基于无机载体的先交联后吸附固定化脂肪酶的方法
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摘要
目的:使用双功能环氧交联剂新戊二醇二缩水甘油醚和硅藻土载体固定化海洋脂肪酶,并探究固定化酶的酶学性质.方法:采用先交联后吸附的方法固定化海洋脂肪酶,交联和吸附分别进行单因素与正交实验结合的实验方案,并测定固定化酶的酶活力.结果:确定最佳交联条件:pH 5.5,温度35℃,交联剂质量分数0.5%,交联时间2 h;最佳吸附条件:温度40℃,载体质量1.5 g,吸附时间5 h.在此工艺条件下,所制备的固定化脂肪酶活力为1.89μkat,酶活力回收率72.89%.与游离的脂肪酶相比,所制备的固定化脂肪酶具有更好的温度、pH、储存及操作稳定性.结论:基于环氧交联剂新戊二醇二缩水甘油醚和硅藻土载体,进行先交联后吸附固定化海洋脂肪酶的技术具有良好的工业应用前景.
Objective:The purpose of this research is to immobilize marine lipase with bifunctional epoxy cross-linking neopentyl glycol diglycidyl ether and kieselguhr, and to explore the enzymatic properties of immobilized enzyme. Methods: The marine lipase was immobilized by pre-crosslinking and adsorption, and the experimental scheme combining single factors and orthogonal experiment were carried out, and then the enzyme activity of the immobilized enzyme was determind. Results: The optimum crosslinking conditions were determined as follows: pH 5.5, temperature 35 ℃, crosslinker concentration 0.5%, crosslinking time 2 h; optimal adsorption conditions were: temperature 40 ℃, carrier quantity 1.5 g, adsorption time 5 h. Under the optimal process conditions, the prepared immobilized lipase activity reached 1.89 μkat, and the enzyme activity recovery rate reached 72.89%. Compared with free lipase, the immobilized lipase exhibited better thermo-stability and pH-stability; meanwhile, it also showed good storage stability, with 87% of its original activity left after being stored at 4 ℃ for 60 days. The immobilized lipase maintains more than 60% of its original activity after 5 consecutive operations. Conclusion:Based on the epoxy cross-linking agent neopentyl glycol diglycidyl ether and kieselguhr carrier, marine lipase adsorbed and immobilized after cross-linking has good industrial application prospects.
Objective:The purpose of this research is to immobilize marine lipase with bifunctional epoxy cross-linking neopentyl glycol diglycidyl ether and kieselguhr, and to explore the enzymatic properties of immobilized enzyme. Methods: The marine lipase was immobilized by pre-crosslinking and adsorption, and the experimental scheme combining single factors and orthogonal experiment were carried out, and then the enzyme activity of the immobilized enzyme was determind. Results: The optimum crosslinking conditions were determined as follows: pH 5.5, temperature 35 ℃, crosslinker concentration 0.5%, crosslinking time 2 h; optimal adsorption conditions were: temperature 40 ℃, carrier quantity 1.5 g, adsorption time 5 h. Under the optimal process conditions, the prepared immobilized lipase activity reached 1.89 μkat, and the enzyme activity recovery rate reached 72.89%. Compared with free lipase, the immobilized lipase exhibited better thermo-stability and pH-stability; meanwhile, it also showed good storage stability, with 87% of its original activity left after being stored at 4 ℃ for 60 days. The immobilized lipase maintains more than 60% of its original activity after 5 consecutive operations. Conclusion:Based on the epoxy cross-linking agent neopentyl glycol diglycidyl ether and kieselguhr carrier, marine lipase adsorbed and immobilized after cross-linking has good industrial application prospects.
引文
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[10] 孟策,戴小敏,刘袖洞.固定化脂肪酶的载体材料[J].辽宁化工,2018,47(12):1244-1249.MENG C,DAI X M,LIU S D.Carrier materials for immobilized lipase[J].Liaoning Chemical Industry,2018,47(12):1244-1249.
[11] 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.
[12] 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].Biology Journal,2011,28(1):19-21.
[13] 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].饲料工业,2006,27(18):24-28.GU X J,LIU L,LOU J,et al.Studies on coimmobilization of α-amylase and glucoamylase on celite[J].Feed Industry,2006,27(18):24-28.
[14] 徐珊,李任强,郑振华,等.脂肪酶的包埋和交联固定化研究[J].云南农业大学学报(自然科学),2017,32(6):959-966.XU S,LI R Q,ZHENG Z H,et al.Encapsulation and cross-linking immobilization of lipase[J].Journal of Yunnan Agricultural University (Natural Sciences),2017,32(6):959-966.
[15] 钱明华,张继福,张云,等.大孔树脂吸附-交联法固定脂肪酶[J].华南农业大学学报,2019,40(2):103-110.QIAN M H,ZHANG J F,ZHANG Y,et al.Immobilization of lipase on macroporous resin by adsorption-crosslinking method[J].Journal of South China Agricultural University,2019,40(2):103-110.
[16] 许超群,万云宝,张小龙,等.吸附交联法固定化黑曲霉β-葡萄糖苷酶[J].中国酿造,2016,35(4):83-87.XU C Q,WAN Y B,ZHANG X L,et al.Immobilization of β-glucosidase from Aspergillus niger by adsorption and crosslinking method[J].Chinese Brewing,2016,35(4):83-87.
[17] 林海蛟,王云鹏,张云,等.无机载体吸附-交联固定化海洋脂肪酶技术研究[J].江西农业大学学报,2019,41(1):186-196.LIN H J,WANG Y P,ZHANG Y,et al.Adsorption-crosslinking immobilization of marine lipase by inorganic carrier[J].Journal of Jiangxi Agricultural University,2019,41(1):186-196.
[18] 袁勤生.酶与酶工程[M].2版.上海:华东理工大学出版社,2012:38-39.YUAN Q S.Enzyme and enzyme engineering[M].2nd ed.Shanghai:East China University of Science and Technology Press,2012:38-39.
[19] 侯爱军,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.
[20] 徐珊,李任强,张继福,等.使用国产环氧树脂LXEP-120固定化脂肪酶研究[J].广西师范大学学报(自然科学版),2018,36(4):108-118.XU S,LI R Q,ZHANG J F,et al.Immobilization of lipase using domestic epoxy resin LXEP-120[J].Journal of Guangxi Normal University(Natural Science Edition),2018,36(4):108-118.
[21] TORRES P,BATISTA-VIERA F.Immobilization of β-galactosidase from bacillus circulans onto epoxy-activated acrylic supports[J].Journal of Molecular Catalysis B:Enzymatic,2012,74(3/4):230-235.
[22] 嵇东情,郑仁朝,郑裕国.环氧树脂固定化拟南芥腈水解酶NIT的研究[J].精细与专用化学品,2015,23(5):12-16.GENG D Q,ZHENG R C,ZHENG Y G.Study on immobilization of arabidopsis nitril hydrolase NIT by epoxy resin[J].Fine and Specialty Chemicals,2015,23(5):12-16.
[23] 崔培梧,黎继烈,肖作为,等.环氧基树脂固定化青霉菌柚苷酶的工艺条件[J].食品与发酵工业,2014,40(5):87-92.CUI P W,LI J L,XIAO Z W,et al.Process conditions of immobilized penicillin naringin by epoxy resin[J].Food and Fermentation Industries,2014,40(5):87-92.
[24] 候丽云.脂肪酶固定化及其在催化合成乙酸香茅酯中的应用研究[D].扬州:扬州大学,2013.HOU L Y.Lipase immobilization and its application in catalytic synthesis of citronellyl acetate[D].Yangzhou:Yangzhou University,2013.
[25] GUERRERO C,VERA C,ARAYA E,et al.Repeated-batch operation for synthesis of lactulose with beta-galactosidase immobilized by aggregation and crossing[J].Bioresource Technology,2015,190:122.
[26] CORTEZ L M,ROMERO J,GONZALEZ V,et al.Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor[J].Materials Science & Engineering:C,2015,52:306.
[27] HAMERSKA-DUDRA,BRYJAK J,TROCHIMCZUK A W.Immobilization of glucoamylase and trypsin on crosslinked thermosensitive carriers[J].Enzyme and Technology,2007,41(3):197.
[28] 孙玉英,张继泉.吸附交联法和包埋法固定化混合酶研究[J].淮海工学院学报(自然科学版),2013,22(3):82-88.SUN Y Y,ZHANG J Q.Study on immobilized mixed enzymes by adsorption cross-linking method and embedding method[J].Journal of Huaihai Institute of Technology(Natural Science Edition),2013,22(3):82-88.
[29] 鱼园.载体吸附交联及无载体交联固定化磷脂酶D方法的研究[D].西安:西北大学,2010.YU Y.Carrier adsorption cross-linking and carrier-free cross-linking immobilization of phospholipase D method[D].Xi′an:Northwest University,2010.
[2] TRAN D N,BALKUS K J.Perspective of recent progress in immobilization of enzymes[J].ACS Catalysis,2011,1(8):956-968.
[3] RODRIGUES R C,ORTIZ C,BERENGUER-MURCIA A,et al.Modifying enzyme activity and selectivity by immobilization[J].Chemical Society Reviews,2013,42(15):6290-6307.
[4] ZHANG Y F,GE J,LIU Z.Enhanced activity of immobilized or chemically modified enzymes[J].ACS Catalysis,2015,5(8):4503-4513.
[5] 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].Storage & Process,2012,12(5):48-53.
[6] 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.
[7] 胡鹏程,陈光.酶固定化研究进展[J].安徽农业科学,2014,42(14):4185-4186,4204.HU P C,CHEN G.Research progress in enzyme immobilization[J].Journal of Anhui Agricultural Sciences,2014,42(14):4185-4186,4204.
[8] JESIONOWSKI T,ZDARTA J,KRAJEWSKA B.Enzyme immobilization by adsorption[J].Adsorption,2014,20(5/6):801-821.
[9] 李阳,韦伟,曹茜,等.脂肪酶固定化新材料[J].中国粮油学报,2014,29(7):122-128.LI Y,WEI W,CAO X,et al.New materials for immobilized lipase[J].Journal of the Chinese Cereals and Oils Association,2014,29(7):122-128.
[10] 孟策,戴小敏,刘袖洞.固定化脂肪酶的载体材料[J].辽宁化工,2018,47(12):1244-1249.MENG C,DAI X M,LIU S D.Carrier materials for immobilized lipase[J].Liaoning Chemical Industry,2018,47(12):1244-1249.
[11] 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.
[12] 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].Biology Journal,2011,28(1):19-21.
[13] 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].饲料工业,2006,27(18):24-28.GU X J,LIU L,LOU J,et al.Studies on coimmobilization of α-amylase and glucoamylase on celite[J].Feed Industry,2006,27(18):24-28.
[14] 徐珊,李任强,郑振华,等.脂肪酶的包埋和交联固定化研究[J].云南农业大学学报(自然科学),2017,32(6):959-966.XU S,LI R Q,ZHENG Z H,et al.Encapsulation and cross-linking immobilization of lipase[J].Journal of Yunnan Agricultural University (Natural Sciences),2017,32(6):959-966.
[15] 钱明华,张继福,张云,等.大孔树脂吸附-交联法固定脂肪酶[J].华南农业大学学报,2019,40(2):103-110.QIAN M H,ZHANG J F,ZHANG Y,et al.Immobilization of lipase on macroporous resin by adsorption-crosslinking method[J].Journal of South China Agricultural University,2019,40(2):103-110.
[16] 许超群,万云宝,张小龙,等.吸附交联法固定化黑曲霉β-葡萄糖苷酶[J].中国酿造,2016,35(4):83-87.XU C Q,WAN Y B,ZHANG X L,et al.Immobilization of β-glucosidase from Aspergillus niger by adsorption and crosslinking method[J].Chinese Brewing,2016,35(4):83-87.
[17] 林海蛟,王云鹏,张云,等.无机载体吸附-交联固定化海洋脂肪酶技术研究[J].江西农业大学学报,2019,41(1):186-196.LIN H J,WANG Y P,ZHANG Y,et al.Adsorption-crosslinking immobilization of marine lipase by inorganic carrier[J].Journal of Jiangxi Agricultural University,2019,41(1):186-196.
[18] 袁勤生.酶与酶工程[M].2版.上海:华东理工大学出版社,2012:38-39.YUAN Q S.Enzyme and enzyme engineering[M].2nd ed.Shanghai:East China University of Science and Technology Press,2012:38-39.
[19] 侯爱军,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.
[20] 徐珊,李任强,张继福,等.使用国产环氧树脂LXEP-120固定化脂肪酶研究[J].广西师范大学学报(自然科学版),2018,36(4):108-118.XU S,LI R Q,ZHANG J F,et al.Immobilization of lipase using domestic epoxy resin LXEP-120[J].Journal of Guangxi Normal University(Natural Science Edition),2018,36(4):108-118.
[21] TORRES P,BATISTA-VIERA F.Immobilization of β-galactosidase from bacillus circulans onto epoxy-activated acrylic supports[J].Journal of Molecular Catalysis B:Enzymatic,2012,74(3/4):230-235.
[22] 嵇东情,郑仁朝,郑裕国.环氧树脂固定化拟南芥腈水解酶NIT的研究[J].精细与专用化学品,2015,23(5):12-16.GENG D Q,ZHENG R C,ZHENG Y G.Study on immobilization of arabidopsis nitril hydrolase NIT by epoxy resin[J].Fine and Specialty Chemicals,2015,23(5):12-16.
[23] 崔培梧,黎继烈,肖作为,等.环氧基树脂固定化青霉菌柚苷酶的工艺条件[J].食品与发酵工业,2014,40(5):87-92.CUI P W,LI J L,XIAO Z W,et al.Process conditions of immobilized penicillin naringin by epoxy resin[J].Food and Fermentation Industries,2014,40(5):87-92.
[24] 候丽云.脂肪酶固定化及其在催化合成乙酸香茅酯中的应用研究[D].扬州:扬州大学,2013.HOU L Y.Lipase immobilization and its application in catalytic synthesis of citronellyl acetate[D].Yangzhou:Yangzhou University,2013.
[25] GUERRERO C,VERA C,ARAYA E,et al.Repeated-batch operation for synthesis of lactulose with beta-galactosidase immobilized by aggregation and crossing[J].Bioresource Technology,2015,190:122.
[26] CORTEZ L M,ROMERO J,GONZALEZ V,et al.Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor[J].Materials Science & Engineering:C,2015,52:306.
[27] HAMERSKA-DUDRA,BRYJAK J,TROCHIMCZUK A W.Immobilization of glucoamylase and trypsin on crosslinked thermosensitive carriers[J].Enzyme and Technology,2007,41(3):197.
[28] 孙玉英,张继泉.吸附交联法和包埋法固定化混合酶研究[J].淮海工学院学报(自然科学版),2013,22(3):82-88.SUN Y Y,ZHANG J Q.Study on immobilized mixed enzymes by adsorption cross-linking method and embedding method[J].Journal of Huaihai Institute of Technology(Natural Science Edition),2013,22(3):82-88.
[29] 鱼园.载体吸附交联及无载体交联固定化磷脂酶D方法的研究[D].西安:西北大学,2010.YU Y.Carrier adsorption cross-linking and carrier-free cross-linking immobilization of phospholipase D method[D].Xi′an:Northwest University,2010.
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