糖酶、蛋白酶脱毛技术及其机理研究
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摘要
制革行业是我国出口创汇的重要行业,但同时也是一个污染严重的行业,尤以脱毛工序为最。随着我国经济的发展,人民生活水平的提高,对环境保护的要求越来越高,在某种意义上,环保问题是关系到皮革产业生死存亡的大问题。
脱毛工序有许多清洁工艺,酶法脱毛无疑是解决问题的最有前景的途径。酶法脱毛技术上的困难是,脱毛条件难以控制,容易造成对皮的损伤。本研究旨在研究蛋白酶、糖酶的脱毛机理,希望在脱毛同时,减少对皮的损伤,以利于酶法脱毛的推广。
蛋白酶在脱毛的同时会对皮造成损伤,国内普遍认为是其中的胶原酶的缘故,本研究首次对2709碱性蛋白酶进行分离、纯化,通过丙酮沉淀、Sephadex G-100柱层析、DEAE-Sephadex A-50柱层析、Sephadex G-75柱层析等方法,分离、纯化了2709碱性蛋白酶中的胶原酶。SDS-聚丙烯酰胺凝胶电泳测定其分子量为56kD,并为金属蛋白酶。该结果证明碱性蛋白酶2709中存在胶原酶,它可能是2709脱毛时,引起皮损伤的一个重要原因。
脱毛液中总糖含量的分析,对研究脱毛机理有重要意义。比较了常用的多种测定糖含量的方法。脱毛液中含有羟基糖和氨基糖,两者各为一半,但常用的方法测定的仅仅是羟基糖,氨基糖部分被忽略了,结果是不完备的。为此,本研究选择对氨基糖选择性强的乙酰丙酮法进行了研究。结果表明,DNS法在酶法脱毛液中测定结果明显偏高,该方法不适用本体系。而硫酸-苯酚法和乙酰丙酮法结果相吻合,说明两种方法均可行。硫酸-苯酚法可测定总的羟基糖,该法简便、快捷,其结果×(?)可以表示总糖含量(以硫酸皮肤素计)。乙酰丙酮法测定氨基糖选择性好,但方法较烦琐。通过比较脱毛液HCl水解前后的氨基糖含量,可以估算脱毛液中的糖链平均大小,该法结果和HPGFC法接近,方法简单。因此,可以用乙酰丙酮法来估算脱毛液中糖链的大小,这为酶法脱毛机理的研究提供了新的思路。
通过分析蛋白酶脱毛液中总蛋白质含量、总糖含量、胶原蛋白含量,表明蛋白酶脱毛效果好,但损伤大。脱毛液中溶出糖的糖链大小在17.8～22.7,说明脱落下来的多糖有断裂,但水解不够彻底。蛋白酶通过水解蛋白多糖中的核心蛋白来破坏粘蛋白(类粘蛋白),从而降低其粘度,达到脱毛的目的,其对多糖的水解是间接的、效能不高的。
由于2709为丝氨酸蛋白酶,而其中存在的胶原酶是金属蛋白酶,用EDTA可抑制2709中的胶原酶活性,10mM EDTA可抑制约50%的胶原酶活性,而对普通蛋白酶活性影响不大。脱毛实验表明,加入10mM EDTA不影响2709的脱毛效果,而胶原蛋白含量可减少15-20%。同时,也揭示2709并非损伤胶原纤维的唯一原因。
探讨了蛋白酶脱毛机理及对皮损伤的原因,提出了新的见解。认为胶原酶和蛋白酶对皮的损伤有协同作用。蛋白酶作用于胶原蛋白的非螺旋区域,由于该区域羟脯氨酸含量较少,导致测定出来的胶原蛋白含量很少,但对皮的实际损伤较大。
糖酶脱毛作用研究的很少,且对其作用认识不一。本研究重点研究了糖酶的脱毛作用。通过分析脱毛液中的组成、氨基酸组成分析、FTIR分析,探讨了糖酶(淀粉酶、纤维素酶)脱毛机理。总蛋白质含量和总糖含量表明糖酶对皮的作用是明显的,糖酶是通过水解粘蛋白(蛋白多糖)中的多糖链来起作用,其对糖的水解能力要强于蛋白酶。脱毛液中溶出糖的糖链大小在8.2～10.2,说明糖酶对脱落下来的多糖水解彻底。脱毛实验表明糖酶有较好的松散胶原作用,有一定的脱毛效果,但其脱毛效果明显弱于蛋白酶。其优点是对皮的损伤小,使用安全性好。氨基酸组成分析表明糖酶对皮的损伤很小。FTIR扫描说明糖酶对皮有作用。影响糖酶脱毛效果的重要因素是糖酶分子量大,酶在皮中渗透困难,影响了其脱毛效果。对碱膨胀和超声波作用对酶在皮中渗透速度的影响,进行了分析,验证了这一观点。
对酶脱毛作用的贡献大小进行分析是一件困难的工作。本章将蛋白酶和糖酶混合后,以实际测得的酶活力作为变量,以脱毛效果(用脱毛液中总蛋白质浓度表征)作为因变量,进行回归分析,同时考虑交互作用(即代表协同作用)。这样可以定量考察糖酶、蛋白酶各自的脱毛能力,以及它们的协同作用大小。
用回归分析方法,对糖酶和蛋白酶混合作用皮的情况进行了定量分析。结果表明糖酶和蛋白酶各自均起作用,并附加有明显的协同作用。尤以淀粉酶和蛋白酶的协同效应特别明显,其中协同作用的标准化系数为0.894,而蛋白酶活的标准化系数仅为0.183。混合酶脱毛实验再次证明蛋白酶对皮有损伤,而蛋白酶中加入淀粉酶对皮的损伤影响不大。
利用淀粉酶先作用于皮,再用蛋白酶作用,可以充分利用淀粉酶的作用和两种酶的协同作用,减少蛋白酶的作用强度,达到既较好脱毛,又减少对皮的损伤的目的。用响应面设计优化了混和脱毛的工艺条件,优化的工艺条件是:淀粉酶活40u/ml、蛋白酶活400u/ml,pH值为6.95～7.39。和普通蛋白酶脱毛方法进行了比较。在脱毛效果相仿的情况下,混合酶法损伤小,成革物理性能较优。
超声波和酶在皮革中的联合应用尚未见诸报道。研究了超声波对蛋白酶活力的影响,发现2709所受影响极小。研究发现,淀粉酶、蛋白酶脱毛时,使用超声波照射,可以明显缩短脱毛所需时间,原因在于超声波能促进酶在皮中的渗透,尤以淀粉酶为最。因为淀粉酶分子量大,较难渗透,故超声波的促进渗透作用反而较大。该实验同时也验证了淀粉酶对胶原蛋白的水解极弱,使用淀粉酶脱毛安全性优势明显。超声波在淀粉酶体系中只起促进渗透作用,而不能促进淀粉酶对胶原的水解。
当用蛋白酶酶法处理未铬鞣的废皮时,采用超声波照射,可以提高反应速率。通过Lineweaver-Burke公式可计算出动力学参数,K_A增加,说明反应速率增加;K_M几乎不变,说明酶和底物的亲和力没有改变。不加超声波的最终转化率为54.7～62.2%,而加超声波则为81.7～88.4%。这说明加超声波,酶水解废皮的回收率提高了46%,效果是很明显的。
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.
脱毛工序有许多清洁工艺,酶法脱毛无疑是解决问题的最有前景的途径。酶法脱毛技术上的困难是,脱毛条件难以控制,容易造成对皮的损伤。本研究旨在研究蛋白酶、糖酶的脱毛机理,希望在脱毛同时,减少对皮的损伤,以利于酶法脱毛的推广。
蛋白酶在脱毛的同时会对皮造成损伤,国内普遍认为是其中的胶原酶的缘故,本研究首次对2709碱性蛋白酶进行分离、纯化,通过丙酮沉淀、Sephadex G-100柱层析、DEAE-Sephadex A-50柱层析、Sephadex G-75柱层析等方法,分离、纯化了2709碱性蛋白酶中的胶原酶。SDS-聚丙烯酰胺凝胶电泳测定其分子量为56kD,并为金属蛋白酶。该结果证明碱性蛋白酶2709中存在胶原酶,它可能是2709脱毛时,引起皮损伤的一个重要原因。
脱毛液中总糖含量的分析,对研究脱毛机理有重要意义。比较了常用的多种测定糖含量的方法。脱毛液中含有羟基糖和氨基糖,两者各为一半,但常用的方法测定的仅仅是羟基糖,氨基糖部分被忽略了,结果是不完备的。为此,本研究选择对氨基糖选择性强的乙酰丙酮法进行了研究。结果表明,DNS法在酶法脱毛液中测定结果明显偏高,该方法不适用本体系。而硫酸-苯酚法和乙酰丙酮法结果相吻合,说明两种方法均可行。硫酸-苯酚法可测定总的羟基糖,该法简便、快捷,其结果×(?)可以表示总糖含量(以硫酸皮肤素计)。乙酰丙酮法测定氨基糖选择性好,但方法较烦琐。通过比较脱毛液HCl水解前后的氨基糖含量,可以估算脱毛液中的糖链平均大小,该法结果和HPGFC法接近,方法简单。因此,可以用乙酰丙酮法来估算脱毛液中糖链的大小,这为酶法脱毛机理的研究提供了新的思路。
通过分析蛋白酶脱毛液中总蛋白质含量、总糖含量、胶原蛋白含量,表明蛋白酶脱毛效果好,但损伤大。脱毛液中溶出糖的糖链大小在17.8～22.7,说明脱落下来的多糖有断裂,但水解不够彻底。蛋白酶通过水解蛋白多糖中的核心蛋白来破坏粘蛋白(类粘蛋白),从而降低其粘度,达到脱毛的目的,其对多糖的水解是间接的、效能不高的。
由于2709为丝氨酸蛋白酶,而其中存在的胶原酶是金属蛋白酶,用EDTA可抑制2709中的胶原酶活性,10mM EDTA可抑制约50%的胶原酶活性,而对普通蛋白酶活性影响不大。脱毛实验表明,加入10mM EDTA不影响2709的脱毛效果,而胶原蛋白含量可减少15-20%。同时,也揭示2709并非损伤胶原纤维的唯一原因。
探讨了蛋白酶脱毛机理及对皮损伤的原因,提出了新的见解。认为胶原酶和蛋白酶对皮的损伤有协同作用。蛋白酶作用于胶原蛋白的非螺旋区域,由于该区域羟脯氨酸含量较少,导致测定出来的胶原蛋白含量很少,但对皮的实际损伤较大。
糖酶脱毛作用研究的很少,且对其作用认识不一。本研究重点研究了糖酶的脱毛作用。通过分析脱毛液中的组成、氨基酸组成分析、FTIR分析,探讨了糖酶(淀粉酶、纤维素酶)脱毛机理。总蛋白质含量和总糖含量表明糖酶对皮的作用是明显的,糖酶是通过水解粘蛋白(蛋白多糖)中的多糖链来起作用,其对糖的水解能力要强于蛋白酶。脱毛液中溶出糖的糖链大小在8.2～10.2,说明糖酶对脱落下来的多糖水解彻底。脱毛实验表明糖酶有较好的松散胶原作用,有一定的脱毛效果,但其脱毛效果明显弱于蛋白酶。其优点是对皮的损伤小,使用安全性好。氨基酸组成分析表明糖酶对皮的损伤很小。FTIR扫描说明糖酶对皮有作用。影响糖酶脱毛效果的重要因素是糖酶分子量大,酶在皮中渗透困难,影响了其脱毛效果。对碱膨胀和超声波作用对酶在皮中渗透速度的影响,进行了分析,验证了这一观点。
对酶脱毛作用的贡献大小进行分析是一件困难的工作。本章将蛋白酶和糖酶混合后,以实际测得的酶活力作为变量,以脱毛效果(用脱毛液中总蛋白质浓度表征)作为因变量,进行回归分析,同时考虑交互作用(即代表协同作用)。这样可以定量考察糖酶、蛋白酶各自的脱毛能力,以及它们的协同作用大小。
用回归分析方法,对糖酶和蛋白酶混合作用皮的情况进行了定量分析。结果表明糖酶和蛋白酶各自均起作用,并附加有明显的协同作用。尤以淀粉酶和蛋白酶的协同效应特别明显,其中协同作用的标准化系数为0.894,而蛋白酶活的标准化系数仅为0.183。混合酶脱毛实验再次证明蛋白酶对皮有损伤,而蛋白酶中加入淀粉酶对皮的损伤影响不大。
利用淀粉酶先作用于皮,再用蛋白酶作用,可以充分利用淀粉酶的作用和两种酶的协同作用,减少蛋白酶的作用强度,达到既较好脱毛,又减少对皮的损伤的目的。用响应面设计优化了混和脱毛的工艺条件,优化的工艺条件是:淀粉酶活40u/ml、蛋白酶活400u/ml,pH值为6.95～7.39。和普通蛋白酶脱毛方法进行了比较。在脱毛效果相仿的情况下,混合酶法损伤小,成革物理性能较优。
超声波和酶在皮革中的联合应用尚未见诸报道。研究了超声波对蛋白酶活力的影响,发现2709所受影响极小。研究发现,淀粉酶、蛋白酶脱毛时,使用超声波照射,可以明显缩短脱毛所需时间,原因在于超声波能促进酶在皮中的渗透,尤以淀粉酶为最。因为淀粉酶分子量大,较难渗透,故超声波的促进渗透作用反而较大。该实验同时也验证了淀粉酶对胶原蛋白的水解极弱,使用淀粉酶脱毛安全性优势明显。超声波在淀粉酶体系中只起促进渗透作用,而不能促进淀粉酶对胶原的水解。
当用蛋白酶酶法处理未铬鞣的废皮时,采用超声波照射,可以提高反应速率。通过Lineweaver-Burke公式可计算出动力学参数,K_A增加,说明反应速率增加;K_M几乎不变,说明酶和底物的亲和力没有改变。不加超声波的最终转化率为54.7～62.2%,而加超声波则为81.7～88.4%。这说明加超声波,酶水解废皮的回收率提高了46%,效果是很明显的。
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.
引文
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