CN113480664A - 一种合成山奈酚的高活性双功能酶及其合成方法和应用 - Google Patents
一种合成山奈酚的高活性双功能酶及其合成方法和应用 Download PDFInfo
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- CN113480664A CN113480664A CN202110794595.2A CN202110794595A CN113480664A CN 113480664 A CN113480664 A CN 113480664A CN 202110794595 A CN202110794595 A CN 202110794595A CN 113480664 A CN113480664 A CN 113480664A
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- kaempferol
- bifunctional enzyme
- leu
- gly
- glu
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Abstract
本发明提出了一种合成山奈酚的高活性双功能酶，将黄烷酮3‑羟化酶基因f3h和黄酮醇合酶基因fls1同时克隆至原核表达载体pET‑32a(+)，再转化大肠杆菌，以诱导表达重组酶，并利用纯化的重组酶建立无细胞合成体系，实现山奈酚的体外酶促合成。本发明在构建一种高活性双功能酶的基础上，通过优化反应体系成分和反应条件，建立了一种体外高效合成目标分子的方法，显著提高了山奈酚的产量，降低了山奈酚的生产成本。
Description
技术领域
本发明涉及一种合成山奈酚的高活性双功能酶及其合成方法和应用，分类号为C12N9，属于生物医药技术领域。
背景技术
山奈酚，又名3,5,7,4′-四羟基黄酮，是一种植物次生代谢产物，具有多种生物活性，广泛存在于植物中，还具有抗癌、抗氧化、抗炎、防治糖尿病和骨质疏松、保护神经等作用，并且安全无毒，因此在食品、化妆品和医药领域的应用前景良好。
目前，制备山奈酚的主要方法是植物组织有机溶剂提取法，利用大量有机溶剂从植物组织中萃取。该方法步骤繁琐、耗时长、成本高。另一种制备山奈酚的方法是化学合成法。该方法合成步骤多、反应条件苛刻、某些试剂毒性大，且难以实现手性合成有活性的黄酮类化合物。因此这些方法难以广泛应用于工业化生产。
近年来，随着合成生物学的发展，学者们在微生物体内发酵合成了山奈酚。然而微生物细胞系统复杂，人工合成元件与宿主间可能存在不兼容性，导致工程菌不稳定，终产物和中间产物还可能抑制宿主细胞的生长，而且并非所有的工程菌都能生产目标产物。
发明内容
本发明所要解决的技术问题是，克服现有技术的不足而提供一种合成山奈酚的高活性双功能酶，并建立一种山奈酚体外合成体系，实现了山奈酚的体外高效合成，这不仅显著提高了山奈酚的产量，而且降低了山奈酚的生产成本，为山奈酚的开发利用提供了重要依据。
本发明提供了一种合成山奈酚的高活性双功能酶，其氨基酸序列如SEQ ID NO.1所示。
本发明在体外酶促合成山奈酚的基础之上，设计了一种同时具有黄烷酮3-羟化酶和黄酮醇合酶活性的双功能酶F3H-(GGGGS)2-FLS1，该双功能酶形成了与天然底物通道类似的结构，不仅能够显著提高山奈酚合成效率，还大大降低关键酶获取的复杂程度，简便了操作过程，显著减少了人力物力。
研究过程中，测试了不同的融合蛋白，包括：FLS1-(GGGGS)2-F3H、F3H-FLS1、F3H-(GGGGS)1-FLS1、F3H-(GGGGS)2-FLS1、F3H-(GGGGS)3-FLS1、F3H-(GGGGS)4-FLS1、F3H-(EAAAK)1-FLS1、F3H-(EAAAK)2-FLS1、F3H-(EAAAK)3-FLS1、F3H-(EAAAK)4-FLS1。结果发现F3H-(GGGGS)2-FLS1的活性明显高于其他融合蛋白，推测可能是由于这些融合蛋白未能有效折叠形成底物通道。本发明中，利用2个柔性连接肽GGGGS依次连接基因f3h和fls1，连接肽的左右两侧分别由BamHI和EcoRI酶切位点连接基因，得到一个特定排列顺序的双功能酶，用于显著提高山奈酚的体外酶促合成产量。
本发明还提供一种高效活性双功能酶的合成方法，该方法包括以下步骤：
步骤1、双功能酶的构建
从拟南芥中克隆黄烷酮-3-羟化酶（F3H）基因f3h和黄烷酮合酶（FLS1）基因fls1至原核表达载体pET-32a(+)，构建重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1，其中f3h和fls1之间由2个柔性连接肽GGGGS连接；
步骤2、双功能酶的诱导表达与纯化
取重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1，转化感受态大肠杆菌BL21(DE3)，IPTG诱导表达、纯化，得到重组双功能酶。
所述步骤1中，克隆f3h基因时，设计一对PCR引物：正向引物为5’- ACGACGACGACAAGGCCAtggctccaggaactttgac-3’，斜体碱基示酶切位点NcoI；反向引物为5’- ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCagcgaagatttggtcgac-3’，斜体碱基示酶切位点BamHI；
克隆fls1基因时，设计一对PCR引物：正向引物为5’- GGTGGCGGGGGCAGTGAATTCatggaggtcgaaagagtcc-3’，斜体碱基示酶切位点EcoRI；反向引物为
5’- GTGGTGGTGGTGGTGCTCGAGtcaatccagaggaagtttattgagc-3’，斜体碱基示酶切位点XhoI。
采用南京诺唯赞生物科技股份有限公司的无缝克隆试剂盒（ClonExpress UltraOne Step Cloning Kit）将2个PCR片段同时克隆至原核表达质粒pET-32a(+)。构建的重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1委托苏州金唯智生物科技有限公司进行测序。
所述步骤2中，取重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1，转化感受态大肠杆菌BL21(DE3)，37 ℃培养过夜；挑取3-5个菌落，接种至含有100 µg/mL氨苄青霉素的LB培养基中，37 ℃、250 rpm培养过夜；按照1:100转接至300 mL含有100 µg/mL氨苄青霉素的LB培养基，37 ℃ 250 rpm培养至菌液OD600nm达0.4-0.6；向菌液中加入IPTG至终浓度0.2 mM，20℃诱导表达3-4 h，4 ℃离心收集菌体；参照碧云天BeyoGold™ His-tag PurificationResin试剂说明书裂解菌体并纯化重组蛋白，重组蛋白中加入甘油至终浓度10%，分装，-80℃保存备用。
本发明提供一种高效活性双功能酶在生物催化柚皮素合成山奈酚中的应用。
利用双功能酶合成山奈酚的具体方法如下：建立体外酶促合成山奈酚的反应体系，该体系包括Tris-HCl、抗坏血酸、α-酮戊二酸、亚铁离子、柚皮素和双功能酶；将反应体系在一定条件下反应得到反应产物。
进一步的，所述反应体系包括0.1 M Tris-HCl（pH 7.2）、0.4%抗坏血酸、8.2 mMα-酮戊二酸、0.1 mM亚铁离子、1.2 mM柚皮素、300 mg/L双功能酶。
将上述反应体系置于恒温震荡仪中开盖反应40-50 min，温度35 ℃，转速600rpm。反应结束后，加入1/10 体积乙酸终止反应，再加入等体积乙酸乙酯萃取反应产物。2 h后，收集有机相，室温挥干后用甲醇溶解反应产物，进行聚酰胺薄层色谱和高效液相色谱质谱分析。
总之，为了克服微生物发酵法的弊端，本发明建立了无细胞体系，用于体外酶促合成山奈酚。简言之，将黄烷酮3-羟化酶（flavanone 3-hydroxylase, F3H）基因f3h和黄酮醇合酶（flavonol synthase, FLS）基因fls1分别克隆至原核表达载体pET-32a(+)，再转化大肠杆菌，以诱导表达重组酶，并利用纯化的重组酶建立无细胞合成体系，实现山奈酚的体外酶促合成。该体系具有明显的优势：耗时短、操作简单、反应过程易控制、反应体系的成分明确且简单、中间产物少、目标产物易分离和纯化。
本发明的优点是高活性双功能酶具有黄烷酮3-羟化酶和黄酮醇合酶的活性，显著提高了山奈酚的产量，降低了山奈酚的生产成本。同时为了进一步提高该合成体系中山奈酚的产量，本发明在构建一种高活性双功能酶的基础上，通过优化反应体系成分和反应条件，建立了一种体外高效合成目标分子的方法。以柚皮素为原材料，在优化后的体系内经过双功能酶催化反应后可获得92.79 ± 5.01 mg/L山奈酚。
由此可知，体外酶促合成山奈酚是目前最具经济效益的生产方式，而同时通过优化反应体系以及反应条件，山奈酚的产量显著提升，明显降低了生产的成本，因此潜在的经济效益将是相当可观的。
附图说明
图1为本发明中SDS-PAGE电泳分析纯化的双功能酶蛋白的电泳图。图中：M表示蛋白质分子量标准品；1表示 His-TrxA-AtF3H-(GGGGS)2-AtFLS1双功能酶蛋白。
图2为本发明中聚酰胺TLC法分析双功能酶活性的结果图。图中：NRN表示柚皮素；DHK表示二氢山奈酚；KMF表示山奈酚；1表示反应产物。
图3为本发明中HPLC法分析双功能酶蛋白活性的结果图。
具体实施方式
下面结合实施例对本发明的技术方案做进一步的详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护权限不限于下述的实施例。
实施例中所涉及材料的来源：
pET-32a(+)购自Novagen公司，NcoI、XhoI等内酶切购自美国纽英伦生物技术，Super Pfx DNA Polymerase、DNA产物纯化试剂盒、胶回收试剂盒等购自康为世纪生物科技有限公司，无缝克隆试剂盒（ClonExpress Ultra One Step Cloning Kit）购自南京诺唯赞生物科技股份有限公司，BeyoGold™ His-tag Purification Resin购自碧云天生物科技有限公司，柚皮素、二氢山奈酚、山奈酚等标准品购自Sigma，聚酰胺6薄层色谱板、甲醇、乙酸乙酯等购自国药。
本实施例所涉及的其余试剂及材料均为市购，此处不再一一列举。本实施例中所涉及的“%”若没有特殊说明，一般为质量百分比。
实施例1
1. 双功能酶的构建
根据GenBank数据库提供的拟南芥黄烷酮-3-羟化酶（F3H）基因f3h和黄烷酮合酶（FLS1）基因fls1的核苷酸序列信息，分别设计两对引物序列，用于将f3h基因和fls1基因的编码区同时克隆至大肠杆菌表达载体pET-32a(+)，其中f3h和fls1之间由2个柔性连接肽GGGGS连接。
扩增f3h基因的正向引物为5’- ACGACGACGACAAGGCCAtggctccaggaactttgac-3’（其核苷酸序列如SEQ ID No.2所示），斜体碱基示酶切位点NcoI；反向引物为5’- ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCagcgaagatttggtcgac-3’（其核苷酸序列如SEQ ID No.3所示），斜体碱基示酶切位点BamHI。
扩增fls1基因的正向引物为5’- GGTGGCGGGGGCAGTGAATTCatggaggtcgaaagagtcc-3’（其核苷酸序列如SEQ ID No.4所示），斜体碱基示酶切位点EcoRI；反向引物为5’- GTGGTGGTGGTGGTGCTCGAGtcaatccagaggaagtttattgagc-3’（其核苷酸序列如SEQ ID No.5所示），斜体碱基示酶切位点XhoI。
参照康为世纪Super Pfx DNA Polymerase操作说明建立如下PCR扩增体系：
| 试剂 | 100μL反应体系 |
| 5×Super Pfx HF Buffer | 20 μL |
| dNTP Mix，10 mM each | 2 μL |
| Forward Primer，10 μM | 5 μL |
| Reverse Primer，10 μM | 5 μL |
| 拟南芥cDNA | 1 μL |
| Super Pfx DNA Polymerase | 1 μL |
| ddH<sub>2</sub>O | To 100 μL |
PCR反应条件如下：
| 步骤 | 温度 | 时间 |
| 预变性 | 98 ℃ | 3 min |
| 变性 | 98 ℃ | 15 s |
| 退火 | 55 ℃ | 15 s |
| 延伸 | 72 ℃ | 1 min 10 s |
| 终延伸 | 72 ℃ | 10 min |
使用常规的DNA产物纯化试剂盒分别纯化PCR产物。
载体pET-32a(+)经NcoI/XhoI双酶切，纯化的PCR产物进行DNA凝胶电泳并对目的条带进行胶回收（结果见图1）。采用南京诺唯赞生物科技股份有限公司的无缝克隆试剂盒（ClonExpress Ultra One Step Cloning Kit）将2个PCR片段同时克隆至原核表达质粒pET-32a(+)，常规转化感受态大肠杆菌DH5α后，均匀涂布于氨苄抗性的LB平板，37 ℃过夜培养。第二天挑单菌落，接种至3 mL氨苄抗性的LB液体培养基中，37 ℃ 250 rpm培养15 h，提取质粒后委托苏州金唯智生物科技有限公司进行测序鉴定。
双功能酶的诱导表达与纯化
构建的重组质粒经42 ℃热击90 s转入大肠杆菌BL21(DE3)，经37 ℃活化5-10min后涂布于含50 mg/L氨苄抗生素的平板，置于37 ℃培养14-16 h后挑取3-5个单菌于含50 mg/L氨苄抗生素的LB培养液中，于37 ℃摇床中培养过夜。次日，将培养过夜的菌按1：100接种于含50 mg/L氨苄抗生素的LB培养液中，于37 ℃摇床中培养，当菌在600 nm处的吸光度达到0.4 - 0.6时加入0.2 mM IPTG，于20 ℃诱导3 -4 h，诱导结束后超声破碎细胞得到上清用于纯化目标蛋白；利用Ni-琼脂糖微珠按照常规操作纯化得到双功能酶,并通过SDS-PAGE检测酶的纯度。
柚皮素在双功能酶的作用下合成山奈酚
反应体系：100μL反应体系中含有0.1M Tris-HCl （pH7.2），0.4%抗坏血酸，8.2 mMα-酮戊二酸，0.1 mM亚铁离子，1.2 mM柚皮素，300 mg/L His-TrxA-AtF3H-(GGGGS)2-AtFLS1双功能酶蛋白。
反应条件：在恒温震荡仪中开盖反应40 min，温度35 ℃，转速600rpm。反应结束后，加入1/10 体积乙酸终止反应，再加入等体积乙酸乙酯萃取反应产物。2 h后，收集有机相，室温挥干后用甲醇溶解反应产物，进行聚酰胺薄层色谱（TLC）和高效液相色谱质谱（HPLC/MS）分析。
检测
取相同体积的反应产物、柚皮素、二氢山奈酚、山奈酚作为样品进行聚酰胺TLC检测以分析双功能蛋白酶的活性。将每个样品逐滴点于聚酰胺6薄层色谱板，待样品室温挥干后，将薄层色谱板置于预先加入了8 mL展开剂（包含氯仿、甲醇、乙酸乙酯和甲酸的比例为5:1.5:1:0.5）的层析缸中展开，待展开剂扩散至距离色谱板顶端1 cm处时，取出色谱板，室温挥干后喷洒1%三氯化铝的乙醇溶液以增强显色，结果见图2。
分析
同样取相同体积的反应产物、柚皮素、二氢山奈酚、山奈酚作为样品进行HPLC/MS检测分析双功能蛋白酶的活性。
样品的处理:甲醇完全溶解样品后用0.22 μm滤器过滤至样品瓶，上样至6460液质联用仪，利用Agilent C18 column (150×4.6 mm, 5 μm)色谱柱行高效液相色谱分析。
色谱条件为：柱温30 ℃，进样量10 μL。流动相为蒸馏水（A）和乙腈（B），以1 mL/min的流速进行梯度洗脱：0-10 min, A: 90%-75%, B: 10%-25%; 10-35 min, 75%-50%,B: 25%-50%; 35-45 min, A: 50%-15%, B: 50%-85%; 45-50 min, A: 15%-90%, B: 85%-10%; 50-60min, A: 90%, B: 10%。检测器为光电二级阵列管检测器（DAD）。检测波长为全波长。
质谱条件为：电喷雾离子源（ESI），正负离子模式；全扫描(m/z)：50-2000；干燥气流速10 L/min，干燥气温度300 °C；鞘气流速7 L/min，鞘气温度250 ℃；毛细管电压在正模式下为4000 V，负模式下为3500 V。
数据分析：MassHunter 4.0数据处理系统。
以上所述，仅为本发明中的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉该技术的人在本发明所揭露的技术范围内，可理解想到的变换或替换，都应涵盖在本发明的包含范围之内，因此，本发明的保护范围应该以权利要求书的保护范围为准。
序列表
<110> 扬州大学
<120> 一种合成山奈酚的高活性双功能酶及其合成方法与应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 867
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
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Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
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Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
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Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
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Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
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Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
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His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
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Ala Pro Gly Thr Leu Thr Glu Leu Ala Gly Glu Ser Lys Leu Asn Ser
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Lys Phe Val Arg Asp Glu Asp Glu Arg Pro Lys Val Ala Tyr Asn Val
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Phe Ser Asp Glu Ile Pro Val Ile Ser Leu Ala Gly Ile Asp Asp Val
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Asp Gly Lys Arg Gly Glu Ile Cys Arg Gln Ile Val Glu Ala Cys Glu
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Asn Trp Gly Ile Phe Gln Val Val Asp His Gly Val Asp Thr Asn Leu
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Val Ala Asp Met Thr Arg Leu Ala Arg Asp Phe Phe Ala Leu Pro Pro
245 250 255
Glu Asp Lys Leu Arg Phe Asp Met Ser Gly Gly Lys Lys Gly Gly Phe
260 265 270
Ile Val Ser Ser His Leu Gln Gly Glu Ala Val Gln Asp Trp Arg Glu
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Ile Val Thr Tyr Phe Ser Tyr Pro Val Arg Asn Arg Asp Tyr Ser Arg
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Trp Pro Asp Lys Pro Glu Gly Trp Val Lys Val Thr Glu Glu Tyr Ser
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Glu Arg Leu Met Ser Leu Ala Cys Lys Leu Leu Glu Val Leu Ser Glu
325 330 335
Ala Met Gly Leu Glu Lys Glu Ser Leu Thr Asn Ala Cys Val Asp Met
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Asp Gln Lys Ile Val Val Asn Tyr Tyr Pro Lys Cys Pro Gln Pro Asp
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Leu Thr Leu Gly Leu Lys Arg His Thr Asp Pro Gly Thr Ile Thr Leu
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Leu Leu Gln Asp Gln Val Gly Gly Leu Gln Ala Thr Arg Asp Asn Gly
385 390 395 400
Lys Thr Trp Ile Thr Val Gln Pro Val Glu Gly Ala Phe Val Val Asn
405 410 415
Leu Gly Asp His Gly His Phe Leu Ser Asn Gly Arg Phe Lys Asn Ala
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Asp His Gln Ala Val Val Asn Ser Asn Ser Ser Arg Leu Ser Ile Ala
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ggtggcgggg gcagtgaatt catggaggtc gaaagagtcc 40
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtggtggtgg tggtgctcga gtcaatccag aggaagttta ttgagc 46
Claims (8)
1.一种合成山奈酚的高活性双功能酶，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
2.一种高效活性双功能酶的合成方法，其特征在于，包括以下步骤：
步骤1、从拟南芥中克隆黄烷酮-3-羟化酶基因f3h和黄烷酮合酶基因fls1至原核表达载体pET-32a(+)，构建重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1；
步骤2、取重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1，转化感受态大肠杆菌BL21(DE3)，IPTG诱导表达、纯化，得到重组双功能酶。
3.根据权利要求2所述一种高效活性双功能酶的合成方法，其特征在于，所述步骤1中，克隆f3h基因时，设计一对PCR引物：正向引物为
5’- ACGACGACGACAAGGCCAtggctccaggaactttgac-3’；反向引物为5’- ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCagcgaagatttggtcgac-3’；
克隆fls1基因时，设计一对PCR引物：正向引物为5’- GGTGGCGGGGGCAGTGAATTCatggaggtcgaaagagtcc-3’；反向引物为5’- GTGGTGGTGGTGGTGCTCGAGtcaatccagaggaagtttattgagc-3’。
4.根据权利要求2所述一种高效活性双功能酶的合成方法，其特征在于，所述步骤2中，取重组质粒pET-32a-AtF3H-(GGGGS)2-AtFLS1，转化感受态大肠杆菌BL21(DE3)，37 ℃培养过夜；挑取3-5个菌落，接种至含有100 µg/mL氨苄青霉素的LB培养基中，37 ℃、250 rpm培养过夜；按照1:100转接至300 mL含有100 µg/mL氨苄青霉素的LB培养基，37 ℃ 250 rpm培养至菌液OD600nm达0.4-0.6；向菌液中加入IPTG至终浓度0.2 mM，20 ℃诱导表达3-4 h，4 ℃离心收集菌体；参照碧云天BeyoGold™ His-tag Purification Resin试剂说明书裂解菌体并纯化重组蛋白。
5.一种高效活性双功能酶在生物催化柚皮素合成山奈酚中的应用。
6.根据权利要求5所述一种高效活性双功能酶的应用，其特征在于，建立包含所述双功能酶的体外酶促合成山奈酚的反应体系。
7.根据权利要求6所述一种高效活性双功能酶的应用，其特征在于，所述体外酶促合成山奈酚的反应体系包括Tris-HCl、抗坏血酸、α-酮戊二酸、亚铁离子、柚皮素和双功能酶；将反应体系在一定条件下反应得到反应产物。
8.根据权利要求7所述一种高效活性双功能酶的应用，其特征在于，所述体外酶促合成山奈酚的反应体系包括0.1 M Tris-HCl、0.4%抗坏血酸、8.2 mM α-酮戊二酸、0.1 mM亚铁离子、1.2 mM柚皮素、300 mg/L双功能酶。
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