黄豆豆渣中脲酶的提取精制及其影响因素研究
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摘要
为拓展脲酶的国产化途径,对黄豆豆渣中脲酶的提取进行了研究。建立了从黄豆豆渣中提取脲酶的全套流程,利用盐析法和有机溶剂沉淀法,通过浸提及离心等试验操作手段,将黄豆豆渣中的脲酶进行提取精制,产率为0.1%。同时确定了脲酶的最佳提取条件:其最佳浸提温度为50℃,最佳丙酮浓度为50%,最适pH为7.0。利用纳氏试剂比色法的原理,测得该脲酶在最佳实验条件下的米氏常数Km值为4.11×10~(-2)mol/L,1 m L脲酶溶液中的酶活力为18.63 U。从黄豆豆渣中提取脲酶,既可解决脲酶的国产化问题,又可提高黄豆豆渣的附加值,研究结果具有良好的工业价值和经济效益。
The research of extracting urease from soybean dregs was carried out in order to expand the way of urease localization.The research had established a full set of extracting urease from soybean dregs. By use of the method of salting out and organic solvent precipitation, the urease in the soybean dregs was extracted and refined through experiments of extraction and centrifuging,etc.,and its yield rate was 0. 1%. Meanwhile,the optimum condition of urease extracting was determined: the optimum extracting temperature was 50℃,the optimum acetone concentration was 50%,and the most suitable pH value was 7.0.By the principle of Nessler's reagent colorimetric method,it was tested that the value of Michaelis constant(K_m) was 4.11×10~(-2) mol/L and the value of enzyme activity in the urease solution of 1 m L was 18. 63 U under the optimum conditions. Extracting urease from soybean dregs not only solves the problem of urease localization,but also improves the added value of soybean dregs.The result of the research brings good industrial values and economic benefits.
The research of extracting urease from soybean dregs was carried out in order to expand the way of urease localization.The research had established a full set of extracting urease from soybean dregs. By use of the method of salting out and organic solvent precipitation, the urease in the soybean dregs was extracted and refined through experiments of extraction and centrifuging,etc.,and its yield rate was 0. 1%. Meanwhile,the optimum condition of urease extracting was determined: the optimum extracting temperature was 50℃,the optimum acetone concentration was 50%,and the most suitable pH value was 7.0.By the principle of Nessler's reagent colorimetric method,it was tested that the value of Michaelis constant(K_m) was 4.11×10~(-2) mol/L and the value of enzyme activity in the urease solution of 1 m L was 18. 63 U under the optimum conditions. Extracting urease from soybean dregs not only solves the problem of urease localization,but also improves the added value of soybean dregs.The result of the research brings good industrial values and economic benefits.
引文
[1]林丽云,董晓洁,陈阿微.黄豆脲酶的提取及其活性测定[J].食品研究与开发,2013,34(9):85-88.
[2]Follmer C.Insights into the role and structure of plant ureases[J].Phytochemistry,2008,69(1):18-28.
[3]Levin D A,Watermeyer G,Mohamed N,et al..Evaluation of a locally produced rapid urease test for the diagnosis of Helicobacter pylori infection[J].S.Afr.Med.J.,2007,97(12):1281-1284.
[4]San Francisco S,Urrutia O,Martin V,et al..Efficiency of urease and nitrification inhibitors in reducing ammonia volatilization from diverse nitrogen fertilizers applied to different soil types and wheat straw mulching[J].J.Sci.Food Agric.,2011,91(9):1569-1575.
[5]杨少斌,刘小冲.等离子体引发PTFE膜固定化脲酶处理废水研究[J].河南化工,2007,24(10):23-24.
[6]周建立,康振,刘庆涛,等.重组酸性脲酶对黄酒中尿素和氨基甲酸乙酯的降解应用[J].生物工程学报,2016,32(1):74-83.
[7]张振山,叶素萍,李泉,等.豆渣的处理与加工利用[J].食品科学,2004,25(10):400-406.
[8]Miyagawa K,Sumida M,Nakao M,et al..Purification,characterization,and application of an acid urease from Arthrobacter mobilis[J].J.Biotechnol.,1999,68(2-3):227-236.
[9]贾琦,李兆祥,杜永霞,等.大豆种皮中大豆脲酶的提取及精制[J].中外食品,2014(2):37-42.
[10]崔有宏,罗侃,吴育凌,等.黄豆脲酶的提取与性质研究[J].甘肃科学学报,2000,12(1):62-66.
[11]Witte C P,Rosso M G,Romeis T.Identification of three urease accessory proteins that are required for urease activation in Arabidopsis[J].Plant Physiol.,2005,139(3):1155-1162.
[12]韦月平,王鹏.有机溶剂沉淀法提取脲酶的条件优化[J].辽东学院学报(自然科学版),2014,21(4):241-243.
[13]罗侃,崔有宏,王绪明.脲酶Km值的比色测定法及其评价[J].西北国防医学杂志,1995(1):1-4.
[14]马锋,李阳,冯佰利.双青豆脲酶提取工艺研究[J].陕西农业科学,2010(5):56-58.
[15]周东凯,刘莹,马学良,等.大豆脲酶的提取及其影响因素研究[J].大豆科学,2008,27(4):704-707.
[16]曹慧,徐斐.豆浆中脲酶活性测定方法的建立及酶学性质的研究[J].食品工业科技,2012(1):106-108.
[17]蒋传葵,金承德,吴仁龙.工具酶的活力测定[M].上海:上海科学技术出版社,1982,117-119.
[18]余云雷,齐德生,张妮娅.大豆脲酶活性测定方法比较研究[J].养殖与饲料,2005(6):19-21.
[19]卢晓凌,王忠艳.大豆脲酶活性的两种不同测定方法的研究[J].饲料工业,2008,29(2):54-56.
[20]郭明,燕冰宇,杨萍.新型方法制备固定化脲酶基质材料及固定化脲酶的性能研究[J].高校化学工程学报,2014,28(5):1147-1153.
[2]Follmer C.Insights into the role and structure of plant ureases[J].Phytochemistry,2008,69(1):18-28.
[3]Levin D A,Watermeyer G,Mohamed N,et al..Evaluation of a locally produced rapid urease test for the diagnosis of Helicobacter pylori infection[J].S.Afr.Med.J.,2007,97(12):1281-1284.
[4]San Francisco S,Urrutia O,Martin V,et al..Efficiency of urease and nitrification inhibitors in reducing ammonia volatilization from diverse nitrogen fertilizers applied to different soil types and wheat straw mulching[J].J.Sci.Food Agric.,2011,91(9):1569-1575.
[5]杨少斌,刘小冲.等离子体引发PTFE膜固定化脲酶处理废水研究[J].河南化工,2007,24(10):23-24.
[6]周建立,康振,刘庆涛,等.重组酸性脲酶对黄酒中尿素和氨基甲酸乙酯的降解应用[J].生物工程学报,2016,32(1):74-83.
[7]张振山,叶素萍,李泉,等.豆渣的处理与加工利用[J].食品科学,2004,25(10):400-406.
[8]Miyagawa K,Sumida M,Nakao M,et al..Purification,characterization,and application of an acid urease from Arthrobacter mobilis[J].J.Biotechnol.,1999,68(2-3):227-236.
[9]贾琦,李兆祥,杜永霞,等.大豆种皮中大豆脲酶的提取及精制[J].中外食品,2014(2):37-42.
[10]崔有宏,罗侃,吴育凌,等.黄豆脲酶的提取与性质研究[J].甘肃科学学报,2000,12(1):62-66.
[11]Witte C P,Rosso M G,Romeis T.Identification of three urease accessory proteins that are required for urease activation in Arabidopsis[J].Plant Physiol.,2005,139(3):1155-1162.
[12]韦月平,王鹏.有机溶剂沉淀法提取脲酶的条件优化[J].辽东学院学报(自然科学版),2014,21(4):241-243.
[13]罗侃,崔有宏,王绪明.脲酶Km值的比色测定法及其评价[J].西北国防医学杂志,1995(1):1-4.
[14]马锋,李阳,冯佰利.双青豆脲酶提取工艺研究[J].陕西农业科学,2010(5):56-58.
[15]周东凯,刘莹,马学良,等.大豆脲酶的提取及其影响因素研究[J].大豆科学,2008,27(4):704-707.
[16]曹慧,徐斐.豆浆中脲酶活性测定方法的建立及酶学性质的研究[J].食品工业科技,2012(1):106-108.
[17]蒋传葵,金承德,吴仁龙.工具酶的活力测定[M].上海:上海科学技术出版社,1982,117-119.
[18]余云雷,齐德生,张妮娅.大豆脲酶活性测定方法比较研究[J].养殖与饲料,2005(6):19-21.
[19]卢晓凌,王忠艳.大豆脲酶活性的两种不同测定方法的研究[J].饲料工业,2008,29(2):54-56.
[20]郭明,燕冰宇,杨萍.新型方法制备固定化脲酶基质材料及固定化脲酶的性能研究[J].高校化学工程学报,2014,28(5):1147-1153.