Use of enzymatic processes in the tanning of leather materials

2.1 Processes in the wet workshop

Soaking allows the skins to reabsorb any water lost after skinning, salting, and transportation. Soaking is also used to clean the hides of feces, blood, and dust and remove fibrous material. The soaking methods used depend on the condition of the skins. The hides are usually pre-soaked to remove salt and dust. The main soaking operation may last from several hours to several days [16]. Putrefying bacteria may develop during soaking. To limit their activity, biocides are added. Depending on the type of raw material, other additives may also be used, e.g., surfactants and enzyme preparations [17]. Unhairing and liming are performed to remove the hair, cuticle, and to some extent proteins fibrillary skins. The skins are thereby prepared for the removal of adhering flesh and fat in the fleshing process. The fibrous structure of the hide is opened in the liming step (by swelling) for cleaning and degreasing [18]. The keratin from the hair and epidermis is also removed from the hide by the action of reducing agents, usually sodium sulfide. After the liming process, the presence of lime or other alkali in the hides is no longer necessary and may have an adverse effect on further tanning. The descaling process involves a gradual lowering of the pH (by rinsing with fresh water or weak solutions of acids or salts, e.g., ammonium chloride, ammonium sulfate, or boric acid) and increasing the temperature. Residues of chemicals and decomposed components are removed from the hide.

2.2 Tannery process

In the tanning process, collagen fibers are stabilized with tannins, increasing their resistance to mechanical factors and high temperatures so that the hide is less prone to decay and rotting. Basic chromium sulfate is added to the pickled hide in drum II, with continuous stirring for 4–6 h to allow for absorption. To promote the final reaction of chromium to collagen, the temperature is increased to around 50°C and the pH of the bath is increased to 3.8–4.0 by the addition of basifying agents, such as sodium basic salts or magnesium oxide [14]. Unfortunately, the presence of these substances leads to high COD, BOD, suspended solids, and conductivity [19].

The tanning process modifies the rawhide by stabilizing the collagen fibers against enzymatic attack, mold, and chemical damage. Chrome tanning is still the most popular method of tanning leather, due to its ability to produce high-quality leather [20], with properties such as excellent hydrothermal stability, better dyeing properties, and softness [21]. The chromium tanning process is not an effective technology, due to the absorption rate of chromium Cr(iii) by skin collagen, which ranges from 55 to 70% [22]. A large amount of chromium is discharged into the wastewater, resulting in large losses of chromium tannin. The process increases environmental pollution and wastewater treatment costs and additionally causes huge losses of chromium [23].

Untreated tannery wastewater contains up to 3,000–3,500 mg/l of chromium, which classifies tannery waste as one of the most dangerous industrial wastes [24]. Under oxidizing environmental conditions, Cr(iii) is easily oxidized to Cr(vi), which is harmful to humans and causes allergies, cancer, or necrosis of the liver and kidneys. Cr(vi) causes significant damage to the environment. It reduces the rate of seed germination, leads to high teratogenic and carcinogenic rates, threatens the growth of aquatic organisms, and finally threatens the ecosphere by affecting the process of mitosis [24].

2.3 Post-tanning or bath finishing

Neutralization and rinsing followed by retanning, dyeing, and greasing are most often carried out in one technological container. Specialized activities can also be carried out at this stage of production, to ensure that the leather has certain properties, e.g., hydrophobic or waterproof properties, oleophobicity, gas permeability, flammability resistance, or antistatic properties [25].

3.1 Soaking

Soaking is the first stage of a wet workshop in leather processing technology. Soaking can be divided into two stages [29]:

1. The first soaking is used for preliminary cleaning of the skin by removing large amounts of dirt and unwanted materials.

2. The second soaking uses water and small quantities of imbibing substances to hydrate the skin proteins and open the contracted fibers of the dried skins, solubilizing the denatured proteins and eliminating the salt used in the preservation step. Residual blood, excrement, and earth are removed from the skin [30].

To produce multifunctional enzyme preparations, multi-enzymatic systems are used. The enzyme preparations most frequently used in the soaking process are alkaline proteases. Alkaline proteases are used to ensure faster absorption of water and to reduce the soaking time [31]. The addition of a small amount of amylase and lipase may assist in the degradation and eventual removal of glycoproteins (resulting in fiber opening) and of lipoproteins in the soaking process itself, by aiding structural opening and allowing the chemicals to penetrate more rapidly [32]. Multi-enzyme systems containing proteases and lipases are more efficient due to the synergistic effects of the different enzymes [33]. Scientists continue to explore new and more effective enzyme preparations for use in soaking hides, and there are reports on the use of enzymes such as amidases, hyaluronidases, phospholipases, chondroitinases, and lignocellulases. However, these enzymes are not widely used due to their low specificity [34].

3.2 Unhairing

The process of hair removal is the next step after the soaking process. Leather makes up 7% of cattle weight, and only 25% of this weight is effectively used in the tanning industry. This results in a large amount of solid waste. Other process intermediates and by-products also have a significant impact on the environment. Hair removal is performed with the use of chemicals and usually generates about 60–70% of the total pollution from leather processing. The unhairing process produces high concentrations of environmentally toxic waste. Enzymatic processes using keratinase (the vast majority of which belong to the group of proteolytic enzymes) are now used for enzymatic environmentally friendly dehairing, replacing the traditional chemicals CaO and Na₂S [35]. Proteases have received special attention, due to their ability to hydrolyze keratin, which is an insoluble fibrous protein. Keratin is a major constituent of hair, feather, wool, and nails [36].

Proteases are increasingly being considered as a reliable alternative to conventional lime sulfide, to avoid problems caused by sulfide contamination. The main advantage of enzymatic unhairing is the complete elimination of lime and sulfide from the wastewater. Hair can be used for making many products such as brushes and carpets, and flesh is a main raw material for the glue/gelatin industry [37]. Hydrolyzed hair can also be used as an agricultural fertilizer and soil improver, in animal/poultry feed, and for the production of cosmetics, pharmaceuticals, and amino acids (e.g., cysteine) [38]. Proteases can be isolated from microorganisms (e.g., Bacillus sp., Aspergillus sp., and Amycolatopsis sp.). The greatest technological advantages of microbial proteases include low cost, high production, and efficient activity [39]. Enzymatic–oxidative unhairing does not contribute to the degradation of the hair and generates less organic waste with a lower pollutant load. The combined use of crude enzymatic extract and hydrogen peroxide has been shown to reduce processing time by hair saving (reducing hair destruction). The enzymatic extract was produced by cultivating the Bacillus subtilis strain BLBc 1 [40].

In the green unhairing cycle used in the leather industry, some keratinolytic proteases are used that exhibit high resistance to reducing agents (S²-, dithiothreitol, and β-mercaptoethanol) and are quite stable under extreme conditions [41]. High-alkalinity and high-concentration detergents are needed in the chemical unhairing process. Most enzymes are not stable under these conditions, except a few keratinolytic proteases [42]. Yongquan examined the enzymatic activity of extracellular alkaline protease from Bacillus cereus TD5B and its potential application as a sheep skin dehairing agent [43]. The Bacillus cereus TD5B strain was screened for extracellular alkaline protease production on skimmed milk agar media. Its alkaline protease activity was measured at concentrations of 1, 1.5, and 2%. The results showed that 2% alkaline protease had the highest enzymatic activity. The highest tensile strength of sheep leather was obtained after dehairing with 1% alkaline protease.

In addition to its undoubted advantages, including greater process efficiency, non-toxic sewage and sludge, and a more stable product, the use of keratinase or keratinolytic protease in the unhairing of hides in the tanning industry has the following three basic disadvantages [44]:

1. High cost of enzyme production with the inability to use the biocatalysts again.

2. The keratinolytic activity/caseinolytic activity (K/C) ratio, which evaluates substrate specificity, is below 0.5 in the case of most keratinolytic proteases and keratinases, which decreases unhairing efficiency and leather quality substantially.

3. There is no enzymatic technology that does not damage the hides in the unhairing process [44].

The latest scientific reports indicate that a novel serine protease is produced with the bio-enzyme G3726, which is heterologously expressed by the B. subtilis strain SCK6. With good stability at alkaline pH, the crude enzyme could efficiently and cleanly complete the dehairing of goatskins without damaging the skin collagen, while the leather obtained shows excellent physical properties. Overall, protease G3726 is an attractive candidate for industrial applications, especially in detergent formulations and leather manufacturing [45].

3.3 Bating

Bating is the process of the removal and hydrolysis of the keratose remaining on the surface of a de-limed pelt, as well as the removal of scud consisting of epidermis, hair roots, pigments, fats, sebaceous glands, and sweat gland debris. Generally, it is the process of removing proteins other than collagen, using proteolytic enzymes such as albumin or globulin [46]. In the conventional bating process, pelt bating is carried out in a deliming solution with the help of enzyme preparations (a mixture of protease and ammonium salt), at 30–37°C and pH 7.0–8.5 [47]. Commercially produced trypsin is considered the best enzyme for pelt bating, but it is mainly extracted from bovine pancreatic tissue, which has many disadvantages, such as purity, poor batch stability, unpleasant odor, potential cross-infection, the limited availability of the raw material, and damage to mammal immunogenicity [48]. The search for new enzyme products for bating processes in the leather industry has led to research into neutral and alkaline microbial proteases, which are a class of enzymes with promising industrial applications in pelt bating. However, in alkaline environments, these enzymes have little effect on hides and do not remove plaque and the remains of hair grains satisfactorily. Therefore, it has been suggested to use enzyme preparations that are active in acid environments [49]. The acid protease from Aspergillus usamii is known to be an effective bating agent for sheep pelts and has been found to be more effective than neutral protease [50]. Recent studies have investigated one-bath pickling bating chrome as a method of replacing the conventional trypsin method. The innovative pickling bating method based on microbial origin acidic protease L80A can provide outstanding performance for pelt bating [51].

3.4 Degreasing

The degreasing process is another stage in tanning. The quantities of natural fats in skin are very high, especially in some types of sheepskins: 20–30% of the total weight of skins is composed of fats [52]. The degreasing process can be broken down into the following three successive stages:

1. breakdown of the protein membrane of the fat-containing;

2. removal of the fat;

3. emulsification of the fat in water or solubilization in solvent.

Three-component processes using two enzymes (proteolysis, lipolysis, and emulsification) are used in the drift-greasing process for effective degreasing. Some authors suggest using alkaline lipase combined with proteinase and pancreatin to improve the degreasing effect and soften pig skin [27]. Kanagaraj et al. claims that a combination of enzymes might be necessary not only to break down products but also to release them from the hide [53]. Hydrolysis of ovine storage lipid using lipid hydrolases was demonstrated in an attempt to understand the mechanism of degreasing. Lipases were used as agents for degreasing. Lipases are known to play a critical role in leather processing and represent an environmentally sound method of removing fat. In the case of bovine hides, tensides can be replaced completely with lipases and surfactants. However, the disadvantages are that lipase does not remove all types of fats in the same way [16].

Ejaz et al. used LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) to degrease sheep skins [54]. The results showed that using only 10 mg of lipase/kg of raw hides enabled successful degreasing in only 15 min at pH 7. Comparative scanning electron microscope, attenuated total reflectance – fourier transform infrared spectroscopy, and physicochemical analyses showed that the enzymatically treated leather had better properties than the chemically treated leather. However, differences were observed in the composition of the wastewater, where the proportion of solid waste was higher for enzymatic degreasing than for conventional degreasing with chemicals.

3.5 Tanning/post-tanning

In the tanning process, collagen fibers are stabilized with tannins and with the help of tannin cross-links, so the hides are less prone to decay or rotting. The resistance of the leather to mechanical factors and to high temperatures increases. There are many different tanning methods and materials that may be chosen, depending on the required properties of the finished leather, the cost of the materials, the technology available in the plant, and type of raw material. Most tannins can be assigned to one of the following groups:

• mineral tannins,

• vegetable tannins,

• syntans,

• aldehydes,

• fatty tannins.

The most commonly used tanning agent is basic chromium sulfate (Cr(OH)SO₄). Currently, 80–90% of all hides are tanned with chromium salts [55]. The present study focused on the use in the tanning process of proteolytic enzymes during the wet finishing of leather and the dyeing of leather. The authors reported improvement in properties such as dye affinity, bath exhaustion, uniformity of color, dye penetration into the hide, wettability of the fiber, dye absorption, shrinkage, and tension in stretched wool fibers. In the study of Rakib et al. [56], an attempt was made to develop an eco-friendly vegetable tanning process combining pickle-free tanning and proteolytic enzymes. Leather dyeing is a post-tanning process, but it is worth noting that ecofriendly dyeing using enzymes to achieve increased uptake of dye can result in uniform dyeing with intense and bright shades. Staining with proteolytic enzymes also improves the absorption of the dye, which results in deeper shades of color. Researchers have attempted to develop an eco-friendly vegetable tanning process combining pickle-free tanning and the application of proteolytic enzymes [28]. The results showed more than 95% tannin exhaustion, an increase of 10% compared with the conventional vegetable tanning process.

3.6 New trends in enzyme treatment in leather industry

Proteases, also called proteinases, peptidases, or proteolytic catalysts, represent important enzymes commonly used in the leather industry. Proteases are a large group of enzymes belonging to the class of hydrolases, associated with the processing of long protein chains into short chains and the degradation of peptide bonds connecting amino acid residues [57].

According to historical data, proteases were classified based on source, molecular size, charge, substrate specificity, and catalytic activity. The division of proteases according to the mechanism of action, the species of microorganisms producing them, and the presence of amino acids in the active site is shown in Figure 5.

Figure 5

Division of proteases according to the mechanism of action, the species of microorganisms producing them, and the presence of amino acids in the active site [57].

The applications of proteases isolated from different microbial sources in different industries are represented in Table 1 [58].

Proteases are widely used as catalysts for various organic transformations; meanwhile, biocatalysis mainly involves the effective use of proteases as process catalysts in specific environments. Enzymatic biocatalysis has remarkable properties under moderate conditions, such as high activity, biodegradability, and specificity. Currently, extensive research focuses on the use of techniques that increase the stability of enzymes through, for example, the process of immobilization, aggregation, or changing their chemical structure [57].

Despite the enormous applications of enzymes as potential biocatalysts and process catalysts in various organic reactions, enzymes are associated with certain limitations such as high cost, lack of effective availability, instability, low substrate specificity, and requirement for multiple cofactors [33].

In the tanning industry, acidic proteases are of great interest in processes such as unhairing, as shown in Figure 6.

Figure 6

Mechanism of enzymatic unhairing at 100 and 400 µm [33].

Meanwhile, it should be noted that in recent years, there has been a huge increase in the use of alkaline protease as an industrial catalyst. The increasing use of alkaline proteases, not only in the tanning industry, is due to their biocatalytic activity in various organic transformations, namely, Knoevenagel condensation, aldol reaction, transesterification reaction, separation of amino acids in organic solvents, kinetic separation of carboxylic acids, and enantioselective hydrolysis of amino acid esters or fragmentation of p-amidobenzyl ethers [57].

Basic proteases constitute the largest group with optimal protease activity at neutral to alkaline pH and are serine or metal proteases. Alkaline proteases owe their success in commercial applications to the following features: compatibility with metal ions, resistance to commercially available detergents, and resistance to pH and temperature changes.

In leather tanning processes, during the chemical removal of hair, lime and sulfide act as a shaving blade, leaving the hair follicles intact [59]. These intact hair roots are then removed in the next skin wiping step. This is an important pre-tanning step where not only are hair follicles and other unwanted proteins removed from the de-haired skin, but they also help split the fiber into fibrils. Efficient rubbing of the skin results in high porosity and thumbprints; smoothness and better elasticity of treated leather can be obtained by using alkaline serine protease from B. subtilis ZMS-2. The enzyme at a concentration of 2% w/w effectively removes hair bulbs and undesirable non-structural proteins (globulin, albumin, elastin) from the skin without affecting the main structural protein – collagen [60]. Moreover, not all proteolytic enzymes due to their properties, they are suitable for combing/combing collagenolytic effect. During the de-bristle/de-combing process, proteases are selectively degraded soft keratin tissues inside the hair follicles, thus causing them to be pulled out intact hairs without disturbing the tensile strength of the processed leather. This hair removal at the root level is a very important stage of leather processing, which ensures the ideal smoothness and suppleness of the processed leather, which is influenced by this enzyme.

Most tanneries in Europe today use wet blue leather for processing. This is due to the avoidance of environmentally harmful leather tanning stages in the first stages of the wet workshop. However, wet blue leather is stored in warehouses and basements for a long time before it is processed further, which may cause the leather to dry out and thus make it more difficult for chemicals and enzymes to penetrate in the further stages of leather processing [61]. Therefore, the enzymatic process (i.e., treating wet blue with acid protease) is more widely used in tanneries to improve the comprehensive properties of leather. Acid proteases can hydrolyze proteins either internally or externally cause cleavage at low pH 2–4 to produce small peptides and amino acids [61]. The wet blue bath environment typically has a pH of around 3.5. Hence, acid proteases are the choice of enzyme preparations for moderate hydrolysis of skin protein and good dispersion of skin collagen fiber optical network. However, this process also has some drawbacks: acid protease proteins slowly penetrate the collagen structure (the process takes about 2 h), which significantly reduces the effectiveness of the process. Additionally, the large size of the enzyme molecules further delays the skin penetration process. A solution to this problem was proposed by Tochetto et al. who found that the surface charge of nanoparticles (NPs) was an important factor that affects their penetration into cancer cells and the penetration effect of NP was improved when NPs were negatively charged [62]. Tamilselvi et al. developed supramolecular nanocarriers with the ability to switch surface charges and found that the reversal of surface charge promotes penetration nanocarriers for skin biofilm [63]. The results suggested a slow penetration of acid protease into wet blue mainly due to the electrostatic attraction between the acid protease (usually negatively charged) and the wet blue surface (positively charged). Moreover, optimizing the regulation of the electrostatic interaction between wet blue and acid protease is a future direction of research to increase the effectiveness of wet blue processing in the wet workshop.

Research on the use of enzymes as leather tanning agents is inextricably linked to the need to reduce the use of chromium sulfate in this process. Although the development of various improved chromium tanning methods has significantly reduced the use of chemicals and water in leather tanning, this change is not sufficient due to the production of wastewater containing harmful chromium content. Additionally, there is strong evidence that when treated leather is disposed in the environment, some of the released trivalent chromium is converted to carcinogenic hexavalent chromium. Over the years, many sustainable alternatives to chromium tanning have been developed, based on chemical and enzymatic cross-linking, various bio-based polymers, enzymes, modified zeolites, and nanostructured materials. Proteases are arguably the most extensively utilized enzymes for dehairing hides and skins. Some researchers have utilized them in soaking, rehydration, pickling, and chrome tanning. The search for new protease sources prompted researchers to focus on enzyme engineering utilizing recombinant and immobilization technology [64].

3.7 Use of enzymes in the textile industry – in comparison with the tanning industry

The tanning industry and the textile industry are two sectors that are responsible for a large amount of chemical pollutants emitted into the environment. The use of enzymatic processes in the tanning and textile industries can save both industries from collapse caused by strict restrictions on the emission of pollutants into the environment. Despite similarities in the area of using enzymes as process-efficient molecules while saving energy, both industrial sectors struggle with different types of problems and use different types of enzymes, replacing chemical processes with enzyme-based technologies. In the tanning industry, the most widely used proteases are responsible for the breakdown of peptide bonds present in proteins and animal cells. In the textile industry, the enzymatic technology has a high application potential from the fiber treatment to products’ finalization [65]. In the textile preparation, the enzymes can modify the fibers’ structure, degrade the gum, remove natural pigments, give the fiber a white color, dye, give shine, and soften the textiles [66]. In terms of type, the global textile enzymes market is divided into enzymes such as cellulase, amylase, catalase, pectinase, and laccase.

Catalase and amylase constitute the greatest demand on the market. The cellulase segment has a significant share due to the increase in demand for cellulase in the so-called bio-polishing. Cellulases are used in the final stage of textile production: they are used to remove starch sizing and to soften and reduce the fabric’s tendency to pill. The use of enzymes in textile production significantly improves the quality of final products, including clothing.

Based on the applications in which enzymes are used quite extensively, the global market is segmented into:

• enzymes for bio-polishing fabrics (i.e., biological enzyme finishing, the purpose of which is to improve the surface finish of cotton fabric with cellulase to obtain a longer-lasting anti-adhesive effect, thereby increasing the overall smoothness and softness of the fabric);

• starch-sizing enzymes (pre-treatment of cotton);

• bleaching enzymes (including cotton enzymes, which are necessary to remove brown and yellow discoloration of cotton fibers caused by cotton flower proteins and pigments);

• bioleather/bioskin enzymes.

Increasing demand for textile enzymes in the apparel industry and increasing application to bring technological and manufacturing improvements in various end-use industries are the key factors in the textile enzyme market. Rapid industrialization and urbanization and growing environmental concerns are expected to drive the demand for textile enzymes in the near future. Similar predictions regarding the demand for enzymes are observed in the leather product market, but the sizes of both industries are significantly different, with an advantage for the textile sector. This does not change the fact that in both these industries, intensive work is underway on the use of enzymes instead of chemical technologies [67]. A representation of enzyme application is shown in Figure 7.

Figure 7

Diagram of textile processing steps where enzymes can be industrially used [67].