氨基载体共价结合固定化海洋假丝酵母脂肪酶
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摘要
共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。
Covalent binding is one important immobilization method of industrial enzyme,which uses stable covalent bonds to immobilize industrial enzymes,creates multi-point covalent connections between carrier and enzyme,and prepares immobilized enzymes with good stability and possesses practical application value. Marine Candida rugosa lipase was immobilized using amino carrier through covalent binding method and relative inexpensive glutaraldehyde was used as the crosslinking reagent. By using single factor and orthogonal experiment, the optimal immobilization conditions were determined as follows: 25℃, pH5. 0,0. 1%glutaraldehyde,0. 25 g carrier,crosslinked 0. 5 h,immobilization time 1 h,enzyme loading 800 U,with the final obtained enzyme activity being 83. 01 U/g. Compared with free enzyme,the optimal p H of immobilized lipase was shifted to alkaline direction,the optimal reaction temperature was increased by 10℃,the thermal stability and acid-alkali stability of immobilized enzyme were better than that of free enzyme,and the reusability and storage stability were better than that of free enzyme. Meanwhile,crosslinking agent was found to be an important factor in the preparation of immobilized lipase. Thus it is of great significance to explore new crosslinking agent to improve immobilization effect. A good foundation for the immobilization technique and industrial application of marine candida rugosa lipase were laid.
Covalent binding is one important immobilization method of industrial enzyme,which uses stable covalent bonds to immobilize industrial enzymes,creates multi-point covalent connections between carrier and enzyme,and prepares immobilized enzymes with good stability and possesses practical application value. Marine Candida rugosa lipase was immobilized using amino carrier through covalent binding method and relative inexpensive glutaraldehyde was used as the crosslinking reagent. By using single factor and orthogonal experiment, the optimal immobilization conditions were determined as follows: 25℃, pH5. 0,0. 1%glutaraldehyde,0. 25 g carrier,crosslinked 0. 5 h,immobilization time 1 h,enzyme loading 800 U,with the final obtained enzyme activity being 83. 01 U/g. Compared with free enzyme,the optimal p H of immobilized lipase was shifted to alkaline direction,the optimal reaction temperature was increased by 10℃,the thermal stability and acid-alkali stability of immobilized enzyme were better than that of free enzyme,and the reusability and storage stability were better than that of free enzyme. Meanwhile,crosslinking agent was found to be an important factor in the preparation of immobilized lipase. Thus it is of great significance to explore new crosslinking agent to improve immobilization effect. A good foundation for the immobilization technique and industrial application of marine candida rugosa lipase were laid.
引文
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[5]Cheng J,Zhuang W,Tang C L,et al.Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.Springer Berlin Heidelberg,2017,40(3):331-340.
[6]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.Progress and trend of adsorption-based enzyme immobilization.Chemicall Engineering(China),2012,40(4):1-5.
[7]Azevedo A M,Vojinovic'V,Cabral J M S,et al.Operational stability of immobilised horseradish peroxidase in mini-packed bed bioreactors.Elsevier B V,2004,28(2):121-128.
[8]Ding L,Yao Z H,Li T,et al.Synthesis of macroporous polymer carrier and immobilization of papain.Iranian Polymer Journal,2003,12(6):491-495.
[9]高贵,韩四平,王智,等.国内脂肪酶研究状况分析.生物技术通讯,2003,14(6):543-545.Gao G,Han S P,Wang Z,et al.Analysis of lipase research situation in China.Letters in Biotechnology,2003,14(6):543-545.
[10]Jakub Z,Anne S.M,Teofil J,et al.A general overview of support materials for enzyme immobilization:characteristics,properties,practical utility.MDPI,2018,8(2):1-27.
[11]尹春丽,曹珊珊,许乐,等.氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究.化学与生物工程,2014,31(9):17-20.Yin C L,Cao S S,Xu L,et al.Reseach of the immobilized enzyme of s-adenosine methionine by amino resin.Chemical and Bioengineering,2014,31(9):17-20.
[12]张丰华,孙宁,张伟,等.环氧基和氨基树脂固定化β-半乳糖苷酶的比较研究.中国农业科技导报,2014,16(5):47-52.Zhang F N,Sun N,Zhang W,et al.Comparison of epoxy and amino resin carrier in immobilization ofβ-galactosidase.Journal of Agricultural Science and Technology,2014,16(5):47-52.
[13]Li R Y,Fu G M,Liu C M,et al.Tannase immobilisation by aminofunctionalised magnetic Fe3O4-chitosan nanoparticles and its application in tea infusion.International Journal of Biological Macromolecules,2018,114:1134-1143.
[14]Huang J,Liu C,Xiao H Y,et al.Zinc tetraaminophthalocyanineFe3O4nanoparticle composite for laccase immobilization.International Journal of Nanomedicine,2008,2(4):84-775.
[15]Adinarayana K,Iraj G,Susana C,et al.Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers.Process Biochemistry,2008,43(2):169-178.
[16]Katja G,Anke K,Thomas M,et al.Novel immobilization routes for the covalent binding of an alcohol dehydrogenase from rhodococcus ruber DSM 44541.Tetrahedron:Asymmetry,2008,19(10):1171-1173.
[17]Dae Y K,Joon S R.A simple and rapid colorimetric method for determination of free fatty acids for lipase assay.Jaocs,1986,63(1):89-92.
[18]李晔.酶的固定化及其应用.分子催化,2008,22(1):86-96.Li Y.Immobilization of enzymes and their applications.Journal of Molecular Catalysis(China),2008,22(1):86-96.
[19]Prlainovic N Z,Knezevic-Jugovic Z D,Mijin D Z,et al.Immobilization of lipase from candida rugosa on sepabeads():the effect of lipase oxidation by periodates.Bioprocess and Biosystems Engineering,2011,34(7):10-803.
[20]闻臻臻.假丝酵母产脂肪酶固定化研究.杭州:浙江工业大学,2011.Wen Z Z.The immobilization of candida lipase.Hangzhou:Zhejiang University of Technology,2011.
[21]罗文,谭天伟,袁振宏,等.多孔玻璃珠固定化脂肪酶及其催化合成生物柴油.现代化工,2007,27(11):40-42.Luo W,Tan T W,Yuan Z H,et al.Immobilization of lipase by controlled pore glass and its applicationin biodiesel synthesis.Modern Chemical Industry,2007,27(11):40-42.
[22]Zang X Z,Xie W L.Enzymatic interesterification of soybean oil and methyl stearate blends using lipase immobilized on magnetic Fe3O4/SBA-15 composites as a biocatalyst.Journal of Oleo Science,2014,63(10):1027-1034.
[23]Mohammad K,Mohammad K,Ayyoob A.Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures.Biochemical Engineering Journal,2013,79(2013):267-273.
[2]魏雪,孙丽超,李淑英,等.脂肪酶的固定化及其在食品领域的应用.生物技术通报,2016,32(11):59-64.Wei X,Sun L C,Li S Y,et al.Immobilization of lipase and its application in food field.Biotechnology Bulletin,2016,32(11):59-64.
[3]张洪真.海绵共附生微生物脂肪酶产生菌筛选、培养基优化、酶学性质及基因克隆研究.上海:上海交通大学,2008.Zhang H Z.Screening,optimization of medium,property and gene cloning of lipase from bacteria associated with marine sponge.Shanghai:Shanghai Jiaotong University,2008.
[4]冯超,蒋丽娟,黎继烈,等.固定化脂肪酶研究进展.食品工业科技,2011,32(2):373-375,378.Feng C,Jiang L J,Ji J J,et al.Research progress in lipase immobilization.Science and Technology of Food Industry,2011,32(2):373-375,378.
[5]Cheng J,Zhuang W,Tang C L,et al.Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.Springer Berlin Heidelberg,2017,40(3):331-340.
[6]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.Progress and trend of adsorption-based enzyme immobilization.Chemicall Engineering(China),2012,40(4):1-5.
[7]Azevedo A M,Vojinovic'V,Cabral J M S,et al.Operational stability of immobilised horseradish peroxidase in mini-packed bed bioreactors.Elsevier B V,2004,28(2):121-128.
[8]Ding L,Yao Z H,Li T,et al.Synthesis of macroporous polymer carrier and immobilization of papain.Iranian Polymer Journal,2003,12(6):491-495.
[9]高贵,韩四平,王智,等.国内脂肪酶研究状况分析.生物技术通讯,2003,14(6):543-545.Gao G,Han S P,Wang Z,et al.Analysis of lipase research situation in China.Letters in Biotechnology,2003,14(6):543-545.
[10]Jakub Z,Anne S.M,Teofil J,et al.A general overview of support materials for enzyme immobilization:characteristics,properties,practical utility.MDPI,2018,8(2):1-27.
[11]尹春丽,曹珊珊,许乐,等.氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究.化学与生物工程,2014,31(9):17-20.Yin C L,Cao S S,Xu L,et al.Reseach of the immobilized enzyme of s-adenosine methionine by amino resin.Chemical and Bioengineering,2014,31(9):17-20.
[12]张丰华,孙宁,张伟,等.环氧基和氨基树脂固定化β-半乳糖苷酶的比较研究.中国农业科技导报,2014,16(5):47-52.Zhang F N,Sun N,Zhang W,et al.Comparison of epoxy and amino resin carrier in immobilization ofβ-galactosidase.Journal of Agricultural Science and Technology,2014,16(5):47-52.
[13]Li R Y,Fu G M,Liu C M,et al.Tannase immobilisation by aminofunctionalised magnetic Fe3O4-chitosan nanoparticles and its application in tea infusion.International Journal of Biological Macromolecules,2018,114:1134-1143.
[14]Huang J,Liu C,Xiao H Y,et al.Zinc tetraaminophthalocyanineFe3O4nanoparticle composite for laccase immobilization.International Journal of Nanomedicine,2008,2(4):84-775.
[15]Adinarayana K,Iraj G,Susana C,et al.Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers.Process Biochemistry,2008,43(2):169-178.
[16]Katja G,Anke K,Thomas M,et al.Novel immobilization routes for the covalent binding of an alcohol dehydrogenase from rhodococcus ruber DSM 44541.Tetrahedron:Asymmetry,2008,19(10):1171-1173.
[17]Dae Y K,Joon S R.A simple and rapid colorimetric method for determination of free fatty acids for lipase assay.Jaocs,1986,63(1):89-92.
[18]李晔.酶的固定化及其应用.分子催化,2008,22(1):86-96.Li Y.Immobilization of enzymes and their applications.Journal of Molecular Catalysis(China),2008,22(1):86-96.
[19]Prlainovic N Z,Knezevic-Jugovic Z D,Mijin D Z,et al.Immobilization of lipase from candida rugosa on sepabeads():the effect of lipase oxidation by periodates.Bioprocess and Biosystems Engineering,2011,34(7):10-803.
[20]闻臻臻.假丝酵母产脂肪酶固定化研究.杭州:浙江工业大学,2011.Wen Z Z.The immobilization of candida lipase.Hangzhou:Zhejiang University of Technology,2011.
[21]罗文,谭天伟,袁振宏,等.多孔玻璃珠固定化脂肪酶及其催化合成生物柴油.现代化工,2007,27(11):40-42.Luo W,Tan T W,Yuan Z H,et al.Immobilization of lipase by controlled pore glass and its applicationin biodiesel synthesis.Modern Chemical Industry,2007,27(11):40-42.
[22]Zang X Z,Xie W L.Enzymatic interesterification of soybean oil and methyl stearate blends using lipase immobilized on magnetic Fe3O4/SBA-15 composites as a biocatalyst.Journal of Oleo Science,2014,63(10):1027-1034.
[23]Mohammad K,Mohammad K,Ayyoob A.Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures.Biochemical Engineering Journal,2013,79(2013):267-273.