紫外辐照改性聚丙烯纤维固定溶菌酶的研究
|
摘要
聚丙烯(Polypropylene,PP)从20世纪60年代初开始纤维级的研究与工业生产,经过90年代的高速发展,现已成为纺织工业的主要纤维之一。生产的各种制品不仅用于纺织和农业,在医疗和卫生等领域也得到大量使用,但由于是疏水性纤维需将其进行改性。国内外关于聚丙烯纤维改性的研究报导,大多集中在其表面接枝上亲水或反应性基团,而对改性后纤维上进行生物酶固定化的研究还尚不多见。
本文经紫外辐照引发接枝甲基丙烯酸甲酯(MMA),在聚丙烯纤维表面引入反应性基团酯基,再通过与己二胺的酰胺化反应后用交联剂—戊二醛将改性后纤维表面的胺基与酶分子偶联,实现了生物酶在改性聚丙烯纤维上的共价固定化。主要研究内容包括光接枝反应因素对MMA接枝效果的影响,酰胺化反应及酶固定化条件对溶菌酶固定化酶活的影响。联合红外光谱(FTIR-ATR)、扫描电镜(SEM)、原子力显微镜(AFM)对聚丙烯纤维改性效果进行分析表征。最后,对聚丙烯膜的光接枝改性进行了初步的研究。
实验结果表明:对聚丙烯纤维光接枝改性的最佳工艺条件为,BP用量为0.3mol/L;预照时间8min;MMA浓度为40%(v/v);紫外辐照时间:60min。改性后纤维进行酰胺化反应的较优条件为,己二胺用量30%;反应时间:2h。当采用共价交联法固定化溶菌酶时,戊二醛用量为:2%;溶菌酶:4mg/ml,固定化时间为10h。MMA的接枝效果会影响到表面胺基含量,进而对固定化溶菌酶的酶活产生影响,当接枝率为1.69%时,固定化的溶菌酶具有较高的酶活,为18U/cm2。而改性纤维在固定化溶菌酶后具有良好的抗菌性能,抑菌率为68.60%。通过对比改性前后聚丙烯纤维的FTIR-ATR、SEM、AFM与考马斯蓝染色,证实MMA确实接枝在了聚丙烯纤维表面、改性聚丙烯纤维实现了溶菌酶的固定化。光接枝改性后的聚丙烯纤维和聚丙烯膜都可以进行漆酶的固定化,且固定化后的漆酶具有一定的脱色效果。而接枝率也会对固定化漆酶的脱色效率和操作稳定性产生影响。
Polypropylene from the 20th century, the beginning of the 60's fiber-class research and industrial production, after 90 years the rapid development has become one of the main fiber of the textile industry. However, due to the non-polar polypropylene fiber material, the lack of polar molecular chain functional group, showed a certain degree of chemical inertness, and thus also to some extent limited the scope of its application. Modification of polypropylene fiber at home and abroad on research reports, most of them concentrated in the graft on the surface hydrophilic or reactive group, while the fibers on modification of immobilized enzyme research is still rare.
In this paper, in order to realize enzymatic functionalization of PP fibers, PP fibers were successively modified by UV photo-induced grafting of methyl methacrylate (MMA), amidation reaction and lysozyme immobilization. The study includes light and other factors on the MMA-gg-response effect, amidation reaction and enzyme immobilization conditions on the activity of immobilized lysozyme activity. The structures and morphologies of the modified fabrics were compared using fourier transform infrared spectroscopy (FTIR-ATR)、scanning electron microscope (SEM)and atomic force microscope (AFM). Finally, we also had a preliminary study of the optical film of polypropylene grafted conducted.
The result demonstrated that the optimal conditions for grafting yield on PP fiber surface were as follows: BP dosage: 0.3mol/L, according to the time pre-8min; MMA concentration of40%(v/v); UV irradiation time 60min. Modified fibers the better amidation reaction conditions was the amount of hexamethylene concentration: 30% (v/v), reaction time: 2h. When cross-linked covalently immobilized lysozyme , the amount of glutaraldehyde: 2%; lysozyme: 4mg/ml, fixed time: 10h. The effect of the grafted MMA will affect the surface amine content, and thus the activity of immobilized lysozyme have an impact, when the grafting yield was 1.69% of the lysozyme immobilized with high activity for 18U/cm2 . Modified fibers after lysozyme immobilized with good antimicrobial properties, inhibitory rate of 68.60%. By comparing before and after modified polypropylene fiber FTIR-ATR, SEM, AFM and Coomassie blue staining confirmed that MMA was grafted in polypropylene fiber surface, and modified polypropylene fiber had to achieve the immobilization of lysozyme . Photografting modified polypropylene fiber and polypropylene membrane could be immobilized laccase and immobilized laccase has some effect of decolorization and grafting yield of the immobilized laccase decolorization efficiency and impact on operational stability.
本文经紫外辐照引发接枝甲基丙烯酸甲酯(MMA),在聚丙烯纤维表面引入反应性基团酯基,再通过与己二胺的酰胺化反应后用交联剂—戊二醛将改性后纤维表面的胺基与酶分子偶联,实现了生物酶在改性聚丙烯纤维上的共价固定化。主要研究内容包括光接枝反应因素对MMA接枝效果的影响,酰胺化反应及酶固定化条件对溶菌酶固定化酶活的影响。联合红外光谱(FTIR-ATR)、扫描电镜(SEM)、原子力显微镜(AFM)对聚丙烯纤维改性效果进行分析表征。最后,对聚丙烯膜的光接枝改性进行了初步的研究。
实验结果表明:对聚丙烯纤维光接枝改性的最佳工艺条件为,BP用量为0.3mol/L;预照时间8min;MMA浓度为40%(v/v);紫外辐照时间:60min。改性后纤维进行酰胺化反应的较优条件为,己二胺用量30%;反应时间:2h。当采用共价交联法固定化溶菌酶时,戊二醛用量为:2%;溶菌酶:4mg/ml,固定化时间为10h。MMA的接枝效果会影响到表面胺基含量,进而对固定化溶菌酶的酶活产生影响,当接枝率为1.69%时,固定化的溶菌酶具有较高的酶活,为18U/cm2。而改性纤维在固定化溶菌酶后具有良好的抗菌性能,抑菌率为68.60%。通过对比改性前后聚丙烯纤维的FTIR-ATR、SEM、AFM与考马斯蓝染色,证实MMA确实接枝在了聚丙烯纤维表面、改性聚丙烯纤维实现了溶菌酶的固定化。光接枝改性后的聚丙烯纤维和聚丙烯膜都可以进行漆酶的固定化,且固定化后的漆酶具有一定的脱色效果。而接枝率也会对固定化漆酶的脱色效率和操作稳定性产生影响。
Polypropylene from the 20th century, the beginning of the 60's fiber-class research and industrial production, after 90 years the rapid development has become one of the main fiber of the textile industry. However, due to the non-polar polypropylene fiber material, the lack of polar molecular chain functional group, showed a certain degree of chemical inertness, and thus also to some extent limited the scope of its application. Modification of polypropylene fiber at home and abroad on research reports, most of them concentrated in the graft on the surface hydrophilic or reactive group, while the fibers on modification of immobilized enzyme research is still rare.
In this paper, in order to realize enzymatic functionalization of PP fibers, PP fibers were successively modified by UV photo-induced grafting of methyl methacrylate (MMA), amidation reaction and lysozyme immobilization. The study includes light and other factors on the MMA-gg-response effect, amidation reaction and enzyme immobilization conditions on the activity of immobilized lysozyme activity. The structures and morphologies of the modified fabrics were compared using fourier transform infrared spectroscopy (FTIR-ATR)、scanning electron microscope (SEM)and atomic force microscope (AFM). Finally, we also had a preliminary study of the optical film of polypropylene grafted conducted.
The result demonstrated that the optimal conditions for grafting yield on PP fiber surface were as follows: BP dosage: 0.3mol/L, according to the time pre-8min; MMA concentration of40%(v/v); UV irradiation time 60min. Modified fibers the better amidation reaction conditions was the amount of hexamethylene concentration: 30% (v/v), reaction time: 2h. When cross-linked covalently immobilized lysozyme , the amount of glutaraldehyde: 2%; lysozyme: 4mg/ml, fixed time: 10h. The effect of the grafted MMA will affect the surface amine content, and thus the activity of immobilized lysozyme have an impact, when the grafting yield was 1.69% of the lysozyme immobilized with high activity for 18U/cm2 . Modified fibers after lysozyme immobilized with good antimicrobial properties, inhibitory rate of 68.60%. By comparing before and after modified polypropylene fiber FTIR-ATR, SEM, AFM and Coomassie blue staining confirmed that MMA was grafted in polypropylene fiber surface, and modified polypropylene fiber had to achieve the immobilization of lysozyme . Photografting modified polypropylene fiber and polypropylene membrane could be immobilized laccase and immobilized laccase has some effect of decolorization and grafting yield of the immobilized laccase decolorization efficiency and impact on operational stability.
引文
[1]赵敏,高俊刚,邓奎林,等.改性聚丙烯新材料[M].北京:化学工业出版社,2002:150-164.
[2]解云川,杨青芳.表面光接枝改善聚合物性能的研究进展[J].高分子材料科学与工程,2008,18(5)7-11.
[3]杨欣华,杨育农,王浩江,等.聚丙烯化学改性与表征[J].国外塑料,2006,24(12)66-70.
[4] Ulbricht M, Oechelect A. Photo-bromination and photo-induced graft polymerization as a two-step approach for surface modification of polyacrylonitrile ultrafiltration membranes[J]. European Polymer Journal,1996,32(9):1045-1054.
[5] Gotoh K, Hayashiya M. Improvement of Serviceability Properties of Synthetic Textile Fabrics using 172 nm Ultraviolet Excimer Lamp[J]. Textile Research Journal , 2008,78(1) :37-44.
[6]刘晓洪,王少军.聚丙烯纤维光接枝改性的研究[J].武汉科技学院学报,2006,19(1):18-20.
[7] Mathias Ulbricht. Advanced functional polymer membranes [J] . Polymer , 2006 , 47 (7) : 2217-2262.
[8] Goddard J M, Hotchkiss J H. Polymer surface modification for the attachment of bioactive compounds[J]. Progress in polymer science,2007,32 (7):698-725.
[9] Hong-Tao Deng, Zhi-Kang Xu, Zheng-Wei Dai,et al. Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor[J]. Enzyme and Microbial Technology 2005,36 (7) 996–1002.
[10] Mathias Ulbricht, Hans-Hartmut Schwarz. Novel high performance photo-graft composite membranes for separation of organic liquids by pervaporation[J]. Journal of Membrane Science 1997,136 (1-2) 25-33.
[11]孙玉凤,宋婷,杨万泰.LDPE/ NVP体系表面光接枝聚合研究[J].北京化工大学学报,1999,26(2)17-20.
[12]张宇东,陈代涛,施鼐,等.PP预辐射接枝丙烯酸酯的研究[J].高分子材料科学与工程,1999,15(5):79-82.
[13] Wantai Yang, Bengt R?nby. Photoinitiation performance of some ketones in the LDPE–acrylic acid surface photografting system[J]. European Polymer Journal,1999,35(8):1557-1568.
[14]甘胜华,邓建元,杨万泰.光接枝表面改性聚丙烯膜固定蛋白质的研究[J].北京化工大学学报,2008,35(4)61-64.
[15]李赛,罗祥林,付皓,等. PET膜表面光固定脲酶的研究[J].高分子材料科学与工程,2001,17(2)113-120.
[16]罗祥林,黄嘉,何斌,等.光化学固定法——医用高分子材料表面改性的一种新方法[J].生物医学工程学杂志, 2000:17 (3) :320～323.
[17]邓红涛,吴健,徐志康,等.酶的固定化及其应用的进展[J].膜科学与技术,2004,24(3):47-53.
[18]赵海峰.传统的酶固定化法及改进方法[J],医药化工,2007,2(8):25-26.
[19]邓红涛.脂肪酶在聚丙烯微孔膜上的固定化研究[D].杭州:浙江大学,2005.
[20]朱奇,陈彦.溶菌酶及其应用[J].生物学通报,1998,33(10):9-10
[21] M.L. Foresti , M.L. Ferreira. Analysis of the interaction of lipases with polypropylene of different structure and polypropylene-modified glass surface[J]. Colloids and Surfaces A, 2007,294 (1-3):147–155.
[22] Andrea Salis, Marcella Pinna, Maura Monduzzi,et al. Comparison among immobilised lipases on macroporous polypropylene toward biodiesel synthesis[J]. Journal of Molecular Catalysis B: Enzymatic 2008,54(1-2):19–26.
[23]贾向志,李元,马文煜.溶菌酶的研究进展[J].生物技术通讯,2002,13(5):374-377.
[24]李洪莲,于秀俊,杨秀艳.漆酶及其应用[J] .沧州师范专科学校学报,2005,21(4) :91-92.
[25] Alessandro D A,Silvia R S,Vittorio V,etc.Characterizat of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment[J].Process Biochem , 1999 , 34(6-7):697-706.
[26]胡平平,付时雨.漆酶催化活性中心结构及其特性研究进展[J].林产化学与工业,2001,21(3):69-75
[27]缪静,姜竹茂.漆酶的最新研究进展[J].烟台师范学院学报(自然科学版),2001,17(2):146-150.
[28]刘梅红.印染废水处理技术研究进展[J].纺织学报,2007,28(1):117-118.
[29] Harald Claus.Laccases:structure,reactions,distribution[J].Micron,2004(35):93—96.
[30]唐丽华,李鑫,江渊,等.等离子体引发丙烯酰胺接枝改性聚丙烯非织造布[J].纺织学报, 2007,28(12):27-29.
[31] Liberata Guadagno, Carlo Naddeo, Marialuigia Raimondo. Structural and morphological changes during UV irradiation of the trans-planar form of syndiotactic polypropylene[J]. Polymer Degradation and Stability 93 (2008):176-187.
[32] Yu Chang Tyan, Jiunn Der Liao, Yi Te Wu, et al. Anticoagulant Activity of Immobilized Heparin on the Polypropylene Nonwoven Fabric Surface Depending upon the pH of Processing Environment[J]. Journal of Biomaterials Applications,2002,17(2):153-178.
[33]田冶,杨菊林,杨媛,等.聚乳酸膜氨等离子处理的表面性能[J].化工进展, 2007,26(8):1155-1158.
[34]彭赛平,苏远,贺全国,等. N2等离子体接枝处理丙纶改性及染色的初步研究[J].核聚变与等离子体物理,2007,27(3):269-272.
[35]鲜海军,王侦祥.以聚丙烯腈纤维为载体制备固定化青霉素G酰化酶的研究[J].微生物学报,2001,41(4):475-480.
[36] Zhen-Mei Liu, Sophie Tingry, Christophe Innocent. Modification of microfiltration polypropylene membranes by allylamine plasma treatment Influence of the attachment route on peroxidase immobilization and enzyme efficiency[J]. Enzyme and Microbial Technology 39 (2006) 868–876.
[37] Kramer K J.Kanost M R,Hopkins T L,et a1.Oxidative conmatinn of oatechols with proteins in insect skdetal systems[J].Tetrahedron,57(2001):385-392.
[38] Koichi Kato, Emiko Uchida, En-Tang Kang,et al. Polymer surface with graft chains[J]. Prog. Polym. Sci. 28(2003) 209–259.
[39] M.M. El-Masry, A. De Maio, P.L. Martelli,et al. Influence of the immobilization process on the activity of_-galactosidase bound to Nylon membranes grafted with glycidyl methacrylate Part 1. Isothermal behavior[J]. Journal of Molecular Catalysis B: Enzymatic 16 (2001) 175-189.
[40]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量[J].中国食品添加剂,2008(2):162-165.
[41]蔡谨,王文序.染色法定量检测氨气等离子体改性膜表面的氨基[J].浙江大学学报(自然科学版),1999,33(1):74-76.
[42] B.施特尔马赫.酶的测定方法[M].北京:中国轻工业出版社,1992:236-241.
[43]赵玉萍,张灏,杨严俊.溶菌酶测定方法的改进[J].食品科学,2002,23 (3):116-119.
[44]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定粗提取液中可溶性蛋白质含量方法的研究[J].苏州大学学报,2006,22(2):82-85
[45]吴萨日娜,武志云.印染废水的常用脱色方法的探讨[J].河北纺织,2007(130):86-89.
[46]杜芬,马立新.印染废水处理工艺工程实例[J].环境工程,2009.27(1):18-19,36.
[2]解云川,杨青芳.表面光接枝改善聚合物性能的研究进展[J].高分子材料科学与工程,2008,18(5)7-11.
[3]杨欣华,杨育农,王浩江,等.聚丙烯化学改性与表征[J].国外塑料,2006,24(12)66-70.
[4] Ulbricht M, Oechelect A. Photo-bromination and photo-induced graft polymerization as a two-step approach for surface modification of polyacrylonitrile ultrafiltration membranes[J]. European Polymer Journal,1996,32(9):1045-1054.
[5] Gotoh K, Hayashiya M. Improvement of Serviceability Properties of Synthetic Textile Fabrics using 172 nm Ultraviolet Excimer Lamp[J]. Textile Research Journal , 2008,78(1) :37-44.
[6]刘晓洪,王少军.聚丙烯纤维光接枝改性的研究[J].武汉科技学院学报,2006,19(1):18-20.
[7] Mathias Ulbricht. Advanced functional polymer membranes [J] . Polymer , 2006 , 47 (7) : 2217-2262.
[8] Goddard J M, Hotchkiss J H. Polymer surface modification for the attachment of bioactive compounds[J]. Progress in polymer science,2007,32 (7):698-725.
[9] Hong-Tao Deng, Zhi-Kang Xu, Zheng-Wei Dai,et al. Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor[J]. Enzyme and Microbial Technology 2005,36 (7) 996–1002.
[10] Mathias Ulbricht, Hans-Hartmut Schwarz. Novel high performance photo-graft composite membranes for separation of organic liquids by pervaporation[J]. Journal of Membrane Science 1997,136 (1-2) 25-33.
[11]孙玉凤,宋婷,杨万泰.LDPE/ NVP体系表面光接枝聚合研究[J].北京化工大学学报,1999,26(2)17-20.
[12]张宇东,陈代涛,施鼐,等.PP预辐射接枝丙烯酸酯的研究[J].高分子材料科学与工程,1999,15(5):79-82.
[13] Wantai Yang, Bengt R?nby. Photoinitiation performance of some ketones in the LDPE–acrylic acid surface photografting system[J]. European Polymer Journal,1999,35(8):1557-1568.
[14]甘胜华,邓建元,杨万泰.光接枝表面改性聚丙烯膜固定蛋白质的研究[J].北京化工大学学报,2008,35(4)61-64.
[15]李赛,罗祥林,付皓,等. PET膜表面光固定脲酶的研究[J].高分子材料科学与工程,2001,17(2)113-120.
[16]罗祥林,黄嘉,何斌,等.光化学固定法——医用高分子材料表面改性的一种新方法[J].生物医学工程学杂志, 2000:17 (3) :320～323.
[17]邓红涛,吴健,徐志康,等.酶的固定化及其应用的进展[J].膜科学与技术,2004,24(3):47-53.
[18]赵海峰.传统的酶固定化法及改进方法[J],医药化工,2007,2(8):25-26.
[19]邓红涛.脂肪酶在聚丙烯微孔膜上的固定化研究[D].杭州:浙江大学,2005.
[20]朱奇,陈彦.溶菌酶及其应用[J].生物学通报,1998,33(10):9-10
[21] M.L. Foresti , M.L. Ferreira. Analysis of the interaction of lipases with polypropylene of different structure and polypropylene-modified glass surface[J]. Colloids and Surfaces A, 2007,294 (1-3):147–155.
[22] Andrea Salis, Marcella Pinna, Maura Monduzzi,et al. Comparison among immobilised lipases on macroporous polypropylene toward biodiesel synthesis[J]. Journal of Molecular Catalysis B: Enzymatic 2008,54(1-2):19–26.
[23]贾向志,李元,马文煜.溶菌酶的研究进展[J].生物技术通讯,2002,13(5):374-377.
[24]李洪莲,于秀俊,杨秀艳.漆酶及其应用[J] .沧州师范专科学校学报,2005,21(4) :91-92.
[25] Alessandro D A,Silvia R S,Vittorio V,etc.Characterizat of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment[J].Process Biochem , 1999 , 34(6-7):697-706.
[26]胡平平,付时雨.漆酶催化活性中心结构及其特性研究进展[J].林产化学与工业,2001,21(3):69-75
[27]缪静,姜竹茂.漆酶的最新研究进展[J].烟台师范学院学报(自然科学版),2001,17(2):146-150.
[28]刘梅红.印染废水处理技术研究进展[J].纺织学报,2007,28(1):117-118.
[29] Harald Claus.Laccases:structure,reactions,distribution[J].Micron,2004(35):93—96.
[30]唐丽华,李鑫,江渊,等.等离子体引发丙烯酰胺接枝改性聚丙烯非织造布[J].纺织学报, 2007,28(12):27-29.
[31] Liberata Guadagno, Carlo Naddeo, Marialuigia Raimondo. Structural and morphological changes during UV irradiation of the trans-planar form of syndiotactic polypropylene[J]. Polymer Degradation and Stability 93 (2008):176-187.
[32] Yu Chang Tyan, Jiunn Der Liao, Yi Te Wu, et al. Anticoagulant Activity of Immobilized Heparin on the Polypropylene Nonwoven Fabric Surface Depending upon the pH of Processing Environment[J]. Journal of Biomaterials Applications,2002,17(2):153-178.
[33]田冶,杨菊林,杨媛,等.聚乳酸膜氨等离子处理的表面性能[J].化工进展, 2007,26(8):1155-1158.
[34]彭赛平,苏远,贺全国,等. N2等离子体接枝处理丙纶改性及染色的初步研究[J].核聚变与等离子体物理,2007,27(3):269-272.
[35]鲜海军,王侦祥.以聚丙烯腈纤维为载体制备固定化青霉素G酰化酶的研究[J].微生物学报,2001,41(4):475-480.
[36] Zhen-Mei Liu, Sophie Tingry, Christophe Innocent. Modification of microfiltration polypropylene membranes by allylamine plasma treatment Influence of the attachment route on peroxidase immobilization and enzyme efficiency[J]. Enzyme and Microbial Technology 39 (2006) 868–876.
[37] Kramer K J.Kanost M R,Hopkins T L,et a1.Oxidative conmatinn of oatechols with proteins in insect skdetal systems[J].Tetrahedron,57(2001):385-392.
[38] Koichi Kato, Emiko Uchida, En-Tang Kang,et al. Polymer surface with graft chains[J]. Prog. Polym. Sci. 28(2003) 209–259.
[39] M.M. El-Masry, A. De Maio, P.L. Martelli,et al. Influence of the immobilization process on the activity of_-galactosidase bound to Nylon membranes grafted with glycidyl methacrylate Part 1. Isothermal behavior[J]. Journal of Molecular Catalysis B: Enzymatic 16 (2001) 175-189.
[40]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量[J].中国食品添加剂,2008(2):162-165.
[41]蔡谨,王文序.染色法定量检测氨气等离子体改性膜表面的氨基[J].浙江大学学报(自然科学版),1999,33(1):74-76.
[42] B.施特尔马赫.酶的测定方法[M].北京:中国轻工业出版社,1992:236-241.
[43]赵玉萍,张灏,杨严俊.溶菌酶测定方法的改进[J].食品科学,2002,23 (3):116-119.
[44]曲春香,沈颂东,王雪峰,等.用考马斯亮蓝测定粗提取液中可溶性蛋白质含量方法的研究[J].苏州大学学报,2006,22(2):82-85
[45]吴萨日娜,武志云.印染废水的常用脱色方法的探讨[J].河北纺织,2007(130):86-89.
[46]杜芬,马立新.印染废水处理工艺工程实例[J].环境工程,2009.27(1):18-19,36.