, leaving an almost neutral peptide fragment (Cys-Gly-Gly-Gly-His-Pro-His-Pro-His-Leu-Ser-Phe, pI 7.4, +0.2 at pH 7.0). The decreased positive charges of the peptide enabled [Ru(NH3)5Cl]2+ to contact the electrode surface, enhancing the current response of the probe. Accordingly, a facile electrochemical method for evaluating chymosin was achieved (Figure 1B).
Figure 1Effect of chymosin on changes in the charge of the peptide substrate (A) and its electrochemical sensing platform for chymosin (B). HT, 1-hexanethiol; Ru(NH3)5Cl, pentaamminechlororuthenium (III) chloride.
Characterization of Assembly of Peptide on Au-Deposited GCE
To characterize the assembly process of the substrate peptide at the GCE surface, scanning electron microscopy and AFM images of the modified GCE were obtained. The surface of GCE was smooth (Figure 2A), but became rough when nanoparticles were attached, as clearly observed (Figure 2A). This result suggested the deposition of Au was successful. After incubation with the substrate peptide, large nanoparticles were observed on the surface of Au-GCE (Figure 2B) because of the assembled peptides on the electrode surface. After blocking with HT, the nanoparticles slightly increased in size because of the bound HT on the electrode (Figure 2C). Interestingly, nanoparticles on the surface of peptide-immobilized Au-GCE blocked with HT were smaller in the presence of chymosin (Figure 2D) because of release of the peptide portion after cleavage by chymosin. Morphological changes of the peptide-immobilized electrode in the absence and presence of chymosin were further observed by AFM. In the absence of chymosin, a relatively large, rough, and fluctuant AFM morphology of the peptide-immobilized electrode was observed (Figure 2E). However, after enzymatic cleavage by chymosin, the surface morphology of the peptide-immobilized electrode was regular and showed a narrower distance (Figure 2F). These results suggested that release of the peptide fragment and the repulsion interaction caused by the positive charge were decreased. These results confirmed successful assembly of the peptide on the electrode and initial effect of chymosin on the peptide.
Figure 2Morphologic characterization of assembly process and effects of chymosin on the electrode surface by electron microscope analysis. Scanning electron microscopy of (A) Au-deposited grassy carbon electrode (Au-GCE); (B) Pep-Au-GCE; (C) HT-Pep-Au-GCE; (D) HT-Pep-Au-GCE + Chy. Pep = peptide substrate; HT = 1-hexanethiol; Chy = chymosin. (E, F) Atomic force microscopy images of peptide-modified electrode incubation without (E) and with (F) chymosin.
Electrochemical impedance spectroscopy is an electroanalytical method used for the evaluation of electron-transfer properties of modified surfaces and for understanding of chemical transformations. An EIS analysis provides mechanistic and kinetic information on a wide range of materials, such as a nanomaterial-modified electrode. In theory, impedance measurements can be used to examine any intrinsic property that influences the conductivity of a nanoparticle's solution interface. In our study, EIS was used to characterize the assembly process of materials with different conductive properties. Compared with the GCE, the charge transfer resistance (Rct) of the Au-deposited GCE decreased significantly, as Au has an excellent charge transfer rate, and thus it enhanced the electroconductivity of the modified electrode (Figure 3). When the peptide was immobilized onto the Au-GCE, the Rct of the electrode increased, which was further increased by incubation with HT. After incubation with chymosin, an obvious decrease in Rct was observed, indicating that the peptide bond between the Met and Phe residues was cleaved by chymosin, leaving a neural peptide fragment that can help the positive electrochemical probe [Ru(NH3)5Cl]2+ easily contact the interface of the electrode, decreasing Rct.
Figure 3Characterization of assembly of modified electrodes characterized by electrochemical impedance spectroscopy. Z′ and Z″ represent the real and imaginary parts of the impedance, respectively. Grassy carbon electrode (GCE; a); Au-deposited GCE (b); Pep-Au-GCE (c); HT-Pep-Au-GCE (d); HT-Pep-Au-GCE + Chy (e). Pep = peptide substrate; HT = 1-hexanethiol; Chy = chymosin.
To confirm the feasibility of the proposed sensor, DPV experiments were performed. As shown in Figure 4A, without chymosin, the DPV peak of the peptide-modified electrode was weak, whereas a remarkable enhancement of DPV peak was observed in the presence of chymosin (20 U mL−1). The obvious differences in DPV between the treatments without and with chymosin confirmed that this method can be used to quantify chymosin. The DPV current response increased as the reaction time increased, and maintained a constant value over 10 min (Figure 4B); therefore, 10 min was chosen as the optimal enzymatic reaction time.
Figure 4Electrochemical characteristics of the sensor. Electrochemical measurement of the proposed sensor evaluated by differential pulse voltammetry. (A) Feasibility of the sensor evaluated by differential pulse voltammetry (DPV) in the absence and presence of chymosin. (B) Effect of reaction time on the current by DPV measurement. The data are the average value of 3 determinations. (C) DPV curves for various activities of chymosin. (D) Calibration plot for activity of chymosin (U mL−1) versus DPV response.
Determination of Chymosin by Fabricated Electrochemical Sensor
Using DPV as an analytical tool offers advantages when compared with other electrochemical techniques. Differential pulse voltammetry is very sensitive, often allowing direct analyses at the parts per billion level. It owes its sensitivity to the relatively short pulse time and its differential nature. The short pulse time increases the measured currents, and the differential measurement discriminates against background processes. In our study, DPV was used to determine the chymosin activity using the peptide-modified electrode. Figure 4C shows the DPV curves of the modified electrode for different chymosin activities. The reduction peak current of [Ru(NH3)5Cl]2+ at approximately −0.30 V proportionally increased with increasing chymosin activity from 2.5 to 25 U mL−1 (Figure 4C). The linear relationship between current (I) and chymosin activity was as follows: I (μA) = 4.45 chymosin concentration (U mL−1) − 2.39 (R2 = 0.994), with a limit of detection of 0.8 U mL−1 (signal/noise = 3; Figure 4D).
Selectivity, Repeatability, and Stability of the Sensor
As shown in Figure 5, in the presence of 15 U mL−1 chymosin, the increased DPV peak current of the electrode was −1.92 μA. The interferent (chymotrypsin, thrombin, lipase, lysozyme, trypsin, papain, or their mixture, 300 μg mL−1) or their mixture minimally affected the current. Notably, the sensor showed excellent anti-interference ability even in the presence of various enzymes, including proteases. The repeatability of the sensor was evaluated by measuring the current of the electrodes to detect 20 U mL−1 of chymosin. Furthermore, it is worthy to note that the current response of the biosensor was hardly affected, even in the presence of whey, which further indicated the excellent anti-interferent ability. Figure 6A shows that the relative standard deviation of the response current was 7.08%, suggesting acceptable repeatability. After storage at 4°C for 20 d, the current response of the modified electrodes was maintained at more than 97.6% of the initial response (Figure 6B), demonstrating the good stability of the sensors.
Figure 5Selectivity of constructed electrochemical sensor, showing change in current (ΔI) with chymosin (Chy) and different interferents. THR, human thrombin; LSP, lipase; PAP, papain; LYS, lysozyme; TYR, trypsin; CTY, chymotrypsin.
Figure 6Repeatability (A) and stability (B) of the sensor. (A) The current values of the six electrodes were measured at the same conditions. (B) The current change of the sensors after storage at 4°C for 0 to 20 d.
Next, the chymosin activity in commercial rennet samples was quantified and compared with a reported standard method. The recovery rate was 92.47 to 106.32%, with relative standard deviations of 2.2 to 5.3% (Table 1). This suggests that the constructed sensor can be applied for chymosin sensing in cheesemaking. Compared with previously reported methods, our assay has a low cost and short operation time, it did not have a very low detection limit (Table 2). Additionally, a short peptide was used as a substrate for chymosin, which is easily synthesized and may provide a more stable platform than an antibody-antigen system.
Table 1Determination of chymosin activity in commercial rennet samples by the proposed electrochemical sensor (n = 4)
For the application of the constructed electrochemical sensor, we think the assembly process of the peptide-modified electrode is simple, and thus has the potential to be used to detect the chymosin activity in rennet product, whey, or other cheesemaking products. For the chymosin determination in a soluble sample, such as rennet product or whey, the pretreatment of the sample is relatively simple: adjustment of the pH of the sample to 7.0. However, for determination of the residual chymosin activity in an insoluble sample, such as cheese, the chymosin must be extracted from the sample. For example, for the pretreatment of cheese, the sample is first mashed and dispersed in a DW, and its pH is adjusted to 7.0 with 1 M NaOH by stirring. After the sample is completely dissolved, the pH of the cheese solution is adjusted to 4.6 followed by centrifugation at 5,000 × g at 4°C for 30 min. Then, the pH of the supernatant (whey) is readjusted to 7.0 to retain the chymosin activity. Finally, the chymosin can be determined using the constructed sensor. In this work, as a merit, the substrate of chymosin was a peptide that could be robustly fixed on the surface of an electrode. The simple process of immobilization makes it easily to use in the production line of cheese manufacturing. Interestingly, we did not use an antibody, enzyme, nor complex substrate (such as casein micelle or protein) for sensor construction. Peptides are more stable than proteins or antibodies, which may lead to further development of the sensor for additional applications. Moreover, only 10 min are required to perform the enzymatic reaction before the electrochemical experiment. Therefore, this sensor is suitable for developing stable and small analytical equipment such as glucose meters for chymosin.
CONCLUSIONS
We developed a simple label-free electrochemical sensor for chymosin using a peptide-based enzyme substrate. The sensor was used to quantify chymosin activity in rennet samples. Furthermore, the sensor had good specificity, outstanding stability, and acceptable reproducibility. This work provided a facile, fast, and effective electrochemical assay for detecting chymosin activity and has potential applications in cheesemaking.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (21764005, 21565029, Beijing, China), Key Projects of Yunnan Natural Science Foundation (2018FA005, Kunming, China), Key Research and Development Projects of Yunnan (2018BC005, Kunming, China), Key Scientific and Technology Project of Yunnan (202002AE320005, Kunming, China), Program for Excellent Young Talents of Yunnan University, and the Program for Donglu Scholars of Yunnan University (Kunming, China). The authors have not stated any conflicts of interest.
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Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases.
Bovine serum albumin as an effective sensitivity enhancer for peptide-based amperometric biosensor for ultrasensitive detection of prostate specific antigen.
Peptide substrates for chymosin (rennin). Interaction sites in κ-casein-related sequences located outside the (103–108)-hexapeptide region that fits into the enzyme's active-site cleft.
Target protein induced cleavage of a specific peptide for prostate-specific antigen detection with positively charged gold nanoparticles as signal enhancer.
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