木薯块根中<i>β</i>-葡萄糖苷酶的分离纯化及酶学性质

常祥祥; 田永莉; 颜娟; 李文超; 刘石生

doi:10.13386/j.issn1002-0306.2022050202

木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质

doi: 10.13386/j.issn1002-0306.2022050202

常祥祥^{1, 2,},
田永莉^{1, 2},
颜娟^{1, 2},
李文超^{1, 2},
刘石生^{1, 2, ,}

1.
海南大学食品科学与工程学院，海南海口 570228
2.
热带多糖资源利用教育部工程研究中心，海南海口 570228

基金项目: 海南省自然科学基金高层次人才项目（2019RC031）。

详细信息

作者简介:
常祥祥（1995−），女，硕士研究生，研究方向：农产品加工与利用，E-mail：18334779198@163.com

通讯作者:
刘石生（1977−），男，博士，教授，研究方向：天然植物资源开发与利用，E-mail：stoneliu77@126.com

中图分类号: TS201.2
计量
- 文章访问数: 60
- HTML全文浏览量: 32
- PDF下载量: 11
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-19
- 网络出版日期: 2022-12-16
- 刊出日期: 2023-01-17

Isolation, Purification and Characterization of β-Glucosidase from Cassava Roots

CHANG Xiangxiang^{1, 2
,},
TIAN Yongli^{1, 2},
YAN Juan^{1, 2},
LI Wenchao^{1, 2},
LIU Shisheng^{1, 2
, ,}

1.
College of Food Science and Engineering, Hainan University, Haikou 570228, China
2.
Engineering Research Center of Utilization of Tropical Polysaccharide Resources, Ministry of Education, Haikou 570228, China

摘要

摘要: 研究了木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质。以缓冲液从木薯块根中获得粗提酶液，粗酶酶活力为9.37 U/g木薯干重；再分别通过丙酮沉淀、离子交换层析和凝胶过滤层析进行纯化，β-葡萄糖苷酶酶活力为1.14 U/g木薯干重，经纯化β-葡萄糖苷酶纯度提高了14.62倍，总活力回收率为12.14%，电泳测得其分子量约70 kDa。该酶米氏常数K_m为3.60 mmol/L，V_max为12.36 µmol/(min·mg protein)；其最适pH为7.0，pH在6.0~8.0之间有较好的稳定性；在40 ℃以内有良好稳定性，在4 ℃存放30 d酶活力剩余81.78%。Mn²⁺和K⁺对酶有一定的促进作用，Al³⁺、Cu²⁺、Mg²⁺、Zn²⁺、Ca²⁺、Ba²⁺、Na⁺、尿素和SDS对酶没有显著影响（P>0.05），而Fe³⁺、Fe²⁺、Ag⁺和EDTA均会不同程度地抑制酶活性，其中Ag⁺的抑制作用最强。研究结果可为今后木薯块根中的β-葡萄糖苷酶的应用提供理论依据。
- 木薯块根 /
- β-葡萄糖苷酶 /
- 分离 /
- 纯化 /
- 酶学性质
Abstract: The isolation, purification and enzymatic properties of β-glucosidase from cassava roots were studied. The crude enzyme extract was obtained from cassava roots with buffer solution, and the activity of crude enzyme was 9.37 U/g cassava dry weight. Purified by acetone precipitation, ion exchange chromatography and gel filtration chromatography, β-glucosidase activity was 1.14 U/g cassava dry weight, purified β-glucosidase purity increased by 14.62 times, the total activity recovery was 12.14%, the molecular weight of β-glucosidase was about 70 kDa. The K_m and V_max of the enzyme were 3.60 mmol/L and 12.36 µmol/(min·mg protein) respectively. The optimum pH was 7.0, and it was stable when the pH was between 6.0 and 8.0. It had good stability within 40 ℃, and 81.78% enzyme activity remained after 30 days at 4 ℃. Mn²⁺ and K⁺ promoted the enzyme to a certain extent. Al³⁺, Cu²⁺, Mg²⁺, Zn²⁺, Ca²⁺, Ba²⁺, Na⁺, urea and SDS had no significant effect on the enzyme activity (P>0.05). Fe³⁺, Fe²⁺, Ag⁺ and EDTA all inhibited the enzyme activity to varying degrees, among which Ag⁺ had the strongest inhibitory effect. The results can provide theoretical basis for the application of β-glucosidase in cassava roots in the future.
- cassava roots /
- β-glucosidase /
- isolation /
- purification /
- enzymatic properties

HTML全文

图 1 离子交换层析洗脱曲线图

Figure 1. Plot of ion exchange chromatography elution

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图 2 葡聚糖凝胶层析洗脱曲线图

Figure 2. Plot of dextran gel chromatography elution

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图 3 木薯β-葡萄糖苷酶的SDS-PAGE电泳

Figure 3. SDS-PAGE electrophoresis of cassava β-glucosidase

注：a：粗酶液；b：标准蛋白；c：纯化后的酶液。

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图 4 β-葡萄糖苷酶的pH稳定性

Figure 4. pH stability of β-glucosidase

注：图中不同小写字母表示组内差异显著，P<0.05；图8同。

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图 5 β-葡萄糖苷酶的热稳定性

Figure 5. Thermostability of β-glucosidase

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图 6 β-葡萄糖苷酶的保存稳定性

Figure 6. Storage stability of β-glucosidase

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图 7 酶水解p-NPG的Lineweaver-Burk关系图

Figure 7. Lineweaver-Burk plot of enzymatic hydrolysis of p-NPG

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图 8 金属离子和有机物对酶活性的影响

Figure 8. Effects of metal ions and organics on enzyme activity

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表 1 木薯β-葡萄糖苷酶的纯化

Table 1. Purification of cassava β-glucosidase

方法	木薯粉（g）	总活力（U）	总蛋白量（mg）	比活力（U/mg）	纯化倍数（倍）	总活力回收率（%）
粗酶液提取	100.00	937.13	60.06	15.60	/	100.00
丙酮沉淀		857.69	31.50	27.23	1.75	91.52
离子交换层析		384.68	2.22	173.32	11.11	41.05
凝胶过滤纯化		113.80	0.50	228.16	14.62	12.14

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