According to authoritative data released by the World Health Organization(WHO), cutaneous disorders affect approximately 1.9 billion people worldwide, representing 25% of the global population (WHO, 2023). This is not just a set of cold numbers; behind each percentage point lies countless individuals struggling with discomfort, self-esteem issues, and the financial burden of treatment. In the Asian region, the situation is equally grim. A mere 10% of the population can claim to have skin that meets the standards of certified health. The vast majority, making up 90% of the Asian populace, find themselves caught in the net of sub-healthy skin conditions or actual skin ailments. These sub-healthy states may manifest as mild itching, dryness, or an uneven complexion, while more serious skin diseases can range from eczema and psoriasis to various forms of dermatitis, significantly impacting people's quality of life.

Skin health challenges are particularly pronounced during adolescence. Research shows that teenagers face an 85% risk of developing acne, driven by hormonal fluctuations, increased sebum production, clogged pores, and bacterial growth. Beyond physical discomfort, acne often triggers low self-confidence and social anxiety, underscoring the urgency of effective skincare solutions.

Against this backdrop, papain has emerged as a promising candidate. Derived from papayas, this proteolytic enzyme offers unique properties that have unlocked new possibilities in skincare, leading to the development of papaya cream products and other formulations. However, its potential remains constrained by critical limitations.

Papain’s key skincare benefit lies in its ability to gently and efficiently break down keratin, the protein forming the outermost layer of dead skin cells. It is widely used in skincare products such as facial masks, moisturizers, cleansers, toners, lotions, and serums. Yet, the enzymatic activity of papain is highly sensitive to environmental factors, particularly temperature and pH. Studies indicate its optimal pH range is 5–7 [2], which poorly aligns with the pH environment of human facial skin, limiting its efficacy [3].

Figure 1. Existing papain-based skincare products on the market

Furthermore, current market prices for non-luxury papain facial skincare products typically range from 35–65 yuan, with luxury alternatives priced even higher. Generation Z (aged 18–25) has become the primary consumer group, heavily influenced by social media. This demographic shows a strong preference for green skincare products, with 37% explicitly favoring such items. Notably, brand awareness and electronic word-of-mouth (e-WOM) collectively account for 52% of the factors driving their purchasing decisions [4,5]. To capture this market, developing reasonable and competitive pricing strategies is imperative.

Another challenge is papain’s optimal temperature range of 55–80°C [6], far exceeding human skin surface temperature. This disparity further compromises its effectiveness in skincare applications. While direct data on papain allergies is limited, the broader prevalence of food allergies highlights the need to address potential sensitivity risks. This underscores the value of exploring alternative raw materials for skincare products-even within the same category enhance safety and drive innovation.

Figure 2. Global Research on Papain

Extensive research on papain has been conducted globally, yielding progress in multiple fields. In the food industry, papain tenderizes meat, making tough cuts more palatable and digestible; facilitates the extraction and modification of plant proteins for high-quality ingredients; produces whey protein hydrolysates with anti-inflammatory and antioxidant properties (enhancing nutritional value and shelf stability); and aids in preparing plant protein particles to stabilize Pickering emulsions, improving food texture. In medicine, papain shows promise for antitumor applications, with potential in targeted drug delivery systems to enhance cancer treatment efficacy while reducing side effects. It also accelerates chronic wound healing by promoting cell migration, mitigating complications from non-healing wounds.

Industrial applications of papain include biofuel production (via enzymatic hydrolysis of biomass for sustainable energy), textile finishing, leather tanning, and cosmetics. Advanced techniques like molecular docking have deepened the understanding of its structure, enabling performance optimization across industries. Despite these advances, critical gaps remain. Extraction and purification methods for papain are inefficient and costly, limiting large-scale production. Selective isolation of specific isozymes remains challenging. Application-wise, while papain is well-established in food and medicine, its potential in biomaterials and tissue engineering is underexplored.

Biologically, the mechanisms underlying papain’s anti-inflammatory and antitumor effects are poorly understood, as are its interactions with cellular signaling pathways and immune regulation. Allergenicity is another concern: while papain and its isozymes can trigger allergic reactions, research on differences in allergenicity among isozymes and their underlying mechanisms is insufficient-hindering safe product development, particularly in regulated skincare and pharmaceuticals.

Most notably, papain isozymes are understudied. Their physiological mechanisms and enzymatic functions in specific contexts lack comprehensive research. Given the links between skin issues and inflammation, cellular regulation, and protein metabolism, breakthroughs in isozyme research could revolutionize skincare-enabling targeted, personalized solutions that address most current skin problems.

Isozymes are encoded by distinct genes or alleles, differing in primary structure but with highly conserved active-center amino acid sequences (explaining similar enzymatic functions). Variations in amino acid sequences, however, lead to differences in catalytic activity and environmental responsiveness, altering optimal pH and temperature [7]. This means isozyme-based products could better match human skin’s pH and temperature, improving efficacy and compatibility.

Though isozymes catalyze identical reactions, structural and specificity differences [8] mean they are unlikely to trigger identical allergic reactions. The immune system may recognize them as distinct, eliciting varied responses [9], a key insight for developing safer papain-based skincare.

Figure 3. Data on skincare product consumption

While the high-end skincare market focuses heavily on “whitening and spot removal” (driven by aggressive marketing and premium pricing), our isozyme-based approach targets the underserved “daily gentle care” segment. This niche addresses universal needs: maintaining skin health via consistent, non-irritating routines that harmonize with natural skin functions. Unlike harsh, cosmetic-focused products, our formulations prioritize long-term resilience, suiting sensitive and acne-prone skin for daily use. Refining enzyme technology for skincare production preserves isozyme activity while simplifying manufacturing, reducing costs, and making enzymatic benefits (gentle exfoliation, natural hydration, antioxidant protection) accessible to more consumers. This redefines “effective skincare” as sustained, gentle care rather than occasional treatments.

Isozyme-based formulations offer two market-altering advantages. First, they cater to papain-allergic consumers, often overlooked in mainstream lines. By leveraging distinct molecular structures, our solutions provide safe alternatives, fostering inclusivity. Second, optimized production lowers entry barriers, enabling competitive pricing against mass-market products. This makes advanced enzymatic skincare accessible to budget-conscious demographics like Generation Z, expanding market reach.

Beyond consumer benefits, this approach drives industry progress. Compared to synthetic chemicals or one-size-fits-all enzymes, isozyme-based formulations advance bioengineering and formulation science, prioritizing skin-compatible, natural ingredients [10]. They also differentiate the market: moving beyond homogenization “whitening” claims to set new standards in gentle care, encouraging innovation in sensitive-skin and eco-friendly products (aligning with demand for sustainable, biologically derived ingredients).

To focus our research, we target specific isozymes: cysteine protease XCP2, cysteine protease XCP2-like, chain A papain, and cysteine protease XCP2-like precursor [11,12,13,14,15]. These show promise due to inherent antioxidant properties—critical for skincare, as antioxidants scavenge free radicals, repair cellular damage, and enhance synergistic effects [16].

As previously discussed, we have successfully identified a series of relatively suitable isoenzymes. The selection of these isoenzymes represents the initial and pivotal phase of our research plan. Through the application of bioinformatics approaches, we employed NCBI BLAST analysis to screen out multiple papain isoenzymes that have a similarity of less than 50% with papain and meticulously explored their potential applications in skincare products. This process not only enables the identification of papain isoenzymes but also ensures to achievement of more efficient and cheaper skincare outcomes.

The second stage of our research involves computer simulation and structural prediction. Leveraging the advanced capabilities of AlphaFold, we predict the protein structures of these isoenzymes and assess their potential to deliver enhanced and economical skincare benefits. This computational analysis provides valuable insights into the molecular characteristics and functional properties of the isoenzymes, guiding subsequent experimental investigations.

Subsequently, we use recombinant expression technology to reconstruct and express the isoenzymes that exhibit promising potential. By expressing these isoenzymes in host organisms such as Escherichia coli, we can produce the enzymes on a large scale, facilitating their translation into practical applications in the skincare industry.

First three steps are done by computer modeling.The fourth step of our research focuses on functional comparison and evaluation, mainly done in laboratory. We conduct studies on the individual effects of each protease and also explore the combined impact of protease mixtures on skincare efficacy. This comprehensive approach aims to uncover potential synergistic effects, thereby enhancing the overall quality and efficiency of skincare products.

After that, we take a thorough assessment of the potential of each isoenzyme and initiate optimization efforts. This involves systematically investigating the optimal operating conditions for different proteases and evaluating their practical application potential in real-world skincare formulations if combining with papain, finding out the most suitable facial cleansing rate for human skin. Should these products reach the mass production stage in the future, this optimization step will prove indispensable for ensuring their commercial viability and success.

Finally, we will employ bioinformatics techniques to predict outcomes and simulate the binding effects of isoenzymes. Subsequently, we will utilize these predicted results to validate the accuracy of our experimental findings.

Figure 4. The main processes of our project

The steps to obtain Escherichia coli colonies are detailed as follows:

1. Amplify the target gene using PCR.
2. Linearize the pET28a plasmid via restriction enzyme digestion.
3. Perform homologous recombination to insert the target gene into pET28a.
4. Introduce the recombinant pET28a into E. coli via heat shock transformation.
5. Cultivate E. coli colonies and verify successful insertion of the target gene.

Based on the experimental theory underpinning our research, our core objective is to systematically determine optimal operating conditions for each target isozyme, including pH tolerance, temperature stability, catalytic efficiency, and compatibility with common skincare ingredients. This optimization ensures isozymes retain biological activity in formulations, delivering consistent results in daily use. Once parameters are established, we will scale production to transform lab discoveries into commercial skincare products-balancing efficiency and quality via advanced biomanufacturing to meet market demand.

Isozyme-based products aim to redefine skincare experiences:

1. Enhanced efficacy: Targeting skin health at the molecular level—via gentle exfoliation (preserving the skin barrier), antioxidant activity (combating environmental stress), and anti-inflammatory effects (soothing irritation). Unlike mismatched papain-based products, these formulations align with skin physiology, maximizing benefits with minimal side effects.
2. Consumer autonomy: Offering products tailored to specific needs (sensitive, acne-prone, etc.), enabling personalized routines over generic solutions.
3. Affordability: Optimized production reduces costs, positioning products as accessible alternatives to papain-based options.

This project also enhances agricultural resource efficiency. Traditional papain production relies on large-scale papaya cultivation—intensive in land, water, and labor—with inefficient extraction leading to waste. In contrast, our microbial fermentation approach for isozyme production eliminates reliance on papaya, freeing manufacturing from crop yield, regional, and climatic constraints. Genetically engineered microbes synthesize target isozymes in controlled environments, avoiding fruit-processing waste. Optimizing fermentation parameters improves production efficiency, establishing a low-resource, high-recycling supply chain that promotes balanced agricultural resource use.

The environmental benefits of bio-enzyme preparations over synthetic exfoliants are profound. Unlike petroleum-derived, non-biodegradable chemicals (which persist in ecosystems), bio-enzymes are naturally occurring proteins that break down harmlessly, leaving no toxic residues. Their low toxicity reduces risks to aquatic life, soil, and air during production, use, and disposal. Replacing harsh chemicals curtails pollution in waterways and landfills, addressing a critical industry environmental issue. Long-term, this supports sustainable development goals by reducing reliance on non-renewable resources and advancing green manufacturing.

In essence, our project demonstrates that effective skincare, affordability, and environmental stewardship can coexist, creating value for consumers and the planet alike.

1. DETERMINATION OF OPTIMUM CONDITION OF PAPAIN ENZYME FROM PAPAYA VAR JAVA (Carica papaya )(by Aline Puspita Kusumadjaja - 2010)
2. Natural skin surface pH is on average below 5, which is beneficial for its resident flora(by H. Lambers - 2006)
3. Trevisol TC, Henriques RO, Cesca K, Souza AJA, Furigo A Jr. In vitro effect on the proteolytic activity of papain with proteins of the skin as substrate. Int J Cosmet Sci. 2022 Oct;44(5):542-554. doi: 10.1111/ics.12805. Epub 2022 Aug 17. PMID: 35892222.
4. Generation Z: The Purchase Intention of Green Skin Care Products(by Ying San, Lim - 2021)
5. Influential Factors Shaping Consumer Behavior in the Skincare Industry(by Ridiya Ningrum - 2024)
6. DETERMINATION OF OPTIMUM CONDITION OF PAPAIN ENZYME FROM PAPAYA VAR JAVA (Carica papaya )(by Aline Puspita Kusumadjaja - 2010)
7. Factors affecting enzyme activity(by D.A.S. Grahame - 2015)
8. Analysis of Tissue-Specific Expression Patterns of Isozymes(by Masataka Takarabe - 2006)
9. Cross‐reactivities of non‐homologous allergens(by Merima Bublin - 2020)
10. Immobilization of enzyme and its applications in the food industry(by QIAO De-liang - 2008)
11. Extremophiles and their enzymatic diversity and biotechnological potential(by Fatima Atif - 2024)
12. Kiwifruit of Actinidia eriantha cv. Bidan has in vitro antioxidative, anti-inflammatory, and immunomodulatory effects on macrophages and splenocytes isolated from male BALB/c mice(by Young-Eun Kim - 2018)
13. In vitro antioxidant activities and antioxidant enzyme activities in HepG2 cells and the main active compounds of endophytic fungus from pigeon pea [Cajanus cajan (L.) Millsp.](by JinTong Zhao - 2014)
14. Antimicrobial activity of Eucalyptus globulus oil, xylitol, and papain: a pilot study(by Valéria de Siqueira Mota - 2015)
15. Unclasping potential chickpea resources for the antioxidant enzyme Superoxide Dismutase(by Amrendra Pratap Singh - 2022)
16. New Insights into Antioxidant Peptides: An Overview of Efficient Screening, Evaluation Models, Molecular Mechanisms, and Applications(by Yuhao Zhang - 2024)