Papaya potion conducted a total of five repeats of the DBTL cycle and successfully synthesized the cysteine protease papain along with four of its isoenzymes.

Figure 1. The Flow of our plan

1. Design: Research and identification of our isoenzyme-coding genes, along with selection of a competent vector and competent host bacterial cells.

2. Build: We obtained the gene sequences from the NCBI database and codon-optimized the sequences for E. coli expression to synthesize our enzymes.

3. Test: Conducted a series of protein tests to experiment with the compatibility and activity of our five enzymes in order to come up with the best combination of enzymes.

4. Learn: Learning from the results and using molecular docking to explain the mechanisms in which our enzyme combinations turned out to rank as accordingly.

Design:

To express the cysteine protease papain for our skincare formulation, the pET28a-papain recombinant plasmid was constructed by first obtaining the isoenzyme gene sequence from the NCBI database. The sequence was codon-optimized for E.coli expression and amplified using PCR to produce adequate quantities for downstream cloning. The expression plasmid used was pET28a, a conventional vector in molecular biology research due to its high-level expression capabilities and His-tag purification compatibility. The isoenzyme coding sequence was placed into the pET28a vector directly downstream of the T7 promoter and a synthetic ribosome binding site, enabling high-level transcription by T7 RNA polymerase. A lac operator element was placed between the promoter and the papain gene to allow LacI-mediated repression that is relieved upon IPTG addition. When IPTG is added to a bacterial culture, it binds to the lac repressor, preventing the repressor from binding to the lac operator, and thus allowing the expression of genes under the control of the lac promoter. The plasmid also confers kanamycin resistance, allowing for selection in E.coli cells.

Figure 3. The map of pET28a plasmid

The pET28a-papain construct enables us to tightly control and efficiently express papain under laboratory conditions. Homologous recombination method was used for the construction of pET28a-papain recombinant plasmids rather than using DNA ligase because of its greater efficiency and accuracy in combining the Papain gene with pET28a.

Figure 4. The map of pET28a-papain

Build:

The pET-28a-papain construct is designed to express the Carica papaya papain protease in E. coli under the control of the T7 promoter, enabling high-level, inducible protein expression. The system utilizes the lac operon-based regulation, where IPTG induction activates T7 RNA polymerase, initiating transcription of the papain coding sequence. A His-tag is fused to the protein to facilitate purification via affinity chromatography. This design enables efficient production and downstream application of recombinant papain for functional assays such as protein degradation or skin exfoliation.

The papain gene fragment (678 bp) was amplified using PCR, and both the gene and the pET-28a(+) vector were digested with NdeI and HindIII restriction enzymes to generate compatible sticky ends. Figure 5 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of Papain is approximately 678 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 5. Gel electrophoresis of PCR amplified papain gene

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E. coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E. coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 6A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 6B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 6C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 6. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-papain

Test:

Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant papain protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 7, distinct protein bands were observed in all lysate samples. A ~24 kDa band was detected in the papain-expressing strain, consistent with the expected molecular weight of papain. These results confirmed that XCP2-papain recombinant enzymes were successfully expressed in E. coli.

Figure 7. SDS-PAGE results of pET28a-Papain

Learn:

Before we start the experiment, we used AlphaFold to predict their three-dimensional structures. The predicted structure provides a solidfoundation for the subsequent molecular docking experiments.That’s the molecular docking results. According to this table, we can reach the first conclusion: the binding force stability values of each isoenzyme are different. Obviously, the negative value of papain is the largest. It indicates that its binding affinity is the strongest.Besides, we may find from Table 1, papain doesn’t only form s hydrogen bonds with substrates but also generates additional π bonds, which can enhance binding stability. It explains the docking results. In our ELISA test, we found that papain has the highest concentration among the 5 isoenzymes which proves the prediction.

Table 1 Indication of the binding affinity between five enzymes and the substrate

Design:

The design is pragmatically like that of cycle 1, except we used XCP2-Abrus Precatorius, an isoenzyme of papain, in place of papain as it may show greater enzyme activity either individually or when in synergy with other isoenzymes.

Figure 8. The map of pET28a-Abrus precatorius

Build:

The pET-28a-XCP2-Abrus precatorius construct is engineered to express the XCP2 cysteine protease derived from Abrus precatorius in E.coli. The gene is placed downstream of the T7 promoter, enabling tightly controlled and high-yield expression upon IPTG induction via the lac operon system. The vector includes an N-terminal His-tag for simplified purification using nickel affinity chromatography. This design allows for efficient production of functional XCP2 protease to evaluate its keratin-degrading and exfoliation potential in vitro.

The XCP2-Abrus gene (1083 bp), a cysteine protease from Abrus precatorius, was amplified by PCR. Both the PCR product and the pET-28a(+) vector were digested with NdeI and HindIII to generate compatible ends. Figure 9 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Abrus is approximately 1083 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 9. Agarose gel electrophoresis results of PCR for XCP2-Abrus

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 10A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 10B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 10C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 10. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Abrus

Test:

Protein expression was induced using 1 mM IPTG. Cells were lysed by sonication at low temperature. The recombinant XCP2-Abrus protein was purified using nickel affinity chromatography. The washing steps included buffer A (low imidazole) and buffer B (high imidazole), followed by SDS-PAGE analysis of elution fractions.Verified clones were transformed into E. coli BL21(DE3) for protein expression. As shown in Figure 11, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Abrus-expressing strain, consistent with the expected molecular weight of XCP2-Abrus. These results confirmed that recombinant enzymes were successfully expressed in E. coli.

Figure 11. SDS-PAGE results of pET28a-XCP2-Abrus

Learn:

Abr forms hydrogen bond with substrates which leads to a high stability. However, it has less affinity caused by its different binding sites. The prediction is proved by our ELISA test. As the same problem as Act, we didn’t succeed to transform the recombinate plasmid, but we improve the plasmid concentration.

Design:

The design is pragmatically like that of cycle 1, except we used XCP2-Actinidia Eriantha, an isoenzyme of papain, in place of papain as it may show greater enzyme activity either individually or when in synergy with other isoenzymes.

Figure 12. The map of pET28a-Actinidia Eriantha

Build:

The pET-28a-XCP2-Actinidia eriantha construct is designed to express the XCP2 cysteine protease derived from Actinidia eriantha (hardy kiwifruit) in E. coli under the control of the T7 promoter. The system employs IPTG-inducible lac operon regulation, ensuring tightly controlled and high-level protein expression. A 6×His-tag is fused to the N-terminus to enable convenient purification via nickel affinity chromatography. This recombinant expression system facilitates functional studies of XCP2’s proteolytic activity and its potential applications in keratin degradation and exfoliation.

The XCP2-like gene from Actinidia eriantha (1089 bp) was PCR-amplified and digested along with the pET-28a(+) vector using NdeI and HindIII. After purification, the digested fragments were ligated using Homologous recombination enzyme. Figure 13 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Actinidia is approximately 1089 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 13. Agarose gel electrophoresis results of PCR for XCP2-Actinidia

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 14A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 14B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 14C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 14. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Actinidia

Test:

The verified plasmid was transformed into E.coli BL21(DE3) for protein expression. Following IPTG induction, the cells were harvested and lysed with sonication at low temperature. The recombinant protein was purified using Ni-NTA affinity chromatography, including stepwise washes with imidazole buffers. As shown in Figure 15, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Actinidia-expressing strain, consistent with the expected molecular weight of XCP2-Actinidia. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 15. SDS-PAGE result of pET28a-XCP2-Actinidia

Learn:

Actinidia has the second largest affinity. We may find that it forms hydrogen bond with substrates. The difference of affinity to other isoenzymes is caused by the different binding sites. In the ELISA test, we proved its high efficiency. However,we made some mistakes in the process of heat shock transformation of recombinate plasmid of Act to E. Coli(DH5α) in the first time, but in the second time, we succeeded and increased the concentration of the plasmid.

Design:

The design is pragmatically like that of cycle 1, except we used XCP2-Cajanus Cajan, an isoenzyme of papain, in place of papain as it may show greater enzyme activity either individually or when in synergy with other isoenzymes.

Figure 16. The map of pET28a-Cajanus Cajan

Build:

The pET-28a-XCP2-Cajanus cajan construct is engineered to express the XCP2 cysteine protease derived from Cajanus cajan (pigeon pea) in E. coli under the control of the T7 promoter. The system utilizes the lac operon for IPTG-inducible expression, enabling tight regulation and high yield of the recombinant protein. An N-terminal His-tag is incorporated to facilitate purification through nickel affinity chromatography. This construct allows for efficient production and activity analysis of XCP2, with the goal of evaluating its keratin-degrading and exfoliation potential for skincare-related applications.

The XCP2-Cajanus gene (1083bp) was obtained by PCR amplification from a codon-optimized synthetic template. Both the insert and pET-28a(+) backbone were digested with NdeI and HindIII to create sticky ends. The digested fragments were ligated using Homologous recombination enzyme to generate the plasmid pET28a-XCP2-Cajanus. Figure 17 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Cajanus is approximately 1083 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 17. Agarose gel electrophoresis results of PCR for XCP2-Cajanus

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 18A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 18B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 18C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 18. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cajanus

Test:

The confirmed plasmid was transformed into E.coli BL21(DE3) for protein expression. IPTG was added to induce expression, and the culture was harvested after 20 hours. Cell lysis was performed using a sonicator under low-temperature conditions to prevent protease denaturation. Purification was carried out using nickel affinity chromatography. The column was washed with buffer A (low imidazole) and eluted with buffer B (high imidazole). Collected fractions including lysate, flow-through, and elution were analyzed via SDS-PAGE. As shown in Figure 19, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Cajanus-expressing strain, consistent with the expected molecular weight of XCP2-Cajanus. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 19. SDS-PAGE result of pET28a-XCP2-Cajanus

Learn:

Similar to other isoenzymes, Caj forms hydrogen bond with substrates which leads to a high stability. However, it has less affinity caused by its different binding sites. The prediction is proved by our ELISA test.

Design:

The design is pragmatically like that of cycle 1, except we used XCP2-Cicer Arietinum, an isoenzyme of papain, in place of papain as it may show greater enzyme activity either individually or when in synergy with other isoenzymes.

Figure 20. The map of pET28a-Cicer Arietinum

Build:

The pET-28a-XCP2-Cicer arietinum construct is designed to express the cysteine protease XCP2 from Cicer arietinum (chickpea) in E.coli. The coding sequence is placed under the control of the T7 promoter, allowing IPTG-inducible expression via the lac operon. An N-terminal His-tag is included to facilitate protein purification using nickel affinity chromatography. This design enables efficient recombinant production of XCP2 for functional assays, with the goal of evaluating its keratin-degrading ability and potential use in skin exfoliation or biological material recycling.

The XCP2-like Cicer gene (1095bp) was PCR-amplified from a synthesized, codon-optimized template. The gene fragment and pET28a(+) plasmid were digested with NdeI and HindIII to generate sticky ends. Using Homologous recombination enzyme, we ligated the insert into the linearized vector to form the recombinant plasmid pET28a-XCP2-Cicer. Figure 21 shows the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of XCP2-Cicer is approximately 1095bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

Figure 21. Agarose gel electrophoresis results of PCR for XCP2-Cicer

The purified insert and backbone were then ligated using Homologous recombination enzyme. The ligation product was transformed into E.coli BL21(DE3) competent cells via heat shock to obtain the recombinant plasmid. After transformation, the cells were recovered at 37°C for 1 hour, plated on LB agar containing kanamycin, and incubated overnight. Colonies were picked, and the plasmid was extracted and verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmids were transformed into chemically competent E.coli BL21(DE3) cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 22A), indicating successful transformation. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 22B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. To confirm the accuracy of plasmid construction, recombinant pET28a plasmids were sent to a commercial sequencing facility for Sanger sequencing. Sequence alignment (Figure 22C) showed that the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 22. Colony growth, gel electrophoresis result and DNA sequencing of pET28a-XCP2-Cicer

Test:

The verified plasmid was transformed into E.coli BL21(DE3). IPTG was added to induce expression of the XCP2-Cicer protein. The cells were lysed using a sonicator under cold conditions to preserve enzyme activity. Purification was conducted using nickel affinity chromatography, with sequential washes using low-imidazole buffer A and high-imidazole buffer B. As shown in Figure 23, distinct protein bands were observed in lysate samples. A ~39 kDa band was detected in the Cajanus-expressing strain, consistent with the expected molecular weight of XCP2-Cicer. These results confirmed that recombinant enzymes were successfully expressed in E.coli.

Figure 23. SDS-PAGE result of pET28a-XCP2-Cicer

Learn:

Similar to other isoenzymes, Cicer forms hydrogen bond with substrates which leads to a high stability. However, it has less affinity caused by its different binding sites. The prediction is proved by our ELISA test.

1. Papain-mediated Casein Clot Dissolution Assay

To evaluate the initial proteolytic potential of the recombinant enzyme papain, we conducted a qualitative assay based on its ability to degrade acid-precipitated casein from milk.

As illustrated in Figure 25, untreated milk containing 6% (w/v) casein appeared uniformly dispersed (A). The addition of dilute hydrochloric acid caused the formation of a dense white precipitate, indicating the aggregation of denatured casein proteins (B). Following incubation with papain at 37 °C for 1 hour, the precipitate visibly diminished and the solution became notably clearer (C), indicating enzymatic breakdown of the protein network.

These results confirm that papain can hydrolyze casein aggregates and exhibits functional caseinolytic activity. While this assay qualitatively supports its protease function, it does not allow for comparison between different enzyme efficiencies.

Figure 24. Result of casein clot dissolution assay

2. FN1 Matrix Dissolution Assay

To investigate whether the recombinant proteases are capable of degrading biologically relevant substrates, fibronectin-1 (FN1) was chosen as the model protein for enzymatic digestion.

As depicted in Figure 25, the FN1 control sample (without enzyme treatment) showed a single, sharp band at approximately 47 kDa, indicating that the protein remained intact in the absence of enzymatic activity. In contrast, samples treated with each of the five enzymes—Papain, Abrus, Actinidia, Cajanus, and Cicer—exhibited a clear decrease in the intensity of the full-length FN1 band. Additionally, prominent new bands emerged near 24 kDa, reflecting the formation of low-molecular-weight fragments resulting from proteolytic cleavage.

The consistent generation of these ~24 kDa bands across all enzyme groups suggests that, despite originating from different species, the recombinant proteases may share similar substrate recognition or cleavage patterns. These findings demonstrate that all five enzymes are active against FN1 and may be effective tools in applications related to extracellular matrix degradation or remodeling.

Figure 25. Result of FN1 matrix dissolution assay

3. ELISA-based Enzyme Activity Assay

To measure and compare the proteolytic activity of different crude enzyme lysates, we carried out an ELISA-based quantification assay. Prior to analysis, total protein concentrations were determined using NanoDrop, and each lysate was diluted to various working concentrations. A standard curve was constructed from serial dilutions of the kit’s reference standard, generating a linear model that allowed us to estimate relative enzymatic activities from OD₄₅₀ values.

To quantitatively evaluate the enzymatic activity of the crude lysates, an ELISA assay was performed. Protein concentrations were first normalized using NanoDrop, and all lysates were diluted to different concentrations.

Figure 26. The standard table of papain concentration

A standard curve was generated based on serial dilutions of the ELISA kit standard, yielding a linear regression model for activity estimation.

Figure 27. The standard curve of papain concentration

We used ELISA kits to individually test and analyze the activity of several enzymes. Based on the above standard curves, we calculated the activity of each enzyme and performed significance analysis using Graphpad to calculate the P-value. As shown in Figure 28, the enzyme activity was Papain > Act > Abr > Caj > Cic.

Figure 28. Single enzyme activity detection data

Single-enzyme activity results showed that Papain had the highest protease activity among all five enzymes, consistent with the qualitative results from the casein assay. No statistically significant differences were observed between Cajanus and Cicer.

Figure 29. Graphs of single-enzyme activity comparison

We used an ELISA kit to analyze the activity of the dual-enzyme combinations and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the dual-enzyme combinations and performed a significance analysis using Graphpad to calculate the P-value. As shown in Figure 30, the optimal combination was Caj+Cic.

Figure 30. Dual-enzyme activity detection data

In the dual-enzyme combinations, the mixture of Cajanus and Cicer displayed the highest activity. Other combinations, such as papain with Cajanus or Cicer, and Abrus with Actinidia, showed moderate activity but no significant enhancement compared to single enzymes.

Figure 31. Graphs of dual-enzyme combination comparison

We used an ELISA kit to analyze the activity of the Three-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the Three-enzyme combination and performed a significance analysis using Graphpad to calculate the P value. The optimal combinations were Papain + Abrus + Actinidia and Abrus + Actinidia + Cajanus.

Figure 32. Three-enzyme activity detection data

Three-enzyme combinations revealed that the groups Papain + Abrus + Actinidia and Abrus + Actinidia + Cajanus produced the strongest activity levels, although their differences were not statistically significant. These combinations outperformed most others in the same group.

Figure 33. Graphs of Three-enzyme combinations comparison

We used an ELISA kit to analyze the activity of the four-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the four-enzyme combination and performed a significance analysis using Graphpad to calculate the P value. The optimal combination was Abrus + Actinidia + Cajanus + Cicer.

Figure 34. Four-enzyme activity detection data

In the four-enzyme combinations, the mixture of Abrus + Actinidia + Cajanus + Cicer showed the highest value, indicating the strongest activity among all tested combinations of four enzymes.

Figure 35. Graphs of four-enzyme combinations comparison

We used an ELISA kit to analyze the activity of the five-enzyme combination and verify whether there was a synergistic effect. Based on the standard curve, we calculated the activity of the five-enzyme combination and performed a significance analysis using Graphpad to calculate the P value.Subsequently, we compared the combinations with the strongest enzyme activity under different combinations. The results showed that the dual enzyme combination had the strongest effect.

Figure 36. Different combinations activity detection data

As shown in Figure 37, the dual combination of Cajanus + Cicer displayed the highest overall activity, even exceeding that of the full five-enzyme mixture. This suggests that specific pairings may exhibit synergistic effects stronger than broader mixtures.

Figure 37. Graphs of optimal combinations comparison

Among the five individual enzymes tested, Papain demonstrated the strongest activity, in line with its rapid casein-clearing effect observed earlier. Cajanus and Cicer showed similar performance, and no statistically significant difference was detected between them.

Interestingly, the Cajanus + Cicer combination outperformed even the full five-enzyme mix, highlighting that targeted pairing of compatible enzymes can deliver higher activity than complex multi-enzyme blends. This observation underscores the value of rational enzyme pairing in optimizing proteolytic systems.