A “wave-riding” biocatalysis: an all-enzyme system for genuinely green and flexible manufacture of machine-washable dyed wool fabrics

Abstract

The well-known phenomenon of felt shrinkage that occurs during home laundry of wool textiles has long bothered consumers. Currently, to address this issue, factories have no choice but to employ the chlorination-Hercosett process, which predominates in the production of machine-washable wool fabrics. However, this method often results in color alterations in dyed fabrics and generates absorbable organic halides (AOX), thereby significantly exacerbating environmental challenges. Enzymatic processing has been consistently recognized as a promising alternative, while most necessitate the assistance of chemical reagents. To accomplish fully green manufacturing of machine-washable wool fabrics, this study has innovatively developed a compound protease system based on a “wave-riding” enzymatic mechanism, achieving localized surface modification of wool fibers. The results demonstrated that dyed wool fabrics treated exclusively with proteases exhibited excellent dimensional stability (3.90% ± 0.12%) and controllable strength loss (11.39% ± 0.61%). Additionally, the wearability of the fabrics, which was quantified through assessing factors such as air and moisture permeability, hand feeling, and wettability, was improved. The original color properties of dyed wool fabrics were also preserved; specifically, there was no adverse effect on color fastness, and minimal color difference was observed (ΔE_CMC < 1.00). This approach further fills the technical gap in the field of genuinely green anti-felting solutions for dyed wool fabrics, offering a new pathway toward sustainable textile manufacturing.

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Green foundation

1. To date, the preparation of machine-washable wool predominantly relies on the chlorination method, which remains one of the few areas in the textile industry that has yet to realize ecological processing. Here, we propose an all-enzyme approach as an alternative, which effectively eliminates AOX pollution and advances the manufacture of machine-washable wool towards a sustainable bio-economy.

2. The proposed system consists of proteases with varying keratin specificities, founded on a “wave-riding” biocatalysis mechanism, achieving anti-felting without any chemical assistance for the first time. This advancement establishes an innovative framework for the genuinely green manufacture of machine-washable dyed wool textiles.

3. Future research will concentrate on designing proteases through AI to enhance their catalytic efficiency on substrates, specifically targeting the degradation of wool scales. This endeavor aims to reduce the processing time and energy consumption, ultimately achieving truly green fabrication of wool textiles.

Introduction

Wool, recognized as a versatile green fiber, possesses various characteristics such as natural biodegradability, warmth, elasticity, deodorization, odor absorption, quick-drying capability, and moisture-wicking properties.^1–3 These attributes have facilitated its expansion from traditional clothing to the outdoor sports sector, especially for young consumers pursuing a high-quality lifestyle, becoming an essential raw material for developing functional textiles. However, the core contradiction currently faced by the industry is that woolen clothing cannot be machine washed after getting dirty outdoors.^4,5 The specific manifestation is that wool fibers are susceptible to felting shrinkage under mechanical action, especially during washing.^6,7 This phenomenon can be attributed to the distinctive fiber structure of wool. Wool is a natural protein fiber composed of over twenty types of α-amino acids, primarily sourced from sheep. Among the various types of wool, Merino wool is the most extensively utilized in the research and development of worsted woolen fabrics, primarily due to its unique properties of softness, long and dense fibers, and notable elasticity. Its structure is mainly composed of three components: the cuticle, the cortex, and the cell membrane complex (CMC). The outermost scale layer is formed by overlapping keratinized flat protein cells,^8,9 with the edges of these keratinocytes oriented towards the tips of the fibers. During the washing process, wool fibers absorb water and expand under certain temperature and solution conditions. When subjected to external friction or mechanical forces, relative displacement occurs between the fibers, the movement shifting from the tip of one fiber to the root of another. Following repeated rubbing and squeezing, gradual tangling and shrinkage of the wool fibers occur, ultimately culminating in the undesired felting shrinkage of the wool fabric. This will cause serious distortion in the dimensions of wool fabrics and an uneven surface, directly impacting both the product value and lifespan. Over recent years, extensive research has been conducted aiming at reducing troublesome felting tendencies to achieve machine-washable wool.

Generally, an industrial shrink-proofing process is carried out on wool tops. Nevertheless, this technique completely removes the scales, which can lead to a reduction in fiber cohesion and potentially cause damage to the fibers. Such alterations may have adverse implications for subsequent procedures such as spinning and weaving. To mitigate the adverse effects, it is more reasonable to transfer the anti-felting treatment of wool from the top to the fabric. In past production practices, factories have predominantly concentrated on the machine-washable processing of grey fabrics rather than dyed fabrics (at least this is the case in China's worsted woolen sector). However, it is worth emphasizing that woolen mills maintain a limited inventory of grey fabrics, while a greater quantity of dyed fabrics is available in their warehouses, as the dyeing is mainly carried out on wool tops or yarns. Accordingly, it is imperative to investigate the anti-felting processing of dyed fabrics, as this approach more closely aligns with the current production situation in most woolen textile enterprises. Meanwhile, in the production process, the anti-shrinkage treatment for dyed wool fabrics is performed at the end of the production line, allowing the finished product to be sold directly upon completion. Conversely, wool tops must undergo additional procedures following the anti-felting treatment, including spinning, weaving, and post-treatment, which entails considerable time investment. Consequently, to address the market demand for anti-felting wool fabrics, the implementation of compound protease treatment facilitates a more flexible and rapid response for the manufacturing factory (Fig. 1).

Fig. 1 (a) Comparison between the application of the traditional chlorination-Hercosett and enzymatic methods for machine-washable fabrication of wool and (b) schematic of synergistic effects among compound proteases for anti-felting processing of dyed wool textiles.

Tracing back to the 1830s, the British were the first to discover that aqueous chlorine could effectively degrade wool scales. Since then, researchers have made abundant efforts to endow wool products with machine-washable properties. The wet chlorine method was initially employed for the anti-felting treatment of wool fabrics. For instance, the Kroy–Hercosett⁵ process involves mixing gaseous chlorine with water to produce hypochlorous acid and hydrochloric acid. These acids are subsequently sprayed onto the wool, utilizing the pressure from the deeply impregnated liquid to facilitate the chlorination reaction. However, hydrochloric acid often remains trapped within the fibers, resulting in an unpleasant odor. Reports indicate that dichloroisocyanurate (DCCA) can serve as a substitute for traditional acid chlorination treatments, enabling Hercosett resin to deposit more uniformly on the surface of wool fibers.¹⁰ Due to its higher cost compared to conventional chlorinating reagents, the practical application of DCCA in industrial production is limited. Additionally, some researchers have developed a dry chlorination treatment method that involves exposing fabrics to chlorine gas at atmospheric pressure. Nevertheless, this approach necessitates a substantial amount of pungent and toxic chlorine gas and has high requirements for process control and equipment. Anyway, the chlorination-Hercosett method has always been dominant. Even if this approach raises considerable environmental issues due to the discharge of adsorbable organic halides (AOX) in the effluent,¹¹ it is still acknowledged as the most commercially viable and effective method for enhancing shrink resistance in machine-washable wool textiles. It needs to be emphasized that this treatment induces other adverse effects such as yellowing of the fabric, deterioration of mechanical properties, poor fabric handle, and excessive water consumption. Moreover, when further applied to dyed wool fabrics, the existence of chlorine can alter the chromophore structure of the reactive dyes, leading to undesirable color changes.¹² To eliminate the ecological risks caused by the chlorination method, non-chlorine anti-felting treatments of wool textiles have been widely studied. For instance, Zhang et al. employed plasma technology to pretreat wool,¹³ generating free radicals or surface etching^14,15 on the fiber surface through energy exchange from particle collisions and further obtaining machine-washable knit goods. Sadeghi-Kiakhani et al. achieved oxidative degradation and scale removal of wool fibers by disrupting covalent bonds via ultraviolet irradiation.¹⁶ Nevertheless, these surface modification technologies are tough to implement in large-scale industrial applications due to equipment limitations. Among non-chlorine methods, enzymatic anti-felting treatments, represented by proteases, are naturally receiving increasing attention. Enzymatic treatment offers advantages over alternative methods, such as high catalytic efficiency, environmental sustainability, and operational simplicity. Proteases, functioning as biological catalysts that possess biodegradability, achieve ecological and environmental friendliness in terms of raw materials. Furthermore, the process of enzymatic treatment does not generate any detrimental by-products, indicating that the application of proteases does not impose a long-term pollution burden on the environment. Meanwhile, this approach significantly alleviates the pressure associated with wastewater treatment. These features make it an ideal and sustainable substitute for the chlorinated anti-felting methods currently employed in commercial applications.^17,18

Proteases facilitate the degradation of proteins and polypeptides through the hydrolysis of peptide bonds. Wool primarily consists of keratin, thereby making it an optimal substrate for proteases. Theoretically speaking, the application of proteases can lead to the moderate hydrolysis of the surface scale layer, thereby contributing to the anti-felting of wool. However, during regular protease treatment, proteases can easily penetrate into the interior of fibers and cause serious damage to the structural integrity of wool. Therefore, it is challenging to confine enzymatic hydrolysis to the cuticle scales and achieve machine washability of wool products without severe strength loss.¹⁹ Due to this limitation inherent in proteases, researchers frequently resort to chemical reagents for pretreatment, facilitating effective hydrolysis of wool scales during subsequent protease treatments.^20–22 For example, thiol-type reducing agents²³ like 2-mercaptoethanol and mercaptoacetic acid can interact with cystine in wool, thereby disrupting disulfide bonds. However, these types of reducing reagents possess a degree of biological toxicity, posing considerable challenges to managing the resulting wastewater. Other commonly used reducing reagents such as thiosulfates and sulfites^24,25 have the potential to release sulfur-containing wastewater that contributes to water contamination. Additionally, oxidants²⁶ such as hydrogen peroxide²⁷ and peroxymonosulfuric acid can act on wool scales. They can oxidatively hydrolyze both disulfide bonds and peptide bonds within the cuticle layer of wool fibers, leading to the formation of hydrophilic groups. These hydrophilic groups cause the fiber scales to fully swell and soften after water absorption. This phenomenon reduces the difference between frictional coefficients in both with-scale and against-scale directions, thus enhancing the shrinkage resistance of wool. Nonetheless, oxidants exhibit a limited capacity in preventing the shrinkage of wool fabrics due to their oxidation mechanism. Furthermore, the byproducts generated from those oxidation processes complicate wastewater treatment efforts. Similarly, because there is a potential risk of color alteration associated with the use of chemical reagents,²⁸ factories rarely consider implementing this process for dyed wool textiles. Therefore, the top priority is developing an efficient and environmentally friendly all-enzymatic process in place of chemo-enzymatic treatments to achieve genuinely green anti-felting of wool fabrics.²⁹

Herein, we utilized Savinase Ultra 16XL (designated as S), which exhibits a high keratin degradation capability (K/C = 0.63), in conjunction with Progress Uno 100L (denoted as U) to modify wool fibers. This compound protease system effectively concentrates protein hydrolysis on the surface of the wool, achieving a “wave-riding” enzymolysis effect that significantly minimizes fiber damage (Fig. 1). It is vital to illustrate that the ratio of relative enzyme activity when using keratin as a substrate (K) to the relative enzyme activity when using casein as a substrate (C) serves as an assessment parameter for evaluating the substrate specificity of the protease towards keratin. When the K/C value surpasses 0.5, it is believed that the protease possesses the capability to degrade keratin. Given that S demonstrates high specificity for keratin, this allows the compound protease system to exhibit localized degradation on keratin-rich wool scales. This hydrolysis occurs along the wave-like scales, thereby realizing controllable depths of enzymolysis on the surface of wool fibers without evident damage. Furthermore, we found that this anti-felting technique applies to various wool fabrics dyed with different types of dyes. This process exerts minimal influence on both the color depth and fastness of the dyed wool fabrics, as well as the structure of the dye. Overall, the all-enzymatic method has enormous market potential, as it allows issues related to machine washability in wool to be effectively solved at the end of production lines, enabling rapid responses to market demands. More importantly, it offers the possibility of thoroughly eliminating the pollution caused by chlorine, which people have long relied on. This approach promotes sustainable development in anti-felting treatments of wool.³⁰

Experimental

Materials

Wool woven fabric (180 g m⁻²) used in the study was supplied by Shandong Nanshan Co., Ltd (Shandong, China). Savinase Ultra 16XL (expressed as S) and Progress Uno 100L (expressed as U) were purchased from Novozymes Biotechnology Co., Ltd (Tianjin, China). The Lanasol series of dyes (containing Lanasol Red CE and Lanasol Blue CE) and the Lanaset series of dyes (containing Lanaset Yellow 4GN and Lanaset Red G) were obtained from Huntsman Corporation (USA) (specific information related to the four dyes is shown in Fig. 5). Methylene blue (AR) and casein (AR) were purchased from Shanghai Aladdin Co. (China). Keratin powder (from wool), bovine serum albumin (referred to as BSA), and 5,5′-dithio bis-(2-nitrobenzoic acid) (referred to as DTNB) were obtained from Macklin Biochemical Technology Co., Ltd (Shanghai, China). O.C.T 4583 was obtained from Sakura Finetek Company (USA).

Dyeing of wool fabrics

The dyeing process commenced at a relatively lower temperature of 50 °C. Once the required reagents were dissolved, the dye bath was heated up to 98 °C at a rate of 1 °C min⁻¹. With a concentration of 3% o.w.f. (based on the weight of the fabric), Lanasol dyes were applied at 98 °C for 30 min, followed by dye fixation with 2 g L⁻¹ of Na₂CO₃ for 15 min. Lanaset dyes were applied at 98 °C for 30 min, accompanied by dyes of 3% o.w.f. Finally, the dyed wool fabrics were rinsed with deionized water and 2 g L⁻¹ of soap powder and then dried at 40 °C.

Assay of proteases

Optimal temperature and pH. The fundamental steps for determining protease activity were outlined as follows.³¹ Under the conditions of using casein as the substrate, 1 mL of diluted enzyme solution was added and incubated for 10 minutes. After that, 2 mL of 10% trichloroacetic acid (TCA) was added to terminate the reaction. The control employed the previously mentioned method, which was adjusted to include TCA before the enzyme. Centrifugation was performed for 10 minutes at a speed of 8000 rpm. The absorbance of the resulting supernatant was recorded at a wavelength of 275 nm. It should be stressed that one unit of activity (U) was defined as the amount of enzyme that liberates one μg tyrosine per min under the assay conditions. On this basis, the relative activity of proteases was evaluated at varying temperature or pH levels to identify the optimal conditions for its performance. Additionally, the enzyme solution was maintained for 2 hours under specific conditions, and then the enzyme activity was measured to compare the stability of the proteases.

Substrate specificity. The substrate specificity of proteases is represented by the K/C value. K is defined as the protease activity using casein as the substrate under standard enzyme activity assay conditions (40 °C and pH 8). C is defined as the protease activity using keratin as the substrate. All other testing conditions remain the same except for the change in the substrate.

Kinetic parameters of the enzymatic reaction. The Michaelis constant (K_m) and the maximum reaction rate (V_max) are important parameters in the study of enzyme-catalyzed reaction kinetics. Using various protein solutions of different concentrations as substrates, the initial reaction rates of the protease-catalyzed reactions were measured. Following the Lineweaver–Burk double reciprocal method, plots were made with 1/[S] and 1/[V] as the x and y axes, respectively. Subsequently, the K_m and V_max values were obtained based on eqn (1).where V represents the rate of the enzymatic reaction (g L⁻¹ min⁻¹) and [S] represents the concentration of the substrate (1, 2, 5, 10, and 20 g L⁻¹).

Molecular weight and particle size of proteases. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was selected to measure the molecular weights of S and U. A particle size analyzer (Brookhaven, UK) was used to determine the particle sizes of S and U.

Anti-felting processing of dyed wool fabrics using proteases. Following preliminary exploration and condition optimization, the dyed wool fabrics were subjected to treatment with the compound protease system for 1 hour, with a protease concentration of 15 U mL⁻¹ (where S : U = 2 : 1). The sample subjected to this treatment was defined as SU-treated. The treatment was conducted at 50 °C and pH 8. For comparison, the dyed wool fabrics were separately incubated with S and U. The treated samples were labeled as S-treated and U-treated, respectively. It should be noted that the treatment conditions for S were at 50 °C and pH 8, whereas for U, they were at 60 °C and pH 8. Furthermore, 1 g L⁻¹ of JFC-6 was incorporated into the process to promote the adsorption of proteases onto the wool fabrics.

Characterization

Anti-felting and mechanical properties. According to ISO 6330:2012, the felting shrinkage of wool fabrics was determined using a Y (B) 089D fully automatic shrinkage testing machine (Darong Co., China). The laundering procedures included 1 × 7A and 3 × 5A programs. The felting shrinkage was calculated using eqn (2).where S₀ represents the area of the original square and S₁ represents the area of the final square after the washing procedures. Each experiment was gauged three times, and the average felting shrinkage was taken.

Different wool samples needed to be equilibrated for 24 h under the standard environmental conditions (20 °C, 65% relative humidity) before tensile strength testing. According to the method described in ISO 13934-1:2013, the strength was tested using a YG(B)026D-250 electronic fabric strength tester (Darong Co., China). Tensile strength loss was evaluated according to eqn (3).

where F₀ and F₁ represent the tensile strengths of untreated and treated wool fabrics, respectively. The average values of three parallel experiments were taken as the test results.

The wear resistance test of the samples was conducted using a Martindale abrasion tester according to ISO 12947-2:1998.

Color properties. Based on the Kubelka–Munk equation, the color depth of dyed wool fabrics was measured and expressed as K/S values. The K/S values could be derived using a Color-Eye 7000A color measurement and matching instrument (Macbeth, USA) under standard observer (10°) and D65 illumination.

To further assess the uniformity of color in the dyed wool fabrics, 10 random points on each sample were selected for K/S measurement. The coefficient of variation of K/S values (referred to as K/S CV values) was calculated according to eqn (4).

where K/S* denotes the average K/S value for each sample. The absorbance of residual liquid treated with various proteases was determined using a P4 UV-visible spectrophotometer (MAPADA, China).

The spectral data measured using the Color-Eye 7000A instrument were converted into CIE Lab color space parameters (L*, a*, and b*), where L*, a*, and b* represent lightness, the red-green axis, and the yellow-blue axis, respectively. The color difference was quantified by the ΔE_CMC value calculated using the following eqn (5).

where ΔL*, Δa*, and Δb* represent the differences between the sample and the standard values, and ΔE_CMC represents the total color difference comprehensively.

Colorfastness was also a crucial criterion for evaluating the color properties of dyed wool fabrics. Rubbing fastness and washing fastness of dyed fabrics, both before and after treatment, were determined with reference to ISO 105-X12:2016 and ISO 105-C06:2010, respectively. Subsequently, the grey scale for color change was employed to rate the levels of color fastness.

Assessment of damage to wool fibers. The alkali solubility of wool fabrics was calculated by assessing the weight loss incurred during the test, referring to ASTM D1283-8. The Allwörden reaction required soaking wool fibers in saturated bromine water for 2 min and then observing the number and size of vesicae on the surface of the fibers using a Nikon Eclipse E100 optical microscope (Nikon, Japan). Cross-sectional views of fibers can reflect the accessibility of enzyme molecules to the fiber interior during the anti-felting process. The wool fibers were embedded in O.C.T. 4583 and frozen for 24 h. Subsequently, these frozen samples were cut into 6 μm slices using a CM1950 microtome (Leica AG, Germany). The sections were transferred onto a glass slide and then stained with 0.005% (w/w) methylene blue at 25 °C for 3 min. Eventually, the cross-sectional images of the fibers were observed, and the dyeing depth and distribution of methylene blue in the fibers were assessed.

Frictional properties of wool fibers. The frictional properties of wool fibers could indicate the hydrolysis of scales resulting from protease treatment. An XCF-1A fiber friction coefficient tester (New Fiber Instrument Co., China) was utilized to determine the friction coefficients of wool fibers, and the value of the directional frictional effect (D.F.E) was computed according to eqn (6), in which μ_a and μ_w denote the friction coefficients against and with scales, respectively.

Morphology of wool fibers. The surface morphologies of wool fibers subjected to various treatments were observed using an SU1510 scanning electron microscope (SEM) (Hitachi, Japan). After wool fibers were sprayed with gold, SEM images were captured at a voltage of 5 kV with magnifications of 1000×, 3000×, and 5000× for assessment.

Analysis of the degradation product of wool fabrics. The amino acid composition of the hydrolysates with different treatments was determined using an Agilent 1100 high-performance liquid chromatography system (Agilent, USA). Following the treatment of wool with various proteases, 4 mL from the residual solution was separately extracted and hydrolyzed by the addition of 4 mL of 6 mol L⁻¹ hydrochloric acid and incubated at 120 °C for 24 h under a nitrogen atmosphere. Subsequently, 4.8 mL of 10 mol L⁻¹ sodium hydroxide solution was introduced to neutralize the hydrolysate, and the final volume was diluted to 25 mL by adding deionized water. The resulting supernatant was obtained after filtration and centrifugation. Ultimately, 400 μL of the supernatant was transferred into a liquid phase bottle for the analysis of amino acid composition.

Content of the sulfhydryl group in wool fibers. The quantification of sulfhydryl groups was conducted utilizing the specific reaction between sulfhydryl groups and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). In brief, 10 mg of wool fibers was immersed in 5 mL of phosphate buffer (pH 8). After the addition of 100 μL of DTNB solution, the mixture was incubated for 1 h at room temperature in the dark. The absorbance was subsequently measured at 412 nm. The sulfhydryl content on the surface of the wool fibers was calculated according to eqn (7), in which m stands for the weight of the wool fibers.^32,33

Content of –SH (μmol g⁻¹) = (A₄₁₂ × 10⁶)/13 600 × m (7)

Elemental composition and structural characterization of wool fibers. The elemental composition of the wool surface was determined utilizing an EX-250 energy dispersive spectrometer (EDS) (Horiba, Japan). Total reflection infrared spectroscopy of the wool fabrics was performed using an IS 10 Fourier transform infrared (FT-IR) spectrometer (Nicolet, USA), with the deconvolution of amide I (1600–1700 cm⁻¹) analyzed using the peak fitting software Peak Fit. The transmittance was scanned in a wavelength range from 500 to 4000 cm⁻¹ at a resolution of 4 cm⁻¹ with 32 scans per sample. The surface structure of the wool fabrics was explored using an InVia Reflex Raman microscope system (Renishaw, UK). The spectral scanning range was from 400 to 3000 cm⁻¹. Crystallinity analysis was performed using a D2 PHASER X-ray diffractometer (Bruker, Germany), covering a scanning range of 5–40° (2θ) and a step length of 0.02°. The crystallinity index (C.I.) was calculated using the empirical formula presented in eqn (8).

C.I. (%) = (I₁ − I₂)/I₁ × 100% (8)

where I₁ represents the maximum intensity observed at around 2θ = 10° and I₂ denotes the minimum intensity observed near 2θ = 14°.

Wearability of wool fabrics. The drape coefficient of the wool fabrics was examined following ISO 9073-9:2008, utilizing a YG811 fabric drape tester (Ningbo Textile Instrument Co., Ltd, China). The air permeability was evaluated under the standard of ASTM D737, employing a YG461E-III fully automatic permeability instrument (Ningbo Textile Instrument Co., Ltd, China). Referring to GB/T 12704.2-2009, the moisture permeability of wool fabrics, quantified as the water vapor transmission rate (WVT), was tested using a YG461E tester (Ningbo Textile Instrument Co., Ltd, China). The hand feeling indexes of wool fabrics, such as smoothness, resilience, and softness, were rated according to AATCC TM 202 using a PhabrOmeter 3 fabric evaluation system (Nu Cybertek, Inc., USA), which also provided a relative hand value. The wettability of the fabrics was carried out using a JC2000DM contact angle tester (Zhong Chen Digital Technology Co., Ltd, China). The anti-static performance, manifested as the peak voltage and half-life period, was conducted following ISO 18080-1:2015 using a YG(B)342D fabric inductive electrostatic tester (Wenzhou Jigao Testing Instrument Co., Ltd, China).

Results and discussion

The fundamental properties of proteases and the compound system

The optimum conditions and stability for the temperature and pH of proteases and the compound system are shown in Fig. 2a and b. The maximum value of enzyme activity observed among S, U, and compound proteases is defined as 100% relative activity. Significantly, the enzymatic activity of the compound protease almost got rid of the influence of changes under temperature and pH conditions, consistently retaining an activity level above 80%. For further analysis, the assessment of protease stability was conducted by measuring enzymatic activity following an incubation for 2 h under specified conditions. It can be seen that the combined use of S and U does not impair the catalytic activity of each protease but rather integrates complementary advantages to some extent. This could be attributed to the mixed system mitigating the impact of the external environment on the enzyme activity by enhancing the binding ability to the substrate through synergistic interactions.³⁴

Fig. 2 Enzymatic properties of proteases. (a) Optimal temperature and thermal stability of S, U, and SU; (b) optimal pH and pH stability of S, U, and SU; (c) K/C values of S, U, and SU; and (d, e and f) Lineweaver–Burk plots and Michaelis–Menten kinetic parameters of S and U using casein, keratin, and BSA as substrates, respectively.

Fig. 2c presents the K/C value of proteases, which is a crucial criterion for distinguishing proteases from keratinases. It can be seen that the K/C value of S was recorded at 0.63 (greater than 0.5), and its keratin-degrading ability was also retained within the SU system, as evidenced by an overall K/C value of 0.52. It means that they demonstrate greater specificity for keratin.³⁵ In summary, when the compound protease acts on wool fibers, the keratin specificity of S makes it tend to remain on the fiber surface. This encourages efficient keratin hydrolysis within the scale layer.

To further explore the characteristics of proteases and assess their catalytic efficiency, the enzyme-catalyzed reaction kinetics were investigated through the determination of the Michaelis constant (K_m) and the maximum reaction rate (V_max) of the protease. Substrates with different sulfur contents, such as casein, keratin, and bovine serum albumin (BSA), were selected for this study. The distinct structures and abundant chemical compositions of these proteins were utilized to model the complex composition of wool, thus judging the affinity and catalytic efficiency of the proteases concerning wool substrates. It should be noted that K_m serves as a measure of the proteases’ affinity for the substrate, with lower K_m values signifying higher affinity. V_max reflects the maximum catalytic capacity of the protease at substrate saturation.³⁶ The results in Fig. 2d–f indicate that U possesses small K_m and V_max values for both low-sulfur and high-sulfur proteins. U demonstrates a higher affinity compared to S, implying a greater ability to bind to the substrate and more accurately identify and act upon the target substrate.³⁷ At the same time, U also exhibits weak catalytic capacity, which indicates that its limited V_m and slower reaction rate restrict the extent of hydrolysis, preventing substantial damage to the fibers. Combined with the greater specificity for keratin of S, this property may facilitate a controllable degradation mechanism of the SU system within the complex wool environment.

Effects of different treatments on the anti-felting and mechanical properties of wool fabrics

The wool scale layer is composed of a complex mixture of proteins, containing abundant disulfide bonds and possessing a tight structure.³⁸ The high sulfur content of the outer scales hinders the hydrolysis effect of proteases, leading them to preferentially target the non-keratinizing proteins located within the inner scale layer. At the same time, when the wool is subjected to thermal swelling, the CMC layer is more susceptible to being hydrolyzed by proteases. This allows the protein degradation not only to be confined to the surface of the fibers but also to infiltrate into their internal structure. This results in considerable deterioration regarding both the weight reduction and tensile strength of wool.

To tackle this challenge and mitigate potential damage during the use of a protease, we introduced the compound protease composed of S and U. This endows the system with broad substrate specificity and indirectly increases the reaction sites between the protease and the substrate.³⁹ During the optimization of the experimental conditions, we documented the felting shrinkage and strength loss of fabrics. For the application of S or U (Fig. S1a and b†), it can be seen that the excellent shrink-resistant effect was inevitably accompanied by severe fiber damage. However, at a concentration of 30 U mL⁻¹ of the compound protease, the fabrics exhibited a felting shrinkage rate of 4.50%, accompanied by a strength loss of merely 6.10% (Fig. S1c†). This phenomenon can be attributed to the synergistic effect between S and U within the complex protease, which possibly shifts the primary action sites of proteases from the CMC layer to the scale layer. This strategic shift avoids not only the severe detachment of scale layers from wool fibers but also the immoderate degradation of the CMC layer. This is consistent with the effect of the modified proteases in previous studies. It is worth noting that while certain protease modification techniques^{25,31,40–44} have been established to mitigate the felting shrinkage of wool fabrics to some extent, these modifications will often lead to increased steric hindrance during the enzymatic hydrolysis reaction, as well as changes in the enzymatic properties. Furthermore, most modification methods are still in the experimental stage owing to their intricate procedures, and their commercial application in industrial contexts remains limited. The compound enzyme one-bath approach that we have proposed achieves a short-process anti-felting treatment, becoming the most rapid and convenient processing method to date. In comparison with the cold pad-batch method,^28,45 which aims to effectively conserve water and energy, the one-bath method with the compound protease offers superior advantages for its convenience and conciseness, making it more appropriate for industrial applications.

Considering that the fiber characteristics were altered following the dyeing process, primarily its hydrophobic properties, these alterations may accelerate the subsequent enzyme hydrolysis when this approach is employed on dyed wool fabrics. Consequently, adjustments were implemented regarding both the protease concentration and the duration of treatment. A total dosage of 15 U mL⁻¹ was found to achieve the desired anti-felting performance after one hour of treatment, yielding a felting shrinkage rate of 3.90%, with a corresponding strength loss of 11.39% (Fig. S1d†). In comparison with grey wool, dyed wool fabrics exhibited a more pronounced degree of strength loss. This phenomenon may be attributed to the enhanced thermal motion during the high-temperature dyeing process, which may lead to a relaxation of the fibrous structure, thereby making it more vulnerable to protease attack. Nevertheless, the strength loss associated with this treatment still remains within an acceptable range. Furthermore, in comparison with the prior all-enzyme approach, whether it is the combination of keratinase¹² and proteases or transglutaminase⁴⁶ and proteases, the compound protease system we proposed demonstrates a superior anti-felting effect. This system effectively establishes an equilibrium between diminished felting shrinkage and minimal strength loss of wool fabrics, thereby enhancing the overall efficacy of the all-enzyme method.

The final measurements of felting shrinkage and friction coefficients for different samples were finally illustrated to evaluate the efficacy of the removal of the scale layer. The felt shrinkage rate (Fig. 3a) observed in dyed wool fabrics after treatments aligns with the requisite standards for machine washability. The wool fibers are wrapped in a layer of scales that are oriented from the fiber tip to the base. When fibers come into contact, relative slip occurs, leading to the occurrence of felt shrinkage. The against-scale friction coefficient is higher than that of the with-scale movement. The disparity between these two coefficients is referred to as the directional friction effect (D.F.E), which serves as an indicator of the anti-shrinkage performance of the fibers. The treatment of wool fabrics with proteases resulted in a reduction in D.F.E values compared to untreated fabrics (Fig. 3b), suggesting that the scale layer was productively removed, thereby enhancing the anti-felting properties of wool. The extent of damage to wool fibers was assessed by determining the tensile strength loss and the alkali solubility of wool fabrics. As depicted in Fig. 3c, the strength loss of dyed wool fabrics treated with S or U is markedly greater than that of SU. After rapidly hydrolyzing the CMC layer, S or U with high concentration enters the cuticle layer, causing the “core rot” of wool fibers. It means serious fiber damage and significant tensile strength loss. However, the compound proteases have a milder effect, with hydrolysis mainly on the fiber surface, thus preserving most of the fiber's tensile strength. Due to the large number of disulfide and peptide bonds within the wool fibers, they are prone to cleaving under alkaline conditions.⁴⁷ Severe damage in fibers is manifested as a marked increase in alkaline solubility (Fig. 3d). The alkali solubility of the dyed wool fabrics treated with SU is comparable to that of the untreated fabrics, and it is notably lower than that of S or U. This phenomenon can be attributed to the concentrated degradation of S or U in the CMC layer, whereas SU achieves controllable hydrolysis on the surface of wool fibers, resulting in reduced damage to the wool fibers. This approach can effectively prevent the felting of wool fabrics with minimal damage by utilizing protease treatment in the absence of chemical reagents. The ecological benefits of this method are significantly superior to those associated with conventional chemo-enzymatic techniques.^21,48–50

Fig. 3 Anti-felting effect and mechanical properties of wool fabrics after different treatments. (a) Area shrinkage; (b) D.F.E values; (c) tensile strength loss; (d) alkali solubility; (e) stress–strain curves; and (f) wear resistance.

The stress–strain curves of the samples, illustrated in Fig. 3e, depict the deformation behavior of the fabric under tensile stress. The initial segment of the curve typically exhibits a linear relationship, signifying that the fibers deform within the elastic range and are capable of returning to their original configuration upon the removal of the applied force. As stress increases, the curve transitions into the plastic deformation phase, during which the fabric begins to experience permanent deformation. Ultimately, the curve reaches a maximum point, referred to as the breaking strength of the fabric, beyond which failure occurs. In our investigation, a comparative analysis of the stress–strain curves of various samples revealed that the fabric treated with SU demonstrated superior tensile strength and elongation at break compared to the fabric treated with S or U. This enhancement can be attributed to the partial degradation of the scale layers by the compound protease, which results in a smoother fiber surface and a consequent reduction in friction. Such modifications facilitate a more uniform distribution of load among the fibers during stretching, mitigate local stress concentrations, and simultaneously preserve adequate cohesion to uphold the integrity of the fabric structure. Consequently, the fabric treated with SU exhibits enhanced strain hardening characteristics throughout the stretching process. This finding aligns with the observed improvement in the wear resistance of the fabric following treatment with SU, as depicted in Fig. 3f. This improvement can be attributed to the ability of SU to facilitate the uniform removal of surface scale layers without severe damage to the fiber structure. The smoother and more uniform fibers consequently result in a reduction in friction and wear during usage, contributing to an overall enhancement of the fabric's durability.

Additionally, the optical microscopy images after the Allwörden reaction and the staining effect of methylene blue dye for the cross-section of wool fibers after different treatments were employed to evaluate the damage degree of wool fibers. When wool fibers are immersed in saturated brominated water, the disulfide bonds within the scale layer are oxidized. They are subsequently broken down into hydrophilic sulfonic acid groups, producing soluble peptides at the same time. This process generates osmotic pressure both internally and externally within the scale layer,⁵¹ which is manifested by dense vesicles on the surface layer of the wool fibers. A greater number of vesicles correlates with a more intact scale structure. This specific situation is illustrated in Fig. 4a_1–5, where raw wool displays a well-organized arrangement of uniformly sized vesicles on the fiber surface. There are limited vesicles on the wool fiber treated with S or U. This phenomenon may derive from the potential partial or complete exfoliation of the scale layers, which impairs its structural integrity. Consequently, the formation of an effective osmotic pressure difference is hindered, thus the number of vesicles is significantly reduced. Conversely, the dyed wool fabric treated with SU exhibits small and densely packed vesicles on the fiber surface. This occurrence is indicative of slight damage to the scale layer's integrity, which prevents the vesicles from fully expanding. The abundant action sites of the compound protease enable it to act evenly on the wool fibers, resulting in a uniform distribution of vesicles across the surface.

Fig. 4 Optical microscopy photographs obtained after the Allwörden reaction and the staining effect of methylene blue dye for the cross-section of wool fabrics after different treatments. (a₁ and b₁) Raw; (a₂ and b₂) dyed (Lanasol Red CE); (a₃ and b₃) S-treated; (a₄ and b₄) U-treated; and (a₅ and b₅) SU-treated.

Under alkaline conditions, the –COOH and –SH groups present in wool fibers acquire a negative charge. This negative charge contributes to the binding of these groups to the positively charged cationic dye of methylene blue. Therefore, the extent of the hydrolytic effect in wool can be assessed by the intensity and distribution of cationic dyes within the fiber. It can be seen that wool fabrics treated with S or U exhibit a deeper appearance compared to their untreated counterparts, whereas the fabric treated with SU displays a relatively lighter and more uniform color (Fig. 4b_1–5). This is because the proteolysis of wool enhances the exposure of carboxyl groups. Among them, the treatments with S or U affect the protein structure both on the surface and within the wool fibers to varying extents. It results in varied color shades through uneven dye affinity in various wool fiber regions. Notably, due to the synergistic interaction between S and U, the application of the compound protease causes less fiber damage and achieves controllable hydrolysis of the scale layer.

Mechanism of the anti-felting processing based on compound proteases

Aiming to analyze the degradation products of samples and provide a concise evaluation of the protease reaction site, the amino acid compositions of treated residual solutions were determined, as shown in Fig. 5a. Glutamate and tyrosine are exclusively found in the CMC layer of wool, with their concentrations in the hydrolytic residual solution being relatively high. This observation suggests that the protease predominantly targets the CMC layer. Furthermore, sulfur-containing amino acids such as aspartic acid and methionine were only detected in the cuticle layer. There is a certain extent of degradation in sulfur-containing amino acids of wool treated with S or U, while the SU treatment causes less of it. This comes from that the compound protease exerts a mild hydrolytic effect on the fiber interior and controllable hydrolysis can effectively prevent proteases from damaging the structure of wool fibers. It is noteworthy that serine, cysteine, and proline, which are mainly present in the scale layer, also occupied a significant proportion in the hydrolysate, proving that the protease manifests a certain degree of degradation on the scale layer as well. This confirms the broad substrate specificity of S and U. Thus, the compound proteases can selectively hydrolyze wool fibers based on their varying affinities for different substrates.

Fig. 5 (a) Amino acid composition of treated residual solutions; (b) particle size distribution of proteases; and (c) content of the sulfhydryl group in wool fibers.

To further investigate the physical properties of proteases involved in the process, the particle size and molecular weight of S and U were determined. Considering the phenomenon of partial aggregation of proteins in solution,⁵² the volume-based size distribution of proteases was selected to eliminate the impact of larger aggregations on the results of particle size analysis. As can be seen in Fig. 5b, the particle size of S is about 1.4 times greater than that of U, indicating a slight difference in their absorption capabilities on wool. Compared with the SDS-PAGE results presented in Fig. S2,† this demonstrates that a band appears at approximately 27 kDa (lanes S and U). However, lane S exhibits a thicker band that also disperses upwards. This indicates that S possesses a larger molecular weight, which may result from a more complex molecular structure of S, leading to larger molecular particle sizes in solution. The smaller particle size of U facilitates its diffusion into the interior of wool fibers, making a more stable binding within the compound protease solution. In contrast, the hydrolytic effect of S tends to occur on the fiber surface, thus degrading the scale layer. During the treatment with SU, a preferentially slight degradation of the wool fiber interior occurs due to the high affinity of U. This contributes to the loosening of the fiber structure, thereby increasing the accessibility of S to the scale layers. As wool keratin is rich in disulfide bonds, the variation of –SH can indirectly reflect the hydrolytic difference of wool treated with S or U (Fig. 5c). Compared with S, there is a significant rise in the –SH content in fibers treated with U. Due to the smaller particle size of U, it can easily penetrate the fiber interior and loosen the keratin structure by hydrolyzing peptide bonds within wool fibers. This process exposes amino acids containing sulfhydryl groups, consequently elevating the sulfhydryl content on the surface of the wool. This conclusion matches the trend in alkali solubility, in which the U-treated sample is more susceptible to keratin structural damage and the hydrolysis of chemical bonds in an alkaline solution.

The FTIR spectra of wool after various treatments are presented in Fig. 6a. The predominant characteristic absorption peaks observed in the infrared spectra of proteins are amide bands, among which amide A, amide I, amide II, and amide III correspond to the stretching vibration peaks near 3273 cm⁻¹, 1628 cm⁻¹, 1513 cm⁻¹, and 1231 cm⁻¹, respectively.³² The observation that the amide band spectra of the treated dyed samples remained largely consistent suggests that the macromolecular structure of the protein has not been compromised. In addition, the peaks at 1023 cm⁻¹ and 1046 cm⁻¹ are associated with the stretching vibrations of S–O bonds in the cysteine residues.⁵³ Notably, the intensities of these characteristic peaks increased relative to those of the untreated wool. This indicates that the protease treatment may expose cysteine residues within the dyed wool fibers, making them more susceptible to oxidation and the formation of S-sulfonic acid residues. Here, the amide I band of the protein IR spectrogram has been deconvoluted into several Gaussian-type bands (Fig. S5†), which are frequently employed to assess and quantify the relative content of protein secondary structures (Fig. 6b). It is mainly α-keratin in wool fibers, and its secondary structure is predominantly α-helix. Thus, the degradation extent of keratin in wool fibers can be judged by the relative content of α-helix. In comparison with the α-helix contents of wool fabrics treated with S or U, the secondary structure content of dyed wool subjected to SU treatment exhibited mild changes. The possible reason may be proteases degrade the cuticle via the CMC layer, significantly reducing the α-helix content. Conversely, the compound proteases have no marked impact on the crystalline structure of wool, thus maximally retaining the tensile strength of wool.

Fig. 6 Difference in the chemical structure of wool fabrics after treatment. (a) FTIR spectra; (b) area percentage of the secondary structure of amide I; (c) XRD spectra; (d) crystallinity index; (e) Raman spectra; and (f) S–S contents of various samples (the treated samples were pre-stained with Lanasol Red CE).

The X-ray diffraction patterns of the dyed wool samples are presented in Fig. 6c. The samples have broad diffraction peaks at 10° and 20°, exhibiting the typical crystalline structure of wool.⁵⁴ The former is the common diffraction peak of the α-helix and β-sheet, while the latter is the overlapping peak of the two crystalline forms. The crystallinity of the material is directly proportional to the crystallinity index (C.I.)⁵⁵ which is shown in Fig. 6d. These data can indirectly reflect the extent of wool scale removal by proteases. After protease treatment, the degree of crystallinity of the wool fabrics is significantly reduced. Compared with the SU-treated, the crystallinity of wool fabrics treated with S or U is marginally higher. This is due to S and U exhibiting extensive damage to wool fibers. They hydrolyze not only the scale layer with high crystallinity but also the amorphous structures of wool fibers like the CMC, especially the CMC layer as the high affinity of proteases for it. However, the compound proteases shift the major hydrolysis effect from the CMC layer to the scale layer with high crystallinity. This forms the relatively low crystallinity of the wool fabrics treated with SU.

Fig. 6e depicts the Raman spectra of wool fabrics after different treatments. The samples exhibit similar characteristic peaks, with Raman signals monitored near 1660 cm⁻¹ and 1244 cm⁻¹, corresponding to the amide I and amide III bands, respectively.⁵⁴ In addition, the stretching vibration of the C–H bond generates a Raman signal at around 1446 cm⁻¹, while the peak at 510 cm⁻¹ is associated with the stretching vibration of the S–S bond. Previous research⁵⁶ has established that the ratio of S–S band intensity to C–H band intensity serves as a macroscopic indicator of the S–S bond of wool fibers. The untreated wool fabric, with its cuticle layer structure intact,³⁰ has a higher relative content of disulfide bonds (Fig. 6f). A comparative analysis reveals that the ratio of dyed wool treated with SU was 0.652, which is lower than that of untreated dyed wool but higher than that of wool subjected to S or U treatment. This difference arises from the peeling of the cuticle layer induced by S or U treatments, which results in a significant reduction in the S–S content. The compound proteases not only hydrolyze the CMC but also degrade the cuticle layer. This moderate hydrolytic effect does not destroy the main structure of wool fibers, reflected in the slight decrease in the content of the disulfide bond.

The surface morphology of various samples was analyzed using SEM, with the findings depicted in Fig. 7a–e. The analysis reveals that the untreated wool fibers displayed intact and well-defined scales that were densely arranged on their surface. Following treatment with S, a significant number of scales appeared to lift without falling off, resulting in an increased gap at the root of the scales. In contrast, the scale layer on wool fibers treated with U displayed a loose structure, indistinct edges, and longitudinal ridges and grooves. This observation suggests that the proteolytic effect of the protease functions from the interior to the exterior of the scales, assisting the hydrolysis of the inner layer and gradual peeling of the scales. Consequently, the penetration channel for the protease enlarges, resulting in more severe damage to the wool fibers, which aligns with the previously documented tensile strength loss. After SU treatment, the edges of the fibrous scales exhibited signs of blunting, and there was almost no lifting. This indicates that the proteolytic action of the protease is mostly confined to the fiber surface, thereby mitigating internal damage. Specifically, although S is a protease with specificity for keratin, it still mainly hydrolyzes the CMC layer when acting on wool fibers. However, when S synergistically cooperates with U, the different specificities and affinities between the two form a controllable hydrolytic effect, with S preferentially acting on the wool scales. Overall, the implementation of the compound protease system in the anti-felting processing of wool demonstrates a degradation effect characterized by “wide breadth and low depth”. It significantly contributes to the retention of greater strength in wool fibers after the protease treatment.

Fig. 7 SEM images of different wool fabrics. (a) Raw; (b) dyed (Lanasol Red CE); (c) S-treated; (d) U-treated; and (e) SU-treated.

In addition, Fig. 8a–e illustrate the elemental composition of various samples as determined by EDS. Following protease treatment, a slight decrease in sulfur content was tracked, likely owing to the degradation of keratin within the scale layer facilitated by the protease. This degradation is expected to disrupt disulfide bonds and indirectly release free sulfhydryl groups. Concurrently, the protease treatment also removes lipid substances from the surface of the wool fibers. The elimination of these lipids further contributes to the reduction of sulfur content on the wool surface. At the same time, lipid removal exposes the lipoprotein peptide chains or cuticle outer layer proteins, further increasing O and N contents while decreasing the C content.

Fig. 8 EDS spectra and elemental mapping maps of different samples. (a) Raw; (b) dyed (Lanasol Red CE); (c) S-treated; (d) U-treated; and (e) SU-treated.

Color properties and wearability of wool fabrics after different treatments

To further expand the utilization of wool anti-felting technology concerning dyed textiles, the study intends to explore a process that slightly affects the color performance of dyed wool fabrics. The Lanasol and Lanaset series dyes are most commonly employed for wool dyeing in industry. The Lanasol series is a reactive dye specifically designed for wool, while the Lanaset series consists of 1 : 2 metal complex dyes and reactive dyes. Given the complex composition of commercial enzyme preparations, the existence of various reagents may adversely affect the internal structure of dyes. For this reason, two representative dyes were selected from each series to record the changes in their UV absorption spectrum under the influence of proteases, alongside the structures of the dyes. As can be seen in Fig. 9, the designations a_1–3, b_1–3, c_1–3, and d_1–3 correspond to Lanasol Red CE, Lanasol Blue CE, Lanaset Yellow 4GN, and Lanaset Red G, respectively. The chromophore, auxochrome, and conjugated system of the dye molecule are critical determinants of alterations in the UV absorption spectrum. As the dosage of protease increases, the UV absorption spectra of the four dyes remain consistent with the original staining solution. The specific performance is that the absorbance exhibits almost no variation, and the maximum absorption wavelength remains constant. This generally indicates that enzymatic preparations maintain the structural integrity of the dyes, and this conclusion may apply to the remaining dyes not explicitly mentioned. In a comparative study, Wei et al.²⁸ investigated the effects of frequently utilized reducing reagents on the structure of dyes. Their findings indicate that the chemical-enzymatic approach poses a potential risk of compromising the dye structure, which may adversely affect the color appearance of dyed wool fabrics. This further underscores the benefits of employing the all-enzyme method in the context of anti-felting treatments for dyed wool textiles.

Fig. 9 Structures of different dyes and the influence of proteases on the UV absorption spectrum of dye solutions. (a₁–a₃) Lanasol Red CE; (b₁–b₃) Lanasol Blue CE; (c₁–c₃) Lanaset Yellow 4GN; and (d₁–d₃) Lanaset Red G.

Taking wool fabrics dyed with Lanasol Red CE as an example, changes in the color properties of dyed wool fabrics after shrink-resistant treatments were observed. The K/S value curve was comprehensively obtained by scanning three distinct points on the fabric, thereby providing the overall color depth of the sample. The maximum absorption peak of the K/S value curves of the dyed fabrics treated with proteases remains unchanged in comparison with the untreated fabrics, and the intensity of the SU-treated K/S value curve exhibits scarce variation (Fig. 10a). Analysis of the cross-sectional images of dyed wool fibers reveals no notable differences in color depth before and after treatment with SU (Fig. S5†), which is consistent with the K/S value. This suggests that there is a negligible impact on the visual characteristics of the textile after SU treatment. The K/S coefficient of variation (K/S CV) was also calculated and employed to compare the uniformity of color distribution in dyed fabrics.⁵⁷ A lower K/S CV value indicates better color consistency. Due to the presence of minor damage to certain fibers during the weaving process, the protease with a high affinity for the CMC layer tends to preferentially attack these compromised areas when acting on the fibers. It may result in the detachment of the scale layers from the root. Dyes are removed from the fibers mainly following this physical detachment of scale layers. In contrast to wool treated with S or U, the compound enzyme can obtain a uniform processing effect and excellent cloth appearance attributable to its controllable hydrolysis effect (Fig. 10b). Analysis of the residual solutions subjected to protease treatment (Fig. 10c) reveals that the maximum absorption wavelength remains consistent when compared to the original staining solution in Fig. 9a. This observation suggests that the impact of protease on dyed fabrics is predominantly manifested through the shedding of bound dye due to the stripping effect, which results in a slight increase in fabric brightness. This alteration subsequently affects the a* and b* values, leading to an increase in color saturation (presented in Fig. 10e as the c* value), while these changes remain within an acceptable range. Besides, the ΔE_CMC of SU-treated can be controlled within 1.00 (Fig. 10d), a level that is not discernible by the naked eye. The consistency of the hue (h*) values further corroborates the stability of the dye structure throughout the degradation process (Fig. 10e).

Fig. 10 Color properties of dyed wool fabrics after different treatments. (a) Color depth (expressed as the K/S value); (b) homogeneity (expressed as the K/S CV value); (c) absorbance of residual solutions; (d) lightness (expressed as L*), chromaticity coordinates (expressed as a* and b*), and color difference values (expressed as ΔE_CMC); (e) chroma and hue (expressed as the c* value and h* value, respectively); and (f) color fastness properties.

It is widely accepted that during the dyeing process of wool fibers, dyes can diffuse into the interior of the wool fibers through gaps formed by the swelling of the scales, ultimately reaching the cortex layer. Reactive dyes utilized for wool can establish covalent bonds through chemical reactions with functional groups such as –OH and –NH₂ present in the wool fibers. Due to the excellent stability of covalent bonds, protease treatment won't damage the combined fastness of covalent bonds between wool and dyes. Coordination bonds between the Lanaset dyes and the fibers are also of outstanding stability. The colorfastness of the dyed fabric shown in Fig. 10f and Table S1† also basically proves this. The decrease in wet rubbing fastness is possibly due to fiber damage caused during protease hydrolysis of the fibers, which may lead to the exposure of dyes. Thus, the risk of dye falling off increases when the fibers are subjected to physical friction. Fig. S5† demonstrates that the fabrics dyed with the three additional representative dyes maintained satisfactory chromatic aberrations following SU treatment. This proves the applicability of the compound protease system to various types of dyed wool fabrics. Collectively, the data support the conclusion that SU-treated dyed wool fabrics possess an aesthetically pleasing appearance and superior color characteristics, meeting the processing requirements of the factory for dyed wool textiles.

The wearability of wool fabrics under various treatments was systematically evaluated. Fig. 11a illustrates the drape coefficient of the fabric under both dynamic and static conditions, revealing an inverse relationship with softness. The application of compound protease effectively eliminates the wool scales, thereby diminishing frictional interactions between the fibers. As fibers are more prone to bending and deformity, there is a marked decrease in the drape coefficient. Improved air and moisture permeability contribute to the overall comfort and functionality of the fabric. The compound protease treatment can either loosen or partially exfoliate the scale structure, leading to increased porosity and pore size among fibers. This structural change is advantageous for the passage of air and water vapor, provoking significantly enhanced air and moisture permeability in the SU-treated fabric (Fig. 11b and c). Furthermore, a PhabrOmeter system was introduced to quantitatively assess the hand feelings of textiles by simulating human tactile perception. These include smoothness, resilience, softness, and relative hand values. As can be seen in Fig. 11d–g, there is a notable increase in the scores across several parameters. Fig. 11h demonstrates a significant advancement in the wettability of the SU-treated wool fabric, confirmed by a lower contact angle compared to the untreated fabric. This suggests that protease treatments can reduce the contents of hydrophobic keratin proteins and lipids. This may expose the internal hydrophilic cortical layer structure (containing polar groups such as –OH and –NH₂), consequently upgrading wettability. Similarly, antistatic properties, consistent with wettability results, were evaluated through measurements of peak voltage and half-life. A reduced half-life of static voltage proclaims superior antistatic properties; in this instance, the half-life of the SU-treated fabric decreased from 3.88 s to 2.93 s (Fig. 11i). In summary, the improvements in wearability remarkably augment the added value of wool textiles.

Fig. 11 Wearability of wool fabrics after different treatments. (a) Drape coefficient; (b) air permeability; (c) water vapor transmission rate; (d) smoothness; (e) resilience; (f) softness; (g) relative hand value; (h) wettability; and (i) anti-static performance (the treated samples were pre-stained with Lanasol Red CE).

Conclusion

A compound protease system characterized by low-depth hydrolysis effects was developed in this study. The high keratin-hydrolyzing capability of Savinase Ultra 16XL enables this system to control fiber damage at minimal levels while making wool products machine washable. In contrast to conventional chemo-enzymatic treatments, the all-enzyme strategy does not require additional chemical reagents, thereby overcoming the issue of color alterations in dyes typically caused by reducing reagents. This approach also contributes to resource recycling and the maintenance of ecological balance, as proteases serve as eco-friendly biocatalysts. Additionally, the compound enzymatic method results in fabrics that demonstrate uniform coloration with no visible color difference detectable by the naked eye (ΔE_CMC < 1) and exhibit encouraging color fastness properties. Practical applications have confirmed that this method is suitable for other fabrics dyed with various routine dyes, underscoring its universality. Meanwhile, previous research has primarily focused on wool tops, where the excessive removal of scales frequently leads to a reduction in fiber cohesion, adversely affecting subsequent spinning and weaving processes. Our study proposes shifting the machine-washable process from top to dyed fabrics, that is, transitioning from an initial stage of production to a final stage. This adjustment not only eliminates the potential risks associated with decreased fiber cohesion and severe fiber damage but also facilitates a more rapid response to market demands. In summary, this study presents an ideal alternative to traditional chlorination methods, promoting the green transformation of the wool spinning industry towards a novel “bioeconomy”. In future research, we will also attempt to apply the compound protease we proposed to factory applications. A detailed analysis of the production costs and the wastewater generated during this process will be conducted to quantify the cost-effectiveness and environmental benefits associated with this approach.

Author contributions

Xinrui Zhang: writing – original draft, methodology, formal analysis, conceptualization, validation, software, and investigation. Jun Wang: methodology, investigation, and formal analysis. Man Zhou: supervision and methodology. Yuanyuan Yu: supervision, methodology, and formal analysis. Ping Wang: investigation, supervision, methodology, and formal analysis. Qiang Wang: writing – review & editing, resources, validation, methodology, project administration, conceptualization, and funding acquisition.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the National Key Research and Development Program of China (2021YFC2104000) is gratefully acknowledged.

References

1. V. Kadam, S. Rani, S. Jose, D. B. Shakyawar and N. Shanmugam, Sustainable Mater. Technol., 2021, 29, e00298.
2. Q. Zhou, W. C. Wang, Y. Y. Zhang, C. J. Hurren and Q. Li, Text. Res. J., 2020, 90, 2175–2183.
3. J. J. Fu, J. Su, P. Wang, Y. Y. Yu, Q. Wang and A. Cavaco-Paulo, Appl. Microbiol. Biotechnol., 2015, 99, 10387–10397.
4. A. Kaur and J. N. Chakraborty, J. Cleaner Prod., 2015, 108, 503–513.
5. M. M. Hassan and C. M. Carr, J. Adv. Res., 2019, 18, 39–60.
6. S. Rani, V. Kadam, N. M. Rose, S. Jose, S. Yadav and D. B. Shakyawar, Int. J. Biol. Macromol., 2020, 163, 1044–1052.
7. W. Bao, J. S. Shen, X. M. Ding and X. Y. Wu, Text. Res. J., 2019, 89, 4702–4709.
8. I. J. Kaplin and K. J. Whiteley, Aust. J. Biol. Sci., 1978, 31, 231–240.
9. P. Wang, Q. Wang, X. Fan, L. Cui and J. Yuan, Dyeing Finish., 2010, 36, 46–49.
10. J. M. Cardamone and J. Yao, Text. Res. J., 2004, 74, 565–570.
11. A. M. Ferreira, J. A. P. Coutinho, A. M. Fernandes and M. G. Freire, Sep. Purif. Technol., 2014, 128, 58–66.
12. Z. Li, J. Luo, J. Wang, Y. Yu, Q. Wang and P. Wang, J. Cleaner Prod., 2023, 427, 139276.
13. R. P. Zhang and A. Y. Wang, J. Cleaner Prod., 2015, 87, 961–965.
14. S. M. Borghei, S. Shahidi, M. Ghoranneviss and Z. Abdolahi, Plasma Sci. Technol., 2013, 15, 37–42.
15. S. Shahidi, A. Rashidi, M. Ghoranneviss, A. Anvari and J. Wiener, Surf. Coat. Technol., 2010, 205, S349–S354.
16. M. Sadeghi-Kiakhani, S. Safapour, F. Sabzi and A. R. Tehrani-Bagha, Fibers Polym., 2020, 21, 179–187.
17. M. Shahid, F. Mohammad, G. Q. Chen, R. C. Tang and T. L. Xing, Green Chem., 2016, 18, 2256–2281.
18. W. Liu, F. Yuan, J. Wang, C. Qin, Z. Pang, Y. Teng, F. Li and T. Liu, J. Cleaner Prod., 2023, 386, 135828.
19. R. Araújo, C. Silva, R. Machado, M. Casal, A. M. Cunha, J. C. Rodriguez-Cabello and A. Cavaco-Paulo, Biomacromolecules, 2009, 10, 1655–1661.
20. P. Wang, Q. Wang, X. Fan, L. Cui, J. Yuan, S. Chen and J. Wu, Enzyme Microb. Technol., 2009, 44, 302–308.
21. K. Li, Z. R. Li, J. Q. Zhang, J. Wang, Y. Y. Yu, M. Zhou, Q. Wang and P. Wang, Int. J. Biol. Macromol., 2024, 273, 133156.
22. C. D. Prajapati, E. Smith, F. Kane and J. S. Shen, J. Cleaner Prod., 2019, 211, 909–921.
23. K. Wang, R. Li, J. H. Ma, Y. K. Jian and J. N. Che, Green Chem., 2016, 18, 476–481.
24. C. Su, J. S. Gong, J. F. Qin, J. M. He, Z. C. Zhou, M. Jiang, Z. H. Xu and J. S. Shi, J. Cleaner Prod., 2020, 270, 122092.
25. J. S. Shen, E. Smith, M. Chizyuka and C. Prajapati, Fibers Polym., 2017, 18, 1769–1779.
26. J. L. Yao, J. Xu, A. Cui, R. Wang and L. Y. Hao, Fibers Polym., 2024, 25, 3369–3377.
27. X. Wang, X. L. Shen and W. L. Xu, Appl. Surf. Sci., 2012, 258, 10012–10016.
28. B. Wei, S. Wang, J. Wang, M. Zhou, P. Wang, Y. Yu and Q. Wang, Int. J. Biol. Macromol., 2024, 282, 136884.
29. A. Madhu and J. N. Chakraborty, J. Cleaner Prod., 2017, 145, 114–133.
30. B. N. Mu, F. Hassan and Y. Q. Yang, Green Chem., 2020, 22, 1726–1734.
31. J. X. Mei, N. Zhang, Y. Y. Yu, Q. Wang, J. G. Yuan, P. Wang, L. Cui and X. R. Fan, Appl. Microbiol. Biotechnol., 2018, 102, 9159–9170.
32. M. Fernandes and A. Cavaco-Paulo, Biocatal. Biotransform., 2012, 30, 10–19.
33. N. Zhang, N. Zhang, J. F. Zhang, M. Zhou, Q. Wang, P. Wang and Y. Y. Yu, J. Text. Inst., 2022, 113, 2491–2501.
34. Y. Yang, B. X. Zhang and J. Zhang, Soft Matter, 2024, 20, 9654–9663.
35. H. Gradisar, J. Friedrich, I. Krizaj and R. Jerala, Appl. Microbiol. Biotechnol., 2005, 71, 3420–3426.
36. S. Schnell, FEBS J., 2014, 281, 464–472.
37. S. J. Zhou, Z. M. Liu, W. C. Xie, Y. Yu, C. Ning, M. X. Yuan and H. J. Mou, Int. J. Biol. Macromol., 2019, 131, 1117–1124.
38. M. Y. Khalid, A. Al Rashid, Z. U. Arif, W. Ahmed, H. Arshad and A. A. Zaidi, Results Eng., 2021, 11, 100263.
39. L. Wang, Z. Duan, P. Fei, Z. Yan, W. Wang, Y. Di and J. Lu, Colloids Surf., A, 2025, 709, 136215.
40. C. J. S. M. Silva, Q. Zhang, J. Shen and A. Cavaco-Paulo, Enzyme Microb. Technol., 2006, 39, 634–640.
41. E. Smith, Q. Zhang, J. Shen, M. Schroeder and C. Silva, Biocatal. Biotransform., 2008, 26, 391–398.
42. R. Chen, J. Yuan, Y. Yu, X. Fan, Q. Wang and Y. Zhu, J. Food Sci. Biotechnol., 2012, 31, 505–510.
43. M. Schroeder, M. Schweitzer, H. B. M. Lenting and G. M. Guebitz, Biocatal. Biotransform., 2004, 22, 299–305.
44. Z. Zhao, Y. B. Di and W. Wang, J. Nat. Fibers, 2020, 17, 1423–1429.
45. J. G. Yuan, Y. Xiang, A. H. Zhou, S. X. Wang, Q. Ji, L. Liu and D. R. Liu, J. Text. Inst., 2025, 116, 880–889.
46. J. Cortez, P. L. R. Bonner and M. Griffin, Enzyme Microb. Technol., 2004, 34, 64–72.
47. M. A. Taleb, S. Mowafi, C. Vineis, A. Varesano, D. O. S. Ramirez, C. Tonetti and H. El-Sayed, J. Nat. Fibers, 2022, 19, 3351–3364.
48. A. Kantouch, H. El-Sayed and A. El-Sayed, J. Text. Inst., 2007, 98, 65–71.
49. R. Li, Q. T. Zhu, M. Zhou, B. Xu, P. Wang, Q. Wang and Y. Y. Yu, Int. J. Biol. Macromol., 2025, 306, 141698.
50. J. Q. Zhang, K. Li, J. Wang, B. Xu, Y. Liu, Q. Wang and P. Wang, Int. J. Biol. Macromol., 2025, 307, 142160.
51. C. L. Ding, J. Y. Yu and W. G. Chen, Text. Res. J., 2018, 88, 254–260.
52. S. Y. Wang, K. M. Chen, L. Li and X. H. Guo, Biomacromolecules, 2013, 14, 818–827.
53. D. P. Harland, J. P. Caldwell, J. L. Woods, R. J. Walls and W. G. Bryson, J. Struct. Biol., 2011, 173, 29–37.
54. N. Zhang, Q. Wang, J. G. Yuan, L. Cui, P. Wang, Y. Y. Yu and X. R. Fan, J. Cleaner Prod., 2018, 192, 433–442.
55. Z. Jiang, Y. Cui, G. Zheng, Y. Wei, Q. Wang, M. Zhou, P. Wang and Y. Yu, Green Chem., 2022, 24, 5904–5917.
56. X. Shang, Q. Wang, Z. Jiang and H. Ma, J. Mol. Liq., 2022, 367, 120426.
57. Z. Jiang, Y. Y. Zhang, N. Zhang, Q. Wang, P. Wang, Y. Y. Yu and M. Zhou, Color. Technol., 2022, 138, 82–89.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5gc01614b

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