【作者】 王小花；

【导师】 陆大年； 朱利民； 洪枫；

【作者基本信息】 东华大学 ， 纺织化学与染整工程， 2006， 博士

【摘要】 涤纶（poly（ethylene terephthalate），PET）是对苯二甲酸（酯）和乙二醇的缩聚物，亲水性和抗静电性较差。因此，当用作服用性纤维时，有必要对其进行改性。作为一种温和、环保且不损害涤纶原有的优良性能的改性方法，近年来，涤纶酶法改性开始受到人们的注意。就目前研究状况来看，涤纶酶法改性主要存在以下问题：一是所用的酶大多购自酶制剂公司，对涤纶的改性不具针对性，酶对涤纶的催化活性不高，改性结果不理想，因此需要寻找新的酶或新的产酶菌株；二是由于改性效果较小，关于涤纶酶改性的机理鲜有报道。针对这些问题，作者认为首先应该有目的地培养用于涤纶改性的酶，研究酶的应用环境和涤纶的性质对酶法改性的影响，这也是本课题的目的所在。本课题从菌株筛选开始，经微生物发酵培养、目标酶的分离纯化等步骤，有针对性地培养了一种用于涤纶改性的脂肪酶；从水解产物、染色性、亲水性和抗静电性等几个方面考察了涤纶酶改性的效果；探讨了酶的应用环境和涤纶的结晶度等因素对改性结果的影响。为了客观评价该脂肪酶对涤纶的改性效果，又采用了另外四种酶处理涤纶。具体的研究方法和路线及本文的主要结论如下述。用平板筛选法和液体复筛法，从Aspergillus oryzae 1、Aspergillusoryzae 2、Aspergillus，niger H1和Aspergillus，niger M57等四种菌株中，筛选出产脂肪酶能力最好的菌株Aspergillus oryzae 1作为工作菌。考察了碳源、氮源、表面活性剂等的种类和浓度及培养温度、培养液初始pH值和摇床转速等因素对菌株Aspergillus oryzae 1产脂肪酶的影响。为了提高目标酶处理涤纶的针对性，考察了对苯二甲酸二乙酯、对苯二甲酸二乙二醇酯和涤纶短丝等三种添加物对菌株Aspergillus oryzae 1及其所产的脂肪酶的性质的影响。实验表明该菌株合成目标脂肪酶的最佳发酵培养条件是2％（v/v）Vogel’s Medium N、0.5％（w/v，除非特别指明，本摘要中均为质量百分比）麦芽糖、0.5％蛋白胨、1.0％橄榄油、0.032％平平加O、0.01％对苯二甲酸二乙二醇酯，发酵初始pH值6.0，培养温度30℃，摇床转速160rpm，培养时间84h。对苯二甲酸二乙酯抑制菌株Aspergillus oryzae 1的生长及脂肪酶的合成；涤纶短丝对该菌株合成脂肪酶的影响不大；对苯二甲酸二乙二醇酯对菌株合成脂肪酶有“刺激”或“诱导”作用。添加对苯二甲酸二乙二醇酯而培养合成的脂肪酶可以催化作用于对涤纶的小分子模拟物对苯二甲酸二乙酯；添加涤纶短丝和无添加物而培养合成的酶液则对对苯二甲酸二乙酯的水解没有催化作用用硫酸铵盐析法和凝胶层析对Aspergillus oryzae 1脂肪酶进行了纯化。较适宜的硫酸铵饱和度是80％，经此饱和度下的硫酸铵盐析后，脂肪酶的比活力达到118，421U/g，纯化收率为72％，纯化倍数为2.92。再经凝胶层析后，脂肪酶的纯化倍数达到11.9，纯化收率保持在25％左右，比活力达到481，500U/g；经SDS-PAGE电泳鉴定，该脂肪酶纯度较高。该脂肪酶的最佳作用pH值为7.0；其在弱碱溶液中的活性稍高于在弱酸溶液中的活性，是一种中性稍偏碱性的脂肪酶35℃～45℃范围内，该酶相对均有较高的反应活性；在pH7.0下保存时该脂肪酶较为稳定；在55℃保存24h，其残余酶活为50％，具有较高的热稳定性。Mg²⁺和Ca²⁺对该脂肪酶有激活作用，EDTA则对其有很强的抑制作用，表明该脂肪酶是一种金属酶；JFC、平平加和麦芽糖对该脂肪酶有保护作用，在处理聚酯纤维时可以共用。用Aspergillus oryzae 1脂肪酶处理涤纶。在处理过程中，对该酶处理液进行吸光度分析，结果表明涤纶在该脂肪酶的作用下，发生了降解；反相液相色谱分析表明在酶液中的涤纶降解物极有可能是对苯二甲酸单乙二醇酯或对苯二甲酸单乙二醇酯和对苯二甲酸的混合物。酶处理后，涤纶纤维能结合更多的阳离子染料，表明其中的羧基官能团增加；纤维红外光谱分析表明酶处理只发生在纤维表面；涤纶的回潮率略有增加，静电半衰期和水接触角下降。以上实验结果表明Aspergillus oryzae 1脂肪酶确实能作用于涤纶，使涤纶的亲水性和抗静电性得到改善。55℃是该脂肪酶处理涤纶的较适宜的温度；在酶工作液中加入表面活性剂JFC或平平加O，或提高酶浓度对酶改性结果有促进作用；涤纶纤维的无定形区更容易被脂肪酶进攻。在优化条件下，即浴比40、9.0U/ml或12.6U/ml酶浓度、0.5g/L JFC、pH7.0、处理温度55℃、处理时间24h、摇床转速100rpm，经Aspergillus oryzae 1脂肪酶处理后，涤纶织物的静电半衰期从8.42s下降至8.00s左右，其经线的水接触角从75°下降至69°。为了客观评价Aspergillus oryzae 1脂肪酶对涤纶的作用效果，研究了Rhizopus oryzae酯酶、Hog pancreas脂肪酶、脂肪酶L和角质酶J对涤纶的改性作用。结果表明，经Hog pancreas脂肪酶、角质酶J或脂肪酶L处理后的涤纶，其回潮率、静电半衰期和水接触角等性质没有变化，说明这三种酶对涤纶没有作用或作用很小。Rhizopus oryzae酯酶对涤纶的作用较为明显，在优化条件下，即浴比40、0.5％酶、0.1％平平加0、pH7.2、在37℃和100rpm的振荡箱中处理6天，涤纶织物经线的水接触角从75°下降至67°，静电半衰期从8.42s下降至7.45 s。通过实验，总结出了涤纶脂肪酶改性的粗略模型：1)涤纶纤维是线性大分子聚合物，结构紧密，酶很难渗入涤纶分子内部，所以酶改性只发生在纤维的表面。2)涤纶聚合物片断在脂肪酶的作用下脱离涤纶聚合物主体后，该聚合物片断和涤纶聚合物一样，可以成为脂肪酶作用的底物，于是在酶作用于涤纶主体的同时，也作用于该涤纶聚合物片断，使其裂解为分子量更小的片断，更小的涤纶聚合物片断进一步裂解，直至成为溶于酶溶液的小分子物质，这种小分子物质极有可能是对苯二甲酸单乙二醇酯或对苯二甲酸单乙二醇酯和对苯二甲酸的混合物。3)在涤纶纤维分子中，无定形区更易受到脂肪酶的进攻，这是因为，与结晶区相比，无定形区的分子有较高的自由度，可以较容易接近酶分子的活性中心。4)提高涤纶纤维的自由度如升高酶处理温度，或提高酶的稳定性有利于涤纶酶改性的进行。更多还原

【Abstract】 Poly（ethylene terephthalate） fiber is a polyester formed by ethyleneglycol reacting with terephthalate, it exhibits some drawbacksinfluencing its wearing comfort, like static charges and hydrophobicity.To improve these drawbacks, it is necessary to modify PET fiber/fabric.As a mild and environmentally friendly way as well asdemonstrating no damages to the original good properties of PETfiber/fabric, enzymatic modification of PET fiber/fabric came into sightin the last several years. According to the stage of enzymaticmodification of PET fiber/fabric up to now, there are several problemsneeded to solve. Firstly, enzymes used were often purchased fromcompanies and had no pertinences for modification of PET fiber/fabric,so activities of enzymes to PET were rather low and treatment resultswere far to industrial application, new more efficient enzymes areneeded to explore. Secondly, mechanism of enzymatic modification ofPET had not been studied. To solve these problems, the authors considerthat it should cultivate enzymes purposely for modification of PET atfirst. In this paper, aiming at enzymatic modification of PET, a lipasewas obtained by screening strains, optimizing culture conditions,purifying and partial characterizing. PET sample was treated with thelipase and the modification results were evaluated by analyzinghydrolysis products, carboxyl groups on PET surface and physicalproperties of PET such as hydrophlicity, anti-static ability and cationic dyeing ability. Effects of temperature, surfactants and crystallinity ofPET on the enzymatic modification of PET were discussed. To estimatethe lipase objectively, four other enzymes were also used to treat PETsample. Detailed research ways, steps and results are as the followings.By solid-plate method first and then liquid incubation, Aspergillusoryzae 1 was screened out for its most potential for lipase productionfrom four strains.Effects of carbon sources and their concentrations, nitrogen sourcesand their concentrations, surfactants and their concentrations,incubation temperature, beginning pH value of the liquid culture mediumand aeration （rotating speed） on lipase production from Aspergillusoryzae 1 were studied. Purposely, three additives, bis（2-hydroxyethyl）terephthalate, diethyl p-phthalate or PET short fiber was added into theculture medium, their effects on lipase production and mycelium weightas well as on hydrolysis of diethyl p-phthalate were studied. It turnedout that diethyl p-phthalate depressed lipase production and growth ofthe strain, PET short fiber had no evident effect on andbis（2-hydroxyethyl） terephthalate stimulated lipase production from thestrain. Lipase preparation cultivated （"induced"） withbis（2-hydroxyethyl） terephthalate could catalyze hydrolysis of diethylp-phthalate, while lipase preparation cultivated with PET short fiber orcultivated with no additives had no effect on diethyl p-phthalate. Theoptimal culture medium for lipase production from Aspergillus oryzae 1contained 2．0%（v/v） Vogel’s Medium N, 0．5% maltose, 0．5% peptone,1．0% olive oil, 0．032% Peregal O, 0．01% bis（2-hydroxyethyl）terephthalate, beginning pH 6．0, culture temperature 30℃, rotatingspeed 160rpm and culture time 84h.Aspergillus oryzae 1 lipase was purified by ammonium sulfateprecipitation and gel filtration. The optimal saturation of ammonium sulfate was 80%, the lipase was purified 2．92 fold after precipitated by80% saturated ammonium sulfate, the yield was 72% and the specificlipase activity came to 118, 421U/g. After gel filtration by a SuperdexG-75 column, the lipase was purified 11．9 fold and the yield was about25%, the specific lipase activity was 481,500U/g. SDS-PAGE identifiedthat the lipase was pretty pure.The optimal pH of the lipase activity was 7．0 and the lipase waspretty stable at pH7．0; the lipase activity was relatively high between 35℃～45℃and the optimal temperature was 40℃; in aqueous solution,the lipase had a high thermal stability, its activity remained about 50%after incubation at 55℃for 24h; Ca²⁺ and Mg²⁺ stimulated whereasEDTA strongly inhibited the lipase activity, from which it could beconcluded that the lipase was a metal enzyme: JFC, Peregal O andmaltose had protective effects on the lipase stability and they could beused together with the lipase during PET treatment.Lipase preparation from Aspergillus oryzae could act on ester bondson the surface of PET fiber. Under catalysis of the lipase preparation,hydrolysis products of PET fibers were probably mono（2-hydroxyethyl）terephthalate or mixture of terephthalic acid and mono（2-hydroxyethyl）terephthalate. After the lipase modification, there were more carboxylgroups on the treated PET fabric surface that resulted in interacting withmore cationic dyes; increased hydrophilicity and antistatic ability ofPET samples were found based on moisture regain, water contact angleand static half decay time.The appropriate temperature for the lipase treatment of PET was 55℃. Adding JFC, Peregal O or increasing the lipase concentrationfacilitated the modification results of PET. Amorphous area in PET fiberwas more susceptible for lipase attack. The optimal condition for thelipase treatment of PET included bath ratio 40, 9．0U/ml or 12．6U/ml lipase concentration, 0．5g/L JFC, pH7．0, treatment temperature 55℃,rotation speed 100rpm and treatment time 24h. After treated under theoptimal condition, water contact angle of PET fabric decreased from 75°to 69°, static half decay time decreased from 8．40s to about 8．00s.In order to estimate effects of Aspergillus oryzae 1 lipase on PETobjectively, Rhizopus oryzae esterase, Hog pancreas lipase, Lipase L andcutinase J were also used to treat PET. It turned out after PET samplewas treated with Hog pancreas lipase, lipase L or cutinase J, itsantistatic ability, hydrophilicity and cationic dyeing ability almostunchanged. After PET was treated by Rhizopus oryzae esterase solution,its water contact angle decreased from 75°to 67°, static half decay timedecreased from 8．40s to 7．45s, dyeing ability with various cationic dyeimproved to different degrees.According to results in this paper, we described a simple model ofPET enzymatic modification with lipase. 1) PET is a linear polymer ofbig molecular, lipase is difficult to penetrate inside of it, so enzymaticmodification only happens on the PET surface and doesn’t affectstrength of PET. 2) As a result of enzymatic catalysis, PET polymersegment is off PET bulk; this PET segment is also a substrate for enzymeas well and can be catalyzed by enzyme to a smaller segment till it turnsto the final hydrolysis product. The hydrolysis products were probablymono（2-hydroxyethyl） terephthalate or mixture of mono（2-hydroxyethyl）terephthalate and terephthalic acid. 3) In PET bulk, amorphous area ismore susceptible for lipase attack, because ester bonds in amorphousarea are more mobile, they can access and bind with the active site oflipase more easily. 4) Enhancing mobility of PET facilitates enzymaticmodification of PET.Wang Xiao-hua （Textile chemistry and dyeing and finishingengineering）更多还原