牛胰脂肪酶交连酶聚体的制备及性能研究
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摘要
以牛胰脂肪酶(cattle pancreatic lipase,CPL)为研究对象,采用交联酶聚体技术,研究交联酶聚体脂肪酶(CPL-CLEAs)的制备条件、催化性能和酶促反应动力学参数。结果表明,交联酶聚体脂肪酶制备的最优制备条件为:硫酸铵饱和度80%,戊二醛终浓度1.5%,交联时间1 h,在此条件下,所得CPL-CLEAs最高酶活回收率为90%。CPL-CLEAs的最适催化温度是60℃,与游离脂肪酶相比,CPL-CLEAs的最适催化温度提高了10℃,且温度稳定性、储存稳定性和操作稳定性均有所提高。重复使用7次后,CPL-CLEAs仍能保持初始酶活的43%。
In this study,cross-linked enzyme aggregates(CLEAs) of cattle pancreatic lipase(CPL-CLEAs) was prepared by cross-linked enzyme aggregates technology,the preparation conditions,catalysis properties,and dynamics parameters of CPL-CLEAs were investigated. The results showed that the optimum conditions of the preparation of CPL- CLEAs were as followed : final saturability of the ammonium sulfate was 80 %, final concentration of glutaraldehyde was 1. 5 %, and corss- link time was 1 h. Under this condition, the highest activity recovery reached 90%. In addition,compared to free lipase,the optimum temperature of CPL-CLEAs was 60 ℃,which was increased about 10 ℃. The thermal stability,operational stability,and storage stablility of CPL-CLEAs were significantly enhanced compared with free lipase. Furthermore,the CPL-CLEAs still retained 43% of its initial activity after consecutive 7 cycles.
In this study,cross-linked enzyme aggregates(CLEAs) of cattle pancreatic lipase(CPL-CLEAs) was prepared by cross-linked enzyme aggregates technology,the preparation conditions,catalysis properties,and dynamics parameters of CPL-CLEAs were investigated. The results showed that the optimum conditions of the preparation of CPL- CLEAs were as followed : final saturability of the ammonium sulfate was 80 %, final concentration of glutaraldehyde was 1. 5 %, and corss- link time was 1 h. Under this condition, the highest activity recovery reached 90%. In addition,compared to free lipase,the optimum temperature of CPL-CLEAs was 60 ℃,which was increased about 10 ℃. The thermal stability,operational stability,and storage stablility of CPL-CLEAs were significantly enhanced compared with free lipase. Furthermore,the CPL-CLEAs still retained 43% of its initial activity after consecutive 7 cycles.
引文
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[2]刘海洲,吴小飞,牛佰慧,等.脂肪酶在食品工业中的应用与研究进展[J].粮食加工,2008,33(5):55-57.
[3]邹彬,初旭明,张洋,等.离子液体修饰的SBA-16材料在猪胰脂肪酶固定化中的应用[J].生物加工过程,2014(12):6-11.
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[11]邢肖肖,王梦凡,齐崴,等.β-半乳糖苷酶交联酶聚体的制备及酶学性质研究[J].食品工业科技,2014,35(23):158-167.
[12]李连连,崔建东.以大孔硅胶为载体的苯丙氨酸解氨酶交联酶聚体的制备及性质研究[J].食品工业科技,2014,35(11):160-165.
[13]Cui J D,Zhang S,Sun L M.Cross-linked enzyme aggregates of phenylalanine ammonia lyase:novel biocatalysts for synthesis of L-Phenylalanine[J].Applied Biochemistry and Biotechnology,2012,167(4):835-844.
[14]Moon II Kim,Jungbae Kim,Jinwoo Lee.One-dimensional crosslinked enzyme aggregates in SBA-15:Superior catalytic behavior to conventional enzyme immobilization[J].Microporous and Mesoporous Materials,2008,101:18-23.
[15]Mengfan Wang,Wei Qi,Qingxin Yu,et al.Cross-linking enzyme aggregates in the macropores of silica gel:A practical and efficient method for enzyme stabilization[J].Biochemical Engineering Journal,2010,52:168-174.
[16]刘娟,杨楠楠,姜爱莉.固定化脂肪酶的制备及其酶学性质的研究[J].油脂化学,2013,38(1):44-47.
[17]石莲莲.固载化脂肪酶交联酶聚集体的制备及应用[D].天津:河北工业大学,2013.
[18]王梦凡.新型交联酶聚体技术及其在蛋白组学与生物催化中的应用[D].天津:天津大学,2011.
[19]常凯,王红英,孙井辉.磁性壳聚糖微球固定化脂肪酶的研究[J].大连工业大学学报,2011,30(1):31-33.
[2]刘海洲,吴小飞,牛佰慧,等.脂肪酶在食品工业中的应用与研究进展[J].粮食加工,2008,33(5):55-57.
[3]邹彬,初旭明,张洋,等.离子液体修饰的SBA-16材料在猪胰脂肪酶固定化中的应用[J].生物加工过程,2014(12):6-11.
[4]梁静鹃,杨立鹏,韩钧,等.固定化脂肪酶在离子液体中催化合成生物柴油[J].可再生能源,2012,30:72-77.
[5]Zhang W W,Yang X L,Jia J Q,et al.Surfactant-activated magnetic cross-linked enzyme aggregates(magnetic CLEAs)of Thermpmyces lanuginosus lipase for biodiesel production[J].Journal of Molecular Catalysis B:Enzymatic,2015,115:3-89.
[6]Xu D Y,Yang Zh.Cross-linked tyrosinase aggregates for elimination of phenolic compounds from wastewater[J].Chemosphere,2013,92(4):391-398.
[7]Yu HW,Chen H,Wang X,et al.Cross-linked enzyme aggregates(CLEAs)with controlled particles:Application to Candida rugosa lipase[J].Molecular Catalysis,2006,43:124-127.
[8]马雷猛,贵莉莉,王飞,等.添加纳米粒子对脂肪酶交联酶聚集体活性及结构的影响[J].北京化工大学学报,2015,42(2):83-88.
[9]Wilson L,Fern'andez-Lorente G,Fern'andez-Lafuente R.CLEAs of lipases and poly-ionic polymers:A simple way of preparing stable biocatalysts with improved properties[J].Enzyme and Microbial Technology,2006,39:750-755.
[10]孙立梅,李连连,崔建东.牛血清白蛋白辅助交联对苯丙氨酸解氨酶交联酶聚体影响的研究[J].食品工业科技,2013,13:44-47.
[11]邢肖肖,王梦凡,齐崴,等.β-半乳糖苷酶交联酶聚体的制备及酶学性质研究[J].食品工业科技,2014,35(23):158-167.
[12]李连连,崔建东.以大孔硅胶为载体的苯丙氨酸解氨酶交联酶聚体的制备及性质研究[J].食品工业科技,2014,35(11):160-165.
[13]Cui J D,Zhang S,Sun L M.Cross-linked enzyme aggregates of phenylalanine ammonia lyase:novel biocatalysts for synthesis of L-Phenylalanine[J].Applied Biochemistry and Biotechnology,2012,167(4):835-844.
[14]Moon II Kim,Jungbae Kim,Jinwoo Lee.One-dimensional crosslinked enzyme aggregates in SBA-15:Superior catalytic behavior to conventional enzyme immobilization[J].Microporous and Mesoporous Materials,2008,101:18-23.
[15]Mengfan Wang,Wei Qi,Qingxin Yu,et al.Cross-linking enzyme aggregates in the macropores of silica gel:A practical and efficient method for enzyme stabilization[J].Biochemical Engineering Journal,2010,52:168-174.
[16]刘娟,杨楠楠,姜爱莉.固定化脂肪酶的制备及其酶学性质的研究[J].油脂化学,2013,38(1):44-47.
[17]石莲莲.固载化脂肪酶交联酶聚集体的制备及应用[D].天津:河北工业大学,2013.
[18]王梦凡.新型交联酶聚体技术及其在蛋白组学与生物催化中的应用[D].天津:天津大学,2011.
[19]常凯,王红英,孙井辉.磁性壳聚糖微球固定化脂肪酶的研究[J].大连工业大学学报,2011,30(1):31-33.