添加剂对大孔吸附树脂固定化脂肪酶的影响
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摘要
利用大孔吸附树脂DA-201为载体对海洋脂肪酶固定化,并探寻添加剂对固定化过程的影响。分别以NH_4Cl、甘露糖和甘氨酸为添加剂,采用单因素和正交实验相结合的方法优化条件。结果显示,以NH_4Cl为添加剂的最优条件:柠檬酸-柠檬酸钠缓冲液pH 6. 0,固定化温度30℃,载体投放量0. 5g,NH_4Cl浓度25mmol/L,固定化时间3. 0h,酶活力达到115. 27U/g;比不含有添加剂的固定化酶固定化效率提高47. 42%。以甘露糖为添加剂最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度35℃,载体投放量0. 5g,甘露糖浓度10mmol/L,固定化时间4. 5h;酶活力达到122. 75U/g,比不含有添加剂的固定化酶固定化效率提高6. 50%。以甘氨酸为添加剂的最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度20℃,载体投放量0. 5g,甘氨酸浓度为25mmol/L,固定化时间7. 5h;酶活力达到141. 69U/g,比不含有添加剂的固定化酶固定化效率提高26. 12%。采用不同添加剂对大孔吸附树脂DA-201的吸附固定化过程有较大影响,可以极大地提高吸附效率;同时发现缓冲液类型、pH、温度、添加剂浓度和固定化时间等对DA-201树脂吸附脂肪酶有很大影响,对后续吸附固定化工业酶研究有较好的参考价值。
Macroporous absorbent resin DA-201 was adopted as a carrier to immobilize marine-derived lipase,and the influence of additives on the immobilization process was explored. NH_4Cl,mannose and glycine were used as the additives,and the conditions were optimized by a combination of single factor and orthogonal experiments. The results showed that the optimal conditions with additive NH_4Cl were: citric acid-sodium citrate buffer pH6. 0,immobilization temperature 30℃,carrier quantity 0. 5 g, NH_4Cl concentration 25 mmol/L,immobilized time 3. 0 h; enzyme activity reached 115. 27 U/g,which was 47. 42% higher than that of the immobilized enzyme without additive. The optimal conditions with additive mannose were: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 35℃,carrier quantity 0. 5 g,mannose concentration 10 mmol/L,the immobilized time 4. 5 h; the enzyme activity reached 122. 75 U/g,which was6. 50% higher than that of the immobilized enzyme without additive. The optimal conditions with glycine additive: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 20℃,carrier quantity 0. 5 g,the glycine concentration 25 mmol/L,the immobilized time 7. 5 h; the enzyme activity reached 141. 69 U/g,which was 26. 12% higher than that of the immobilized enzyme without additive. The addition of different additives exhibited great effects on the immobilization through absorbtion by macroporous absorbent resin DA-201 and could greatly improve the adsorption efficiency. Additionally,buffer type,pH,temperature,additive concentration and immobilization time were found to have great influence on the adsorption of lipase by resin DA-201,which provides good reference for subsequent research of the immobilization of industrial enzymes.
Macroporous absorbent resin DA-201 was adopted as a carrier to immobilize marine-derived lipase,and the influence of additives on the immobilization process was explored. NH_4Cl,mannose and glycine were used as the additives,and the conditions were optimized by a combination of single factor and orthogonal experiments. The results showed that the optimal conditions with additive NH_4Cl were: citric acid-sodium citrate buffer pH6. 0,immobilization temperature 30℃,carrier quantity 0. 5 g, NH_4Cl concentration 25 mmol/L,immobilized time 3. 0 h; enzyme activity reached 115. 27 U/g,which was 47. 42% higher than that of the immobilized enzyme without additive. The optimal conditions with additive mannose were: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 35℃,carrier quantity 0. 5 g,mannose concentration 10 mmol/L,the immobilized time 4. 5 h; the enzyme activity reached 122. 75 U/g,which was6. 50% higher than that of the immobilized enzyme without additive. The optimal conditions with glycine additive: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 20℃,carrier quantity 0. 5 g,the glycine concentration 25 mmol/L,the immobilized time 7. 5 h; the enzyme activity reached 141. 69 U/g,which was 26. 12% higher than that of the immobilized enzyme without additive. The addition of different additives exhibited great effects on the immobilization through absorbtion by macroporous absorbent resin DA-201 and could greatly improve the adsorption efficiency. Additionally,buffer type,pH,temperature,additive concentration and immobilization time were found to have great influence on the adsorption of lipase by resin DA-201,which provides good reference for subsequent research of the immobilization of industrial enzymes.
引文
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[17]Chang C S,Hsu C S.Enhancement of enantioselectivity on reaction rate of the synthesis of(S)-ketoprofen hydroxyalkyl ester in organic solvents via isopropanol-dried immobilized lipase.Jonrnal of Chemical Technology and Biotechnology,2005,80(5):537-544.
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[19]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.皮革科学与工程,2011,21(1):22-27.Hou A J,Xu B B,Liang L,et al.Improved copper soapspectrophotometry for determination of lipase activity.Leather Science and Engineering,2011,21(1):22-27.
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[21]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.扬州:扬州大学,2013.Hou L Y.Lipase immobilization and its application in catalytic synthesis of citronellyl acetate.Yangzhou:Yangzhou University,2013.
[22]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.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.Journal of Guangxi Normal University(Natural Science Edition),2018,36(4):108-118.
[2]蒋振华,于敏,任立伟,等.有机相中固定化脂肪酶催化合成植物甾醇酯.催化学报,2013,34(12):2255-2262.Jiang Z H,Yu M,Ren L W,et al.Catalytic synthesis of phytosterol esters by immobilized lipase in organic phase.Chinese Journal of Catalysis,2013,34(12):2255-2262.
[3]赵天涛,高静,张丽杰,等.有机相中脂肪酶催化合成乳酸乙酯.催化学报,2006,27(6):537-540.Zhao T T,Gao J,Zhang L J,et al.Catalytic synthesis of ethyl lactate by lipase in organic phase.Chinese Journal of Catalysis,2006,27(6):537-540.
[4]相欣然,黄和,胡燚.纳米复合材料固定化酶的研究进展.无机化学学报,2017,33(1):1-15.Xiang X R,Huang H,Hu Y.Advances in research on immobilized enzymes of nanocomposites.Journal of Inorganic Chemistry,2017,33(1):1-15.
[5]Tran D N,Balkus K J.Perspective of recent progress in immobilization of enzymes.ACS Catalysis,2011,1(8):956-968.
[6]Rodrigues R C,Oritiz C,Berenguer-Murcia A,et al.Modifying enzyme activity and selectivity by immobilization.Chemical Society Reviews.,2013,42(15):6290-6307.
[7]Zhang Y,Ge J,Liu Z.Enhanced activity of immobilized or chemically modified enzymes.ACS Catalysis,2015,5(8):4503-4513.
[8]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.保鲜与加工,2012,12(5):48-53.Han Z P,Ye J Z,Luo R Q.Advances in immobilized enzymes and their applications in foods.Preservation and Processing,2012,12(5):48-53.
[9]徐珊,李任强,张继福,等.乙二醇缩水甘油醚交联海藻酸钠-羧甲基纤维素钠固定化脂肪酶.中国生物工程杂志,2017,37(12):77-83.Xu S,Li R Q,Zhang J F,et al.Ethylene glycol diglycidyl ether cross-linked with sodium alginate-carboxymethyl cellulose to immobilized lipase.China Biotechnology,2017,37(12):77-83.
[10]游金坤,余旭亚,赵鹏.吸附法固定化酶的研究进展.化学工程,2012,40(4):1-5.You J K,Yu X Y,Zhao P.Advances in adsorption of immobilized enzymes.Chemical Engineering,2012,40(4):1-5.
[11]王跃生,王洋.大孔吸附树脂研究进展.中国中药杂志,2006,31(12):961-965.Wang Y S,Wang Y.Research progress of macroporous adsorption resin.Chinese Journal of Traditional Chinese Medicine,2006,31(12):961-965.
[12]黄永林,阮俊,沈晓琳,等.大孔吸附树脂分离提取大叶钩藤总生物碱.广西科学,2006,13(2):127-129.Huang Y L,Ruan J,Shen X L,et al.Separation and extraction of total alkaloids from Uncaria macrophylla by macroporous adsorption resin.Guangxi Science,2006,13(2):127-129.
[13]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用.中药材,2005,28(3):239-242.Lv J L,Yang Z H,Yuan K.Application of new gel resin and macroporous resin in separation and purification of Chinese herbal medicine components.Chinese Medicinal Materials,2005,28(3):239-242.
[14]谢雪凤,张朝晖,陈培策.AB-8大孔吸附树脂固定化过氧化氢酶的研究.材料导报,2009,23(18):50-53.Xie X F,Zhang Z H,Chen P C.Research on immobilization of catalase by AB-8 macroporous adsorption resin.Materials Review,2009,23(18):50-53.
[15]赵庆节,余莹,周文瑜,等.有机相中大孔吸附树脂固定化酶催化拆分消旋萘普生.华东理工大学学报,2001,27(4):423-426.Zhao Q J,Yu Y,Zhou W Y,et al.Catalytic resolution of racemic naproxen by macroporous adsorption resin immobilized enzyme in organic phase.Journal of East China University of Science and Technology,2001,27(4):423-426.
[16]王海雄,吴侯,翁新楚.大孔吸附树脂固定猪胰脂酶的初步研究.生物技术,2004,14(3):27-30.Wang H X,Wu H,Weng X C.Preliminary study on fixation of porcine pancreatic lipase by macroporous adsorption resin.Biotechnology,2004,14(3):27-30.
[17]Chang C S,Hsu C S.Enhancement of enantioselectivity on reaction rate of the synthesis of(S)-ketoprofen hydroxyalkyl ester in organic solvents via isopropanol-dried immobilized lipase.Jonrnal of Chemical Technology and Biotechnology,2005,80(5):537-544.
[18]彭维,欧爱芬.金属离子对酶活性影响研究.酿酒,2013,40(1):60-62.Peng W,Ou A F.Research on the effect of metal ions on enzyme activity.Wine Making,2013,40(1):60-62.
[19]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.皮革科学与工程,2011,21(1):22-27.Hou A J,Xu B B,Liang L,et al.Improved copper soapspectrophotometry for determination of lipase activity.Leather Science and Engineering,2011,21(1):22-27.
[20]袁勤生.酶与酶工程.第二版.上海:华东理工大学出版社,2012:38-39.Yuan Q S.Enzyme and enzyme engineering.2nded.Shanghai:East China University of Science and Technology Press,2012:38-39.
[21]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.扬州:扬州大学,2013.Hou L Y.Lipase immobilization and its application in catalytic synthesis of citronellyl acetate.Yangzhou:Yangzhou University,2013.
[22]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.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.Journal of Guangxi Normal University(Natural Science Edition),2018,36(4):108-118.