Enzymatic removal of proteoglycans in hides promotes opening-up of collagen fibre bundles

Introduction
When a hide loses moisture, the proteoglycans present in the hide stick the collagen fibres together. This negatively impacts the penetration of water and other chemicals into the hides during processing, and also the opening-up of the collagen fibres and hence the softness of the leather. It is therefore ideal to remove proteoglycans from the hides as soon as possible in the leather making process.
Proteoglycans are chemically stable compounds, and only about half of them are removed during the liming process.
Glycosidase enzymes have great application in the tanning industry. These enzymes hydrolyse glycosidic bonds and the sugar chain, thus removing proteoglycans from hides. However, there are a wide variety of these enzymes available commercially, and they differ in the way they affect proteoglycans. There has been a lack of research carried out investigating the application of glycosidase enzymes to the actual processing stages when making leather.
In this research paper, 7 typical commercially available glycosidases were used to remove proteoglycans from hides. They were applied in the soaking process and the relationship between the dosage of enzyme and the degree of proteoglycan removal and leather properties were investigated.

Experimental
Wet salted hides were used in this study. 7 different commercially available glycosidase enzymes were chosen to determine their effect on proteoglycan removal from hides.
After pre-soaking and fleshing, wet salted hides were soaked in 100% water, 0.2% of a soaking auxiliary and differing amounts of glycosidase enzyme. The drum was run for 1 hour, then intermittently for 2 hours, and then allowed to stand overnight. The following day, the total sugar, total protein, hydroxyproline and glycosaminoglycan concentrations in the float were measured after a further 30 minutes of running the drum.
The physical properties of the leather produced were carried out according to standard methods.

Results
7 different commercially available glycosidase enzymes that showed potential to remove proteoglycans from collagen hide powder in prior laboratory studies were tested for their ability to remove proteoglycans from actual wet salted hides. These 7 glycosidase enzymes were ß-Mannase-AKT, Cellulase-AKT, Hyaluronate lyase-AKT, ß-Glucosidase-AKT, Xylanase-AKT, Xylanase-LKT and ß-Glucanase-AKT.

The effect of pH on the activity of glycosidases
Most glycosidase enzymes function optimally at acidic pH’s. This can be a problem as most beamhouse processes take place at alkaline pHs. The enzyme activity of the 7 glycosidase enzymes was measured over the pH range 4 – 9.
The results in Figure 1 show that although the optimum activity of the 7 glycosidases is acidic, most of the enzymes still maintain more than 50% of activity in the pH range 7 – 8. Therefore, they have the potential to be applied in weakly alkaline processes, such as soaking or bating.

Figure 1. Effect of pH on the glycosidase activity (30°C)

Effect of temperature on the activity of glycosidases
Collagen is sensitive to heat, and high temperatures can cause collagen denaturation. The variation of glycosidase activity was investigated in the range 20 - 40°C. The results in Figure 2 show that as the temperature increases, the activity of the glycosidase enzymes increases. At the soaking temperature of 25°C, the enzymes are active, but the tanner may need to increase the concentration of the enzyme used to get optimal proteoglycan removal.

Figure 2. Effect of temperature on the glycosidase activity (pH 7.5)

Produced amount of total sugars and glycosaminoglycans in the soaking float
At the end of the soaking process, the total amount of sugar found in the soaking float was used to determine the removal effect of polysaccharides by the glycosidase enzymes. The glycosidases specifically destroy the polysaccharide chains (proteoglycans), producing small sugar chains. Proteoglycans consist of a core protein chain and one or more glycosaminoglycan (GAG) chains. GAG is therefore also an indicator of proteoglycan destruction in the hide. Figure 3 shows that ß-Mannase-AKT and Xylanase-AKT produced the highest amounts of sugar and GAG in the soaking float. 

Figure 3. Total sugar and glycosaminoglycan content in the soaking floats

The amounts of soluble protein and hydroxyproline in the soaking floats after being treated with the different glycosidase enzymes are shown in Figure 4. The results show that the content of soluble protein in the glycosidase-soaked hides is much higher than the control. The breakdown of the proteoglycan structure results in soluble protein being produced, and this was evidence that these enzymes were destroying the proteoglycan in the hide.
Hydroxyproline is an indicator of collagen destruction. Figure 4 shows that the concentration of hydroxyproline in the soak floats was slightly higher for the hides soaked with the glycosidase enzymes compared to the control, but that overall, the concentration was low, indicating that the glycosidases only have a weak negative effect on the collagen. 

Figure 4. Concentration of soluble protein and hydroxyproline in the soaking floats

The grain surface of the wet blue and crust leather produced was observed after being soaked with different concentrations of glycosidases, and there was no difference in the grain surface of the leather treated with the glycosidase enzymes compared with the control. This indicated that the application of glycosidase enzymes in the soaking process was a safe and efficient method of removing proteoglycans from hides as the enzymes have very little effect on the collagen fibres.

Physical and organoleptic properties of the crust leather
The physical and organoleptic properties of the crust leather produced by using the two most promising of the glycosidase enzymes at different concentrations in the soaking process are shown in Table 1. The results show that, compared with the control, the softness of the crust leather was improved after being treated with the two different glycosidases. The main physical properties of the crust leather were also improved. The improvement is caused by the removal of proteoglycan and better opening up of the collagen fibres.  

Table 1. Physical and organoleptic properties of the crust leather

Conclusion
The results of this research showed that of the 7 glycosidase enzymes tested, 2 of them, ß-Mannase-AKT and Xylanase-AKT, were most effective in removing proteoglycans from the hide. This removal significantly improves the opening-up of collagen fibres, the softness and the physical properties of the crust leather. The use of glycosidase in the soaking process does not cause excessive damage to collagen. Through optimal selection and optimization of glycosidase enzyme concentration in the soaking process, it is possible to safely remove proteoglycans from hides.

This article is a summary of the paper “Removal of Proteoglycans in Animal Hides by Glycosidase to Promote Opening-Up of Collagen Fiber Bundles: Optimization of Glycosidase and its Application in the Soaking Process.” Journal of the American Leather Chemists Association (JALCA) Vol 118, 75 - 86 (2023).