东莨菪素是一种香豆素类化合物,有消肿抗炎、镇痛、抗菌、杀螨等生物活性。从植物中提取东莨菪素由于东莨菪苷及其他成分的干扰常导致提取率低、纯化困难。本研究对黑曲霉(
Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase
东莨菪素(scopoletin)又称东莨菪内酯,属香豆素类化合物,化学名为6-甲氧基-7-羟基香豆素[
相关研究表明,植物组织中同时存在东莨菪苷和东莨菪素两种结构类似物,且东莨菪苷的含量大于东莨菪素的含量[
β-葡萄糖苷酶(β-glucosidase, EC 3.2.1.21)是一类水解末端的非还原性β-d-葡萄糖苷键进而释放葡萄糖和相应配基的糖苷水解酶[
在前期研究中,我们对16个黑曲霉(
大肠杆菌DH5α菌株(
PrimeSTAR HS DNA Polymerase、
Agilent 1260 Infinit高效液相色谱仪,安捷伦科技公司;Biometra TAdvanced基因扩增仪,耶拿有限公司;DK-8D数显恒温水浴锅,常州国华电器有限公司;FE28pH计,METTLER TOLEDO公司;SW-CJ-2FD双人单面超净工作台,苏州净化设备有限公司;Centrifuge 5417R小型高速冷冻离心机,Eppendorf有限公司;K10金属浴,杭州奥盛有限公司;GEAI 600凝胶成像仪,GE公司;1652100电穿孔仪,BIO-RAD有限公司;Avanti J-26S XP立式高速冷冻离心机,BECKMAN公司;YXQ-LS-30SH立式压力蒸汽灭菌器,上海博讯实业有限公司医疗设备厂。
利用SignalP 4.0 Server (
以PUC-57-
将菌株接入BMGY培养基中,30 ℃、200 r/min培养24 h,使
纯化过程中总活力、比活力、纯化倍数、回收率的计算方法如下:
总活力=活力单位数/mL酶液×总体积(mL);
比活力=活力单位数/mg蛋白=总活力单位数/总蛋白mg;
纯化倍数=每次比活力/第一次比活力;
回收率(产率)=(每次总活力/第一次总活力)×100%。
蛋白浓度的测定:根据全式金生物的Easy Ⅱ Protein Quantitative Kit说明书配制工作液,以牛血清白蛋白溶液为标准溶液,测定样品的蛋白浓度。重组蛋白的分子量通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)测定。
质谱鉴定:重组蛋白的鉴定是使用蛋白质内切酶trypsin对蛋白质多肽样品进行酶解,然后使用液相色谱质谱联用仪(liquid chromatograph mass spectrometer, LC-MS/MS)对酶解后的样品进行分析。再通过MASCOT等质谱匹配软件对LC-MS/MS数据进行分析,获得目标蛋白质多肽分子的定性鉴定信息。
先配制浓度为5 mmol/L东莨菪苷的母液,再稀释成一系列浓度的东莨菪苷溶液,通过Agilent 1260 Infinit型高效液相色谱仪(high performance liquid chromatography, HPLC)测定东莨菪苷标准曲线。检测条件为:色谱柱Agilent ZORBAX SB-C18,5 μm (4.6 mm×150 mm),柱温28 ℃,进样量10 μL,检测波长为347 nm;以乙腈(A)和0.1%磷酸缓冲液(B)为流动相进行梯度洗脱,流速为1.0 mL/min,梯度洗脱条件为(0−10 min, 15%−34% A; 10−20 min, 34% A; 20−30 min, 34%−15% A)。
酶活的测定:600 μL的pH 4.0的2 mmol/L的东莨菪苷溶液与100 μL的
酶活定义:在最适反应条件下,1 min内转化1 μmoL东莨菪苷时所需的β-葡萄糖苷量为一个酶活单位(IU)。
分别测定重组β-葡萄糖苷酶
测定重组β-葡萄糖苷酶
在pH 5.0条件下,通过测定重组β-葡萄糖苷酶
用50 mmol/L的不同pH (pH 3.0−6.0)的柠檬酸盐缓冲液与(pH 6.0−8.0)磷酸盐缓冲液配制2 mmol/L的东莨菪苷底物溶液。参照酶活的测定方法测定重组β-葡萄糖苷酶
在最适反应条件下,分别采用1 mmol/L和10 mmol/L的Mg2+、Na2+、Mn2+、Al3+、Fe3+、K+、Cu2+、Cd2+、Fe2+、Ba2+、Zn2+、Ca2+等金属离子,在4 ℃条件下反应24 h,测定重组β-葡萄糖苷酶An-bgl3的残余酶活力来探究金属离子对酶活力的影响,以未被金属离子处理的酶活力为100%。
在最适反应条件下,分别采用1 mmol/L和10 mmol/L的EDTA、SDS、CTAB、Urea、Triton X-20、Tween-80和Triton-X 100等抑制剂和表面活性剂,在4 ℃条件下反应24 h,测定重组β-葡萄糖苷酶An-bgl3的残余酶活力来探究抑制剂和表面活性剂对酶活力的影响,以未被抑制剂和表面活性剂处理的酶活力为100%。
在最适反应条件下,分别测定该酶在不同浓度(10%、20%、40%、60%)的甲醇、乙醇存在时的残余酶活力来探究有机试剂对于重组β-葡萄糖苷酶
使用RobeTTaFold (
取丁公藤(
所有实验均重复平行3次,使用Excel 2016 (微软公司,美国)进行绘图,采用SPSS 21.0进行数据分析。
将葡萄糖苷酶
β-葡萄糖苷酶(
Purification parameters of beta-glucosidase (
Purification steps | Total activity (U) | Total protein (mg) | Specific activity (U/mg) | Purified fold | Recovery rate (%) |
Hydrophobic column | 61 272.6 | 402.3 | 152.3 | 1.2 | 75.2 |
Superdex 200 Increase 10/300 GL | 41 347.2 | 58.1 | 310.8 | 2.4 | 50.7 |
Q sepharose | 14 261.8 | 4.5 | 3 183.4 | 25.0 | 17.5 |
Superdex 200 Increase 10/300 GL | 9 723.6 | 2. 6 | 3 740.0 | 29.4 | 11.9 |
纯化蛋白的SDS-PAGE图
SDS-PAGE profile of the purified proteins.
重组β-葡萄糖苷酶
β-葡萄糖苷酶
Substrate specificity of β-glucosidase
Substrate type | Specific activity of |
Relative activity of |
ND: No enzyme activity was detected. | ||
P-nitrophenyl-β-d-glucopyranoside | ND | ND |
P-nitrophenyl-α-d-glucopyranoside | ND | ND |
P-nitrophenyl-N-acetyl-β-d-glucopyranoside | ND | ND |
P-nitrophenyl-β-d-galactopyranoside | ND | ND |
gGeniposide | ND | ND |
Salicin | ND | ND |
Polydatin | ND | ND |
Esculin | 29.64±0.1 | 76.5 |
Scopolin | 38.72±0.1 | 100.0 |
Amygdalin | ND | ND |
Liquiritin | ND | ND |
Cellbiose | ND | ND |
Sucrose | ND | ND |
Maltose | ND | ND |
β-葡萄糖苷酶
Lineweaver-Burk plot of β-glucosidase
以东莨菪苷为底物,探究重组β-葡萄糖苷酶
β-葡萄糖苷酶
Effect of temperature and pH on recombinant β-glucosidase
以东莨菪苷为底物,测试金属离子、抑制剂和表面活性剂对酶活性的影响(
金属离子对
Effect of metal ions on
Type of metal ions | Relative activity (%) | |
1 mmol/L | 10 mmol/L | |
Mg2+ | 88.3±0.1 | 111.90±0.2 |
Na2+ | 93.2±0.1 | 83.10±0.2 |
Mn2+ | 101.0±0.1 | 222.40±0.1 |
Al3+ | 95.9±0.3 | 70.80±0.1 |
Fe3+ | 93.7±0.2 | 67.58±0.1 |
K |
95.0±0.1 | 84.00±0.6 |
Cu2+ | 95.4±0.2 | 98.20±0.1 |
Cd2+ | 94.6±0.1 | 99.50±0.3 |
Fe2+ | 102.8±0.2 | 282.20±0.1 |
Ba2+ | 92.2±0.2 | 95.00±0.2 |
Zn2+ | 89.5±0.2 | 79.50±0.1 |
Ca2+ | 94.5±0.6 | 103.70±0.1 |
抑制剂、表面活性剂对
Effect of inhibitors and surfactants on
Reagent type | Relative activity (%) | |
1 mmol/L | 10 mmol/L | |
EDTA | 112.5±0.4 | 92.5±0.2 |
SDS | 108.0±0.4 | 98.4±0.2 |
CTAB | 102.4±0.3 | 97.0±0.2 |
Urea | 110.8±0.2 | 97.2±0.4 |
Tween-20 | 103.6±0.3 | 67.8±0.2 |
Tween-80 | 104.2±0.2 | 67.1±0.2 |
Triton-X 100 | 107.8±0.3 | 62.6±0.1 |
有机试剂对
Effect of organic reagents on
Reagent concentration (%) | Relative activity (%) | |
Ethanol | Methanol | |
10 | 101.8±0.2 | 88.3±0.1 |
20 | 86.4±0.4 | 76.0±0.2 |
40 | 23.8±0.3 | 42.3±0.3 |
60 | 17.6±0.5 | 31.8±0.3 |
通过RobeTTaFold对构建蛋白模型,利用POCASA服务器计算口袋的体积,葡萄糖苷酶
葡萄糖苷酶
Pocket conformation diagram of glucosidase
将葡萄糖苷酶
东莨菪苷与葡萄糖苷酶
Docking effect diagram (A) and binding mode (B) of scopolin with glucosidase
七叶苷与葡萄糖苷酶
Docking effect diagram (A) and binding mode (B) of esculin with glucosidase
用β-葡萄糖苷酶
酶解液相结果分析图
HPLC analysis of enzymatic hydrolysis results.
本研究克隆、表达了黑曲霉来源的β-葡萄糖苷酶
β-葡萄糖苷酶的作用底物非常多样,可水解对硝基苯基-α-d-吡喃葡萄糖苷、对硝基苯基-N-乙酰基-β-d-吡喃葡萄糖苷、对硝基苯基-β-d-吡喃半乳糖苷、麦芽糖、蔗糖[
10 mmol/L的Fe2+和Mn2+离子对重组
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