无机载体吸附-交联固定化海洋脂肪酶技术研究
|
摘要
使用硅藻土载体和乙二醇缩水甘油醚进行吸附-交联固定海洋脂肪酶。采用单因素与正交实验结合的实验方案,确定最佳吸附条件为20℃,pH 5.0的0.1 mol/L柠檬酸钠缓冲液,载体投放量为2 g,吸附固定化时间为6 h。交联最佳温度条件为30℃,交联时间为6 h,乙二醇缩水甘油醚浓度为1.2%。吸附交联后酶活达124.83 U/g,酶活回收率高达67.31%。固定化脂肪酶连续操作5次依然保持良好的酶活性,对比游离酶,最适反应pH没有变化,但是最适反应温度固定化酶为50℃,提升5℃。同时,固定化脂肪酶显示出较好的机械稳定性和储存稳定性。结果证明硅藻土吸附-交联固定化海洋脂肪酶在工业酶固定化上有良好的应用前景。
This study carried out the immobilization of marine lipase through adsorption/cross?linking method using kieselguhr and ethylene glycol glycidyl ether.Through single?factor and orthogonal experiments,the optimal adsorption conditions were determined as follows:0.1 mol/L sodium citrate at 20 ℃,pH 5.0,2 g car?rier dosage,adsorption time being 6 h;the optimal cross?linking conditions were determined as follows:30 ℃,cross?linking time being 6 h,and the concentration of ethylene glycol glycidyl ether being 1.2 %.The activity of prepared immobilized lipase reached 124.83 U/g,and the recovery rate of lipase activity was as high as 67.31 %.The immobilized lipase still maintained good enzyme activity after continuous operation for 5 times.Compared with free lipase,the optimal reaction pH of immobilized lipase was the same,but the optimal reaction tempera?ture for the immobilized lipase was 50 ℃,5 ℃ higher than that for free lipase.At the same time,the immobilized lipase showed better mechanical stability and better storage stability.These results show that the immobilization of lipase by adsorption-crosslinking method using kieselguhr as the absorbant possesses good application pros?pect in industry.
This study carried out the immobilization of marine lipase through adsorption/cross?linking method using kieselguhr and ethylene glycol glycidyl ether.Through single?factor and orthogonal experiments,the optimal adsorption conditions were determined as follows:0.1 mol/L sodium citrate at 20 ℃,pH 5.0,2 g car?rier dosage,adsorption time being 6 h;the optimal cross?linking conditions were determined as follows:30 ℃,cross?linking time being 6 h,and the concentration of ethylene glycol glycidyl ether being 1.2 %.The activity of prepared immobilized lipase reached 124.83 U/g,and the recovery rate of lipase activity was as high as 67.31 %.The immobilized lipase still maintained good enzyme activity after continuous operation for 5 times.Compared with free lipase,the optimal reaction pH of immobilized lipase was the same,but the optimal reaction tempera?ture for the immobilized lipase was 50 ℃,5 ℃ higher than that for free lipase.At the same time,the immobilized lipase showed better mechanical stability and better storage stability.These results show that the immobilization of lipase by adsorption-crosslinking method using kieselguhr as the absorbant possesses good application pros?pect in industry.
引文
[1]Gandi N N.Application of lipase[J].Journal of the American Oil Chemists’Society,1997,74(6):621-634.
[2]相欣然,黄和,胡燚.纳米复合材料固定化酶的研究进展[J].无机化学学报,2017,33(1):1-15.Xiang X R,Huang H,Hu Y.Research progress on enzyme immobilized on nanocomposites[J].Chinese Journal of InorganicChemistry,2017,33(1):1-15.
[3]Tran D N,Balkus K J.Perspective of recent progress in immobilization of enzymes[J].ACS Catalysis,2011,1(8):956-968.
[4]Rodrigues R C,Oritiz C,Berenguer-Murcia A,et al. Modifying enzyme activity and selectivity by immobilization[J]Chemical Society Reviews,2013,42(15):6290-6307.
[5]Zhang Y,Ge J,Liu Z.Enhanced activity of immobilized or chemically modified enzymes[J].ACS Catalysis,2015,5(8):4503-4513.
[6]韩志萍,叶剑芝,罗荣琼.固定化酶的方法及其在食品中的应用研究进展[J].保鲜与加工,2012,12(5):48-53.Han Z P,Ye J Z,Luo R Q.Progress of immobilized enzymes in preparation and application in food processing field[J].Stor?age and Process,2012,12(5):48-53.
[7]乔德亮,胡冰,曾晓雄.酶固定化及其在食品工业中应用新进展[J].食品工业科技,2008(1):304-308.Qiao D L,Hu B,Zeng X X.Immobilization of enzyme and its applications in food industry[J].Science and Technology ofFood Industry,2008(1):304-307.
[8]Hartmeier W.Immobilisierte biokatalysatoren[M].Springer Berlin Heidelberg,1986:18-20.
[9]冯超,蒋丽娟,黎继烈,等.固定化脂肪酶研究进展[J].食品工业科技,2011,32(2):373-375.Feng C,Jiang L J,Li J L,et al.Research progress in lipase immobilization[J].Science and Technology of Food Industry,2011,32(2):373-375.
[10]游金坤,余旭亚,赵鹏.吸附法固定化酶的研究进展[J].化学工程,2012,40(4):1-5.You J K,Yu X Y,Zhao P.Progress and trend of adsorption-based enzyme immobilization[J].Chemical Engineering,2012,40(4):1-5.
[11]贾鹏翔,刘雪莹.固定化酶及其在化工等领域中的应用[J].皮革科学与工程,2004,14(5):31-36.Jia P X,Liu X Y.Immobilized enzyme and its application in fields including chemical engineering[J].Leather Science andEngineering,2004,14(5):31-36.
[12]Jesionowski T,Zdarta J,Krajewska B.Enzyme immobilization by adsorption[J].Adsorption-journal of the InternationalAdsorption Society,2014,20(5/6):801-821.
[13]李勤.固定化技术在食品工业中的应用和发展研究[J].四川食品与发酵,2006,42(132):16-19.Li Q.Study of application and development of immobilization technique in food industry[J].Sichuan Food and Fermentaion,2006,42(132):16-19.
[14]秦胜利,于建生.酶固定化技术研究进展[J].河南化工,2011,28(3):24-32.Qin S L,Yu J S.Research progress of enzyme immobilization techniques[J].Henan Chemical Industry,2011,28(3):24-32.
[15]Yang J J,Ma X X,Zhang Z S,et al.Lipase immobilized by modification-coupled and adsorption-cross-linking methods:acomparative study[J].Biotechnology Advances,2010,28(5):644-650.
[16]Cunha A G,Fernández-Lorente G,Bevilaqua J V,et al.Immobilization of Yarrowia lipolytica lipase—a comparison ofstability of physical adsorption and covalent attachment techniques[J].Applied Biochemistry and Biotechnology,2008,146(1/2/3):49-56.
[17]Jones P O,Vasudevan P T.Cellulose hydrolysis by immobilized Trichoderma reesei cellulase[J].Biotechnology Letters,2010,32(1):103-106.
[18]杨杰,张玉彬,吴梧桐.固定化酶技术及其在医药上的应用新进展[J].药物生物技术,2013,20(6):553-556.Yang J,Zhang Y B,Wu W T.Immobilized enzyme technology and its application in medicine[J].Pharmaceutical Biotechnol?ogy,2013,20(6)549-552.
[19]肖海军,贺筱蓉.固定化酶及其应用研究进展[J].生物学通报,2001,36(7):9-10.Xiao H J,He X R.Immobilized enzyme and its application research progress[J].Bulletin of Biology,2001,36(7):9-10.
[20]陈冬梅.固定化酶及在食品工业中的应用[J].现代农业科技,2010(19):330-332.Chen D M.Immobilized enzymes and ITS application in the food industry[J].Modern Agricultural Science and Technology,2010(19):330-332.
[21]候爱军,徐冰斌,梁亮,等.改进铜皂-分光光度法测定脂肪酶活力[J].皮革科学与工程,2011,21(1):22-27.Hou A J,Xu B B,Liang L,et al.A modified colorimetric assay of lipase activity using emulsified olive oil as the substrate[J].Leather Science and Engineering,2011,21(1):22-27.
[22]GB/T 23535-2009,脂肪酶制剂[S].北京:中国标准出版社,2009.GB/T 23535-2009,Lipase prepatations[S].Beijing:Standards Press of China,2009.
[23]杨宇翔,吴介达,黄忠良,等.几种硅藻土的表面电化学性质的研究[J].无机化学学报,1997,13(1):12-15.Yang Y X,Wu J D,Huang Z L,et al.Study on surface electrochemical properties of several kieselguhr[J].Chinese Journal ofInorganic Chemistry,1997,13(1):12-15.
[24]高贵,韩四平,王智,等.硅藻土固定化脂肪酶[J].吉林大学学报(理学版),2002,40(3):324-326.Gao G,Han S P,Wang Z,et al.Kieselguhr immobilized lipase[J].Journal of Jilin University(Science Edition),2002,40(3):324-326.
[25]Bellino M G,Regazzonia A E,Soler-Illia G J A A.Amylase-functionalized mesoporous silica thin filmsas robust biocatalystplatforms[J].Acs Applied Materials&Interface.2010,2(2):360-365.
[26]杨艳红,吴克,王华林,等.硅藻土固定中性脂肪酶的制备及其性质[J].生物学杂志,2011,28(1):19-21.Yang Y H,Wu K,Wang H L,et al.Preparation and properties of neutrophil lipase immobilized by diatomine[J].Journal ofBiology,2011,28(1):19-21.
[27]顾旭炯,刘璘,楼坚,等.硅藻土共固定化淀粉酶和糖化酶的研究[J].生物技术,2006,27(18):24-28.Gu X J,Liu L,Lou J,et al.Studies on coimmobilization ofα-Amylase and glucoamylas on celite[J].Biotechnology,2006,27(18):24-28.
[28]郑毅,郑楠,吴松刚,等.固定化脂肪酶选择性富集鱼油ω-3多不饱和脂肪酸甘油酯[J].化工学报,2006,57(2):353-358.Zheng Y,Zheng N,Wu S G,et al.Selective concentration of PUFAs in glyceride from fish oil with immobilized lipase[J].CI?ESC Journal,2006,57(2):353-358.
[2]相欣然,黄和,胡燚.纳米复合材料固定化酶的研究进展[J].无机化学学报,2017,33(1):1-15.Xiang X R,Huang H,Hu Y.Research progress on enzyme immobilized on nanocomposites[J].Chinese Journal of InorganicChemistry,2017,33(1):1-15.
[3]Tran D N,Balkus K J.Perspective of recent progress in immobilization of enzymes[J].ACS Catalysis,2011,1(8):956-968.
[4]Rodrigues R C,Oritiz C,Berenguer-Murcia A,et al. Modifying enzyme activity and selectivity by immobilization[J]Chemical Society Reviews,2013,42(15):6290-6307.
[5]Zhang Y,Ge J,Liu Z.Enhanced activity of immobilized or chemically modified enzymes[J].ACS Catalysis,2015,5(8):4503-4513.
[6]韩志萍,叶剑芝,罗荣琼.固定化酶的方法及其在食品中的应用研究进展[J].保鲜与加工,2012,12(5):48-53.Han Z P,Ye J Z,Luo R Q.Progress of immobilized enzymes in preparation and application in food processing field[J].Stor?age and Process,2012,12(5):48-53.
[7]乔德亮,胡冰,曾晓雄.酶固定化及其在食品工业中应用新进展[J].食品工业科技,2008(1):304-308.Qiao D L,Hu B,Zeng X X.Immobilization of enzyme and its applications in food industry[J].Science and Technology ofFood Industry,2008(1):304-307.
[8]Hartmeier W.Immobilisierte biokatalysatoren[M].Springer Berlin Heidelberg,1986:18-20.
[9]冯超,蒋丽娟,黎继烈,等.固定化脂肪酶研究进展[J].食品工业科技,2011,32(2):373-375.Feng C,Jiang L J,Li J L,et al.Research progress in lipase immobilization[J].Science and Technology of Food Industry,2011,32(2):373-375.
[10]游金坤,余旭亚,赵鹏.吸附法固定化酶的研究进展[J].化学工程,2012,40(4):1-5.You J K,Yu X Y,Zhao P.Progress and trend of adsorption-based enzyme immobilization[J].Chemical Engineering,2012,40(4):1-5.
[11]贾鹏翔,刘雪莹.固定化酶及其在化工等领域中的应用[J].皮革科学与工程,2004,14(5):31-36.Jia P X,Liu X Y.Immobilized enzyme and its application in fields including chemical engineering[J].Leather Science andEngineering,2004,14(5):31-36.
[12]Jesionowski T,Zdarta J,Krajewska B.Enzyme immobilization by adsorption[J].Adsorption-journal of the InternationalAdsorption Society,2014,20(5/6):801-821.
[13]李勤.固定化技术在食品工业中的应用和发展研究[J].四川食品与发酵,2006,42(132):16-19.Li Q.Study of application and development of immobilization technique in food industry[J].Sichuan Food and Fermentaion,2006,42(132):16-19.
[14]秦胜利,于建生.酶固定化技术研究进展[J].河南化工,2011,28(3):24-32.Qin S L,Yu J S.Research progress of enzyme immobilization techniques[J].Henan Chemical Industry,2011,28(3):24-32.
[15]Yang J J,Ma X X,Zhang Z S,et al.Lipase immobilized by modification-coupled and adsorption-cross-linking methods:acomparative study[J].Biotechnology Advances,2010,28(5):644-650.
[16]Cunha A G,Fernández-Lorente G,Bevilaqua J V,et al.Immobilization of Yarrowia lipolytica lipase—a comparison ofstability of physical adsorption and covalent attachment techniques[J].Applied Biochemistry and Biotechnology,2008,146(1/2/3):49-56.
[17]Jones P O,Vasudevan P T.Cellulose hydrolysis by immobilized Trichoderma reesei cellulase[J].Biotechnology Letters,2010,32(1):103-106.
[18]杨杰,张玉彬,吴梧桐.固定化酶技术及其在医药上的应用新进展[J].药物生物技术,2013,20(6):553-556.Yang J,Zhang Y B,Wu W T.Immobilized enzyme technology and its application in medicine[J].Pharmaceutical Biotechnol?ogy,2013,20(6)549-552.
[19]肖海军,贺筱蓉.固定化酶及其应用研究进展[J].生物学通报,2001,36(7):9-10.Xiao H J,He X R.Immobilized enzyme and its application research progress[J].Bulletin of Biology,2001,36(7):9-10.
[20]陈冬梅.固定化酶及在食品工业中的应用[J].现代农业科技,2010(19):330-332.Chen D M.Immobilized enzymes and ITS application in the food industry[J].Modern Agricultural Science and Technology,2010(19):330-332.
[21]候爱军,徐冰斌,梁亮,等.改进铜皂-分光光度法测定脂肪酶活力[J].皮革科学与工程,2011,21(1):22-27.Hou A J,Xu B B,Liang L,et al.A modified colorimetric assay of lipase activity using emulsified olive oil as the substrate[J].Leather Science and Engineering,2011,21(1):22-27.
[22]GB/T 23535-2009,脂肪酶制剂[S].北京:中国标准出版社,2009.GB/T 23535-2009,Lipase prepatations[S].Beijing:Standards Press of China,2009.
[23]杨宇翔,吴介达,黄忠良,等.几种硅藻土的表面电化学性质的研究[J].无机化学学报,1997,13(1):12-15.Yang Y X,Wu J D,Huang Z L,et al.Study on surface electrochemical properties of several kieselguhr[J].Chinese Journal ofInorganic Chemistry,1997,13(1):12-15.
[24]高贵,韩四平,王智,等.硅藻土固定化脂肪酶[J].吉林大学学报(理学版),2002,40(3):324-326.Gao G,Han S P,Wang Z,et al.Kieselguhr immobilized lipase[J].Journal of Jilin University(Science Edition),2002,40(3):324-326.
[25]Bellino M G,Regazzonia A E,Soler-Illia G J A A.Amylase-functionalized mesoporous silica thin filmsas robust biocatalystplatforms[J].Acs Applied Materials&Interface.2010,2(2):360-365.
[26]杨艳红,吴克,王华林,等.硅藻土固定中性脂肪酶的制备及其性质[J].生物学杂志,2011,28(1):19-21.Yang Y H,Wu K,Wang H L,et al.Preparation and properties of neutrophil lipase immobilized by diatomine[J].Journal ofBiology,2011,28(1):19-21.
[27]顾旭炯,刘璘,楼坚,等.硅藻土共固定化淀粉酶和糖化酶的研究[J].生物技术,2006,27(18):24-28.Gu X J,Liu L,Lou J,et al.Studies on coimmobilization ofα-Amylase and glucoamylas on celite[J].Biotechnology,2006,27(18):24-28.
[28]郑毅,郑楠,吴松刚,等.固定化脂肪酶选择性富集鱼油ω-3多不饱和脂肪酸甘油酯[J].化工学报,2006,57(2):353-358.Zheng Y,Zheng N,Wu S G,et al.Selective concentration of PUFAs in glyceride from fish oil with immobilized lipase[J].CI?ESC Journal,2006,57(2):353-358.