芦荟叶皮β-葡萄糖苷酶的分离纯化及酶学性质

李蕊伽,廖海君,白亚娟,陶敏,唐菁,唐云明

doi:10.13718/j.cnki.xsxb.2017.04.009

西南师范大学学报(自然科学版)

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芦荟叶皮β-葡萄糖苷酶的分离纯化及酶学性质

李蕊伽,廖海君,白亚娟,陶敏,唐菁,唐云明. 芦荟叶皮β-葡萄糖苷酶的分离纯化及酶学性质[J]. 西南师范大学学报(自然科学版), 2017, 42(4). doi: 10.13718/j.cnki.xsxb.2017.04.009

引用本文:

LI Rui-jia,LIAO Hai-jun,BAI Ya-juan,TAO Min,TANG Jing,TANG Yun-ming. On Isolation, Purification and Characterization of β-Glucosidase from Leaf Skin of Aloe Vera[J]. Journal of Southwest China Normal University(Natural Science Edition), 2017, 42(4). doi: 10.13718/j.cnki.xsxb.2017.04.009

Citation:

LI Rui-jia,LIAO Hai-jun,BAI Ya-juan,TAO Min,TANG Jing,TANG Yun-ming. On Isolation, Purification and Characterization of β-Glucosidase from Leaf Skin of Aloe Vera[J]. Journal of Southwest China Normal University(Natural Science Edition), 2017, 42(4). doi: 10.13718/j.cnki.xsxb.2017.04.009

芦荟叶皮β-葡萄糖苷酶的分离纯化及酶学性质

李蕊伽,廖海君,白亚娟,陶敏,唐菁,唐云明

西南大学生命科学学院/重庆市甘薯工程研究中心/三峡库区生态环境教育部重点实验室,重庆,400715

On Isolation, Purification and Characterization of β-Glucosidase from Leaf Skin of Aloe Vera

LI Rui-jia,LIAO Hai-jun,BAI Ya-juan,TAO Min,TANG Jing,TANG Yun-ming

摘要: 库拉索芦荟叶皮经匀浆、柠檬酸-柠檬酸钠缓冲液抽提, 硫酸铵分级沉淀, DEAE-Sepharose fast flow层析和Superdex-200 prep grade层析, 获得电泳纯的β-葡萄糖苷酶(β-Glucosidase, BGL). 纯化结果是β-葡萄糖苷酶比活力为98.48 U/mg, 纯化倍数为328.27倍, 酶活性回收率为9.87%, 全酶分子质量约为69.3 kD, 亚基分子质量约为69.7 kD. 酶学性质研究表明: β-葡萄糖苷酶的最适反应温度和最适反应pH值分别为40 ℃和5.0;在20~30 ℃及pH 4.0~8.0范围内稳定性较好;最适条件下, 以pNPG为底物的Km值为2.21 mmol/L,Vmax为1.381 μmol/(min·L). 乙醇,抗坏血酸,Mn2+,K+对该酶活性具有激活作用;SDS,Cu2+对该酶活性具有强烈抑制作用;异丙醇,EDTA,尿素,Mg2+,Li+对该酶活性影响较小;甲醇对该酶有双重作用.
- 芦荟叶皮 /
- β-葡萄糖苷酶 /
- 分离纯化 /
- 酶学性质
Abstract: Electrophoresis-purity β-Glucosidase from leaf skin of Aloe vera has been obtained through homogenization, citrate buffer extraction, ammonium sulfate fractionation, DEAE-Sepharose fast flow chromatography and Superdex-200 prep grade chromatography.After purification, the result shows that the specific activity of β-Glucosidase is 98.48 U/mg, with a 328.27-fold purification and a 9.87% activity recovery.The relative molecular weight of the β-Glucosidase is approximately 69.3 kD, in which the subunit molecular mass is roughly 69.7 kD.The enzymatic properties illustrated that the optimum temperature and pH for the β-Glucosidase are 40 ℃ and 5.0, respectively.This enzyme is stable at 20-30 ℃ and at pH 4.0-8.0.Its apparent Km and Vmax are 2.21 mmol/L and 1.381 μmol/(min·L) towards pNPG.The enzyme activity of β-Glucosidase could be activated by ethanol, ascorbic acid, Mn2+, K+, and strongly inhibited by SDS, Cu2+.Isopropanol, EDTA, urea, Mg2+, Li+ had little activation for it.Methanol has a dual effect on this enzyme.
- leaf skin of Aloe vera；β-Glucosidase；isolation and purification；enzymatic properties /

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芦荟叶皮β-葡萄糖苷酶的分离纯化及酶学性质

李蕊伽,廖海君,白亚娟,陶敏,唐菁,唐云明

西南大学生命科学学院/重庆市甘薯工程研究中心/三峡库区生态环境教育部重点实验室,重庆,400715

关键词:

摘要: 库拉索芦荟叶皮经匀浆、柠檬酸-柠檬酸钠缓冲液抽提, 硫酸铵分级沉淀, DEAE-Sepharose fast flow层析和Superdex-200 prep grade层析, 获得电泳纯的β-葡萄糖苷酶(β-Glucosidase, BGL). 纯化结果是β-葡萄糖苷酶比活力为98.48 U/mg, 纯化倍数为328.27倍, 酶活性回收率为9.87%, 全酶分子质量约为69.3 kD, 亚基分子质量约为69.7 kD. 酶学性质研究表明: β-葡萄糖苷酶的最适反应温度和最适反应pH值分别为40 ℃和5.0;在20~30 ℃及pH 4.0~8.0范围内稳定性较好;最适条件下, 以pNPG为底物的Km值为2.21 mmol/L,Vmax为1.381 μmol/(min·L). 乙醇,抗坏血酸,Mn2+,K+对该酶活性具有激活作用;SDS,Cu2+对该酶活性具有强烈抑制作用;异丙醇,EDTA,尿素,Mg2+,Li+对该酶活性影响较小;甲醇对该酶有双重作用.

English Abstract

On Isolation, Purification and Characterization of β-Glucosidase from Leaf Skin of Aloe Vera

西南大学生命科学学院/重庆市甘薯工程研究中心/三峡库区生态环境教育部重点实验室,重庆,400715

Keywords:

Abstract: Electrophoresis-purity β-Glucosidase from leaf skin of Aloe vera has been obtained through homogenization, citrate buffer extraction, ammonium sulfate fractionation, DEAE-Sepharose fast flow chromatography and Superdex-200 prep grade chromatography.After purification, the result shows that the specific activity of β-Glucosidase is 98.48 U/mg, with a 328.27-fold purification and a 9.87% activity recovery.The relative molecular weight of the β-Glucosidase is approximately 69.3 kD, in which the subunit molecular mass is roughly 69.7 kD.The enzymatic properties illustrated that the optimum temperature and pH for the β-Glucosidase are 40 ℃ and 5.0, respectively.This enzyme is stable at 20-30 ℃ and at pH 4.0-8.0.Its apparent Km and Vmax are 2.21 mmol/L and 1.381 μmol/(min·L) towards pNPG.The enzyme activity of β-Glucosidase could be activated by ethanol, ascorbic acid, Mn2+, K+, and strongly inhibited by SDS, Cu2+.Isopropanol, EDTA, urea, Mg2+, Li+ had little activation for it.Methanol has a dual effect on this enzyme.