【作者】 刘畅；

【导师】 曾运航；

【作者基本信息】 四川大学 ， 轻工技术与工程， 2023， 硕士

【摘要】 面对制革准备及鞣制工段污染负荷高、处理难度大的问题,近年来采购蓝湿革直接进行成品革生产的制革企业逐年增加。但是,不同厂商、批次的蓝湿革往往纤维分散程度存在明显差异,甚至贮存过程中蓝湿革可能出现纤维板结的缺陷。通过酸性蛋白酶适度水解蓝湿革的蛋白质,可达到松散胶原纤维、打开粒面以及减小蓝湿革批次差异的目的。然而,因为皮组织结构的复杂性和酶促反应的高效性致使蛋白酶在蓝湿革内传递慢且反应快,鞣后软化过程不仅耗时长,而且容易出现革表面和内层作用不均匀的问题。针对上述问题,本论文聚焦研究表面电荷对酸性蛋白酶在蓝湿革中传质行为和软化效果的影响,以期为制革过程更加合理地选用及应用酸性蛋白酶进行快速、均匀的蓝湿革软化提供科学指导。论文第一部分选取胃蛋白酶(从猪胃粘膜中提取)、酸性蛋白酶制剂537(生产菌为宇佐美曲霉,记为AP-1)和酸性蛋白酶制剂T(生产菌为里氏木霉,记为AP-2)进行蓝湿革软化实验。这三种酶在35~40°C、pH 3.5和Cr3+浓度0~500mg/L的溶液中稳定性较高;等电点(pI)分别为1.9、2.4和3.6,pH 3.5时溶液的Zeta电位分别为-4.0、-2.5和0.4 m V;相对分子质量分别为35、40和35 k Da。考察了这三种酸性蛋白酶在蓝湿革中的渗透效果,结果表明:与粒径和酶活力相比,酸性蛋白酶的表面电荷是影响酶传质行为的重要因素。AP-2酶在蓝湿革中的渗透效果最佳,因此它分散胶原纤维及使蓝湿革变软的性能也优于胃蛋白酶和AP-1。这是因为带负电荷的胃蛋白酶和AP-1酶在软化pH为3.5时容易与带正电荷的蓝湿革静电结合而停留在革表层中,难以向革内渗透。由此可见,选择pI高于软化pH的酸性蛋白酶对于获得良好的渗透效果和软化性能是必要的。进一步选用pI 8.75(明显高于软化pH 3.5)且在酸性条件下也有催化活性的木瓜蛋白酶作为研究模型,通过丁二酸酐、柠檬酸、邻苯二甲酸酐,马来酸酐修饰木瓜蛋白酶制备了在pH 3.5时正电荷量不同的4种修饰蛋白酶,然后用其软化蓝湿革,探究了酸性蛋白酶所带正电荷数量对其传质行为和软化效果的影响。结果表明,所选的酸酐修饰剂对木瓜蛋白酶活力和粒径影响较小,随着木瓜蛋白酶正电荷数量的增加,木瓜蛋白酶在蓝湿革中的渗透率反而下降,这说明较强的酶-革静电斥力也不利于酶的渗透。因此,选择pH略高于软化pH的酸性蛋白酶或尽量减小酶-革静电相互作用应该是强化酶传质的有效途径。论文的第二部分考察了制革助剂甲酸钠和中和单宁(含酚羟基和磺酸基)对酸性蛋白酶在蓝湿革中传质行为和软化效果的影响。结果表明:甲酸钠通过提高体系的pH,中和单宁通过与酶分子、胶原纤维的相互作用引入阴离子基团,均能增加酸性蛋白酶分子的负电荷数量,降低蓝湿革的正电荷量。使用中和单宁更利于减少酶溶液Zeta电位(负值)与蓝湿革Zeta电位(正值)的差值,即更利于减小酶分子与蓝湿革胶原纤维之间的静电引力。用荧光示踪技术继续考察上述助剂对酸性蛋白酶在蓝湿革内传质行为的影响,实验数据进一步证实了酶溶液与蓝湿革的Zeta电位之差越小,酶在蓝湿革中渗透越快且分布越均匀。酸性蛋白酶溶液在蓝湿革中的快速渗透和均匀分布也确实提升了皮革的酶软化效果。综上所述,酸性蛋白酶的表面电荷性质是影响其在蓝湿革内传质行为的重要因素,减小酸性蛋白酶与蓝湿革之间的静电相互作用,对提升酸性蛋白酶在蓝湿革内的渗透速率和分布均匀性,改善酶软化效果和皮革品质具有重要意义。通过外加助剂或对酶表面进行化学修饰是调节酶-皮革静电相互作用的有效方法,值得进一步研究。更多还原

【Abstract】 Faced with the high pollution load and difficult pollution treatment in the beamhouse and tanning processes,the tanneries that directly purchase wet blue for finished leather production has increased year by year.However,there are usually great differences in the fiber dispersion degree of wet blue among different tanneries and batches,and the wet blue may even have defects in fiber bonding during storage.Loosening collagen fibers,opening grain surfaces,and reducing batch difference of wet blue can be achieved by moderately hydrolyzing the protein of wet blue with acid protease.However,because the complexity of tissue structure of hides/skins and the high efficiency of protease catalysis result in the slow transfer and rapid reaction of protease in wet blue,the bating in the post-tanning process takes a long time and is prone to uneven reaction between the surface layer and the inner layer of leather.To solve the issues above,this thesis focused on investigating the effect of surface charge on the mass transfer behavior and bating performance of acid proteases in wet blue,aiming at providing scientific guidance for more reasonable selection and application of acid proteases for rapid and uniform wet blue bating in the leather manufacturing.In the first part of this thesis,pepsin(extracted from pig gastric mucosa),acid protease preparation 537(produced by Aspergillus,recorded as AP-1),and acid protease preparation T(produced by Trichoderma reesei,recorded as AP-2)were used for wet blue bating experiments.The three proteases exhibited high stabilities in solutions with pH of 3.5 and Cr3+ concentration of 0–500 mg/L at 35–40℃.Their isoelectric points(pI)were 1.9,2.4,and 3.6,respectively,and the zeta potentials of their solution at pH 3.5 were-4.0,-2.5,and 0.4 m V,respectively.Their relative molecular weights were 35,40,and 35 k Da,respectively.The penetration of the three acid proteases in wet blue were studied,and the results indicated that the surface charge of acid protease is a more important factor affecting the mass transfer behavior of protease compared with the particle size and protease activity.AP-2 had the best penetration performance in the wet blue,therefore its performances in dispersing collagen fibers and softening wet blue were better than those of pepsin and AP-1.This was because the negatively charged pepsin and AP-1 could combine easily with the positively charged wet blue at the bating pH of 3.5 and stayed in the surface layer rather than transferred into the inner layer of the wet blue.Therefore,it is necessary to choose an acid protease with a pI higher than the bating pH for deep penetration and satisfactory bating performance.Further,papain with pI 8.75(much higher than the bating pH of 3.5)and catalytic activity under acidic condition was selected as the research model.Four modified proteases with different positive charges at pH 3.5 were prepared by modifying papain with succinic anhydride,citric acid,phthalic anhydride,and maleic anhydride,and then were used to bate wet blue.The effect of the number of positive charges carried by acid proteases on the mass transfer behavior and bating performance of acid protease was investigated.The results showed that the selected anhydride modifier had little effect on the activity and particle size of papain.The penetration rate of papain in the wet blue decreased with increasing number of positive charges of papain,indicating that the strong electrostatic repulsion between protease and leather also had an adverse effect on the penetration of protease.Therefore,choosing an acid protease with pI slightly higher than the bating pH or minimizing electrostatic interaction between protease and leather should be effective methods for enzyme mass transfer enhancement.The second part of this thesis investigated the effects of leather auxiliaries such as sodium formate and neutralizing syntan(containing phenolic hydroxyl and sulfonic groups)on the mass transfer behavior and bating performance of the acid protease in wet blue.The results showed that the negative charges of protease molecules were increased and the positive charges of wet blue were reduced by using sodium formate or neutralizing syntan,because sodium formate can increase the pH of the system,and neutralizing syntan can introduce anionic groups into protease molecules and collagen fibers through interactions.Neutralizing syntan was more conducive to reducing the difference between the zeta potential(negative value)of protease solution and the zeta potential(positive value)of wet blue.This meant that neutralizing syntan was more beneficial to reducing the electrostatic attraction between protease molecules and collagen fibers in wet blue.Then,the effect of the above auxiliaries on the mass transfer behavior of acid protease in wet blue was studied by using a fluorescence tracing technology.The experimental data further confirmed that slighter zeta potential difference between the protease solution and the wet blue could lead to more rapid penetration and more uniform distribution of the protease in the wet blue.The rapid penetration and uniform distribution of acid protease in wet blue indeed improved the bating performance of leather.In summary,the surface charge of acid proteases is an important factor affecting their mass transfer behavior in wet blue.Reducing the electrostatic interaction between acid protease and wet blue is of great significance for increasing the penetration rate and distribution uniformity of acid protease in wet blue,improving the enzymatic bating performance and the leather quality.Addition of auxiliaries and chemical modification of protease surface are effective methods for regulating the electrostatic interaction between protease and leather,which deserves further study.更多还原