Studies On The Depilation By Carbohydrase And Protease And Their Mechanism

Leather industry is an important industry for China, which earns a lot of money from international market. At the same time, it's a heavy pollution industry. Especially, unhairing process produces the heaviest pollution among the whole leather industry. With the development of China, the environmental pressure is increasing. To some degree, environmental problem is a fatal problem to the leather industry.Enzymatic unhairing is a promising solution among many clean techniques. The main difficulty is that proteolysis can not be controlled and thus the extension of the enzyme activity towards the desired reticular structure of the dermis can not be avoided, notably impairing the properties of the elaborated leather. This research aims at reducing the impairment.It is believed that the collagenases which exist in proteases result in the impairment. One collagenase was separated from 2709 alkaline protease and purified. The molecular weight is 56kD, and the collagenase was found to be a metalloprotease. The result implies that collagenase may contribute to the impairment.The analysis of carbohydrates in enzymatic unhairing bath is important to the study of unhairing mechanism. There are 50% of hydroxypolysaccharide and 50% of aminopolysaccharide in the bath. But the previous methods are only effective for hydroxypolysaccharide. Therefore, acetyl acetone method was chosen to determine aminopolysaccharide. According to our research, phenol-sulfuric acid method is simple, rapid and suitable. Since phenol-sulfuric acid is only effective for 2-hydroxypolysaccharide, the results obtained from this method should be corrected. Moreover, acetyl acetone method can be used to calculate the molecular mass of the carbohydrates in the bath. It is helpful for the mechanism study of enzymatic unhairing.By analysis of protein, carbohydrate and collagen in the enzymatic bath, it was found that protease has good unhairing capacity, but it also impairs pelt greatly. By attacking the core protein in the mucoid, protease fulfills the depilation. The hydrolysis of carbohydrates is indirect and inefficient.2709 alkaline protease is a serine protease, and the presence of collagenase is a metalloprotease. Therefore, 10mM EDTA can inhabit half of collagenase activity, while it has little effect on ordinary protease activity. It was found that the unhairing capacity was unaffected, while the impairment was reduced by 15-20%. This phenomenon also implies that collagenase is not the only reason for the impairment.At last, some new explanations about the causes of impairment were put forward. The collagenase and protease have a synergistic effect on the impairment of pelt. The protease can attack the non-helical regions of collagen. Since the contents of hydroxyproline are low in this region, the data of collagen calculated from hydroxyproline were low. In fact, the impairment is heavy.Little was known about the depilation by carbohydrases, and the function of carbohydrases was disputed. The main subject of this paper was the depilation by carbohydrases. By analysis of the composition of the bath from enzymatic unhairing, amino acid analysis and FTIR analysis, the mechanism of carbohydrase(amylase and cellulase) was investigated. Carbohydrase can hydrolyse mucoids (proteoglycans) by attacking the carbohydrate chains in mucoids. The action of carbohydrases on the carbohydrate chains in mucoids is stronger than that of proteases. It was found that carbohydrases can open up the skin fibril and have some unhairing activity. But their unhairing capacity is inferior to proteases. Due to their large volume, it's difficult for them to penetrate the skin. The diffusion difficulty leads to their lower unhairing activity. On the other hand, they do little impairment to pelt. The composition of amino acid proved that carbohydrases do little harm on the collagen, and FTIR again testified that the action of carbohydrases is obvious.It is difficult to assess the contribution of enzyme to depilation. In this paper, protease and carbohydrases were mixed, and the actual activities were used as independents. The amount of protein released from the skin tissue was determined and used as an indicator of depilatory activity. At the same time, the synergistic effect was concerned. By mathematical analysis of the depilation by combinations of carbohydrase and protease, it was found that both carbohydrase and protease play an important role in unhairing, and the two kinds of enzymes have a significant synergistic effect. Especially, in the dual system of amylase and protease, the standardized coefficient of synergistic effect was 0.894, while that of protease was 0.183. The depilation by combinations of carbohydrase and protease again proved that it is protease that impairs the pelt, not carbohydrase.At last, amylase and neutral protease were used to unhair jointly. The pelt was treated with amylase for four hours, then protease was added, and the process continued for another two hours. The optimal condition is: amylolytic activity 40u/ml; proteolytic activity 400u/ml; pH 6.95-7.35. Compared with the conventional protease depilation, the leather produced by this method has better physical properties.Ultrasound is another clean technology. The joint application of enzyme and ultrasound has not been reported. According to our experiments, ultrasound has little effect on the enzyme activity of 2709 alkaline protease. Ultrasound can accelerate the enzymatic unhairing by enhancing the enzymatic diffusion through skins. Moreover, the effect of acceleration is more obvious to amylase than to protease. The reason is that the volume of amylase is larger, and the diffusion of amylase is more difficult. In these experiments, the less impairment caused by amylase was testified again.Ultrasound also can accelerate the hydrolysis of the untanned leather waste. According to Lineweaver-Burke equation, the kinetic parameters were obtained. The increasing of K_A proved the acceleration of ultrasound, while K_M which remained constant implied that the affinity of enzyme to the substrate was unchanged. The ultimate conversion ratio without ultrasound was 54.7-62.2%, whereas, with ultrasound, was 81.7-88.4%. It indicated that the enzymatic hydrolysis of skin with ultrasound gives about 46% increase in the ultimate conversion ratio.