CN110563852A - 一种高效简便的酶固定化的方法 - Google Patents

一种高效简便的酶固定化的方法 Download PDF

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CN110563852A
CN110563852A CN201910921844.2A CN201910921844A CN110563852A CN 110563852 A CN110563852 A CN 110563852A CN 201910921844 A CN201910921844 A CN 201910921844A CN 110563852 A CN110563852 A CN 110563852A
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霍毅欣
赵璐瑶
陈振娅
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Abstract

本方法公开了一种高效简便的酶固定化方法及应用该方法来循环利用酶，并实现酶的连续催化。本发明所提供的生产方法是按照包括以下步骤进行：这种方法使用了CipA包涵体蛋白，并将其与反应体系中的几种酶分别融合,且使这几种融合蛋白在同一质粒中表达。并利用CipA的自组装功能得到三种融合蛋白的聚集体，其存在形式为沉淀包涵体，通过破碎、离心弃去上清，从而获得纯度较高的沉淀包涵体，将其填充至底部装有垫片的层析柱中，将配置好的反应液通过蠕动泵注入层析柱以此使反应可以不断进行。本发明提出了一种高效简便的固定化酶的方法来循环利用酶，并实现产物的体外生产，该方法有潜力应用于一些产物的工业化生产。

Description

一种高效简便的酶固定化的方法
技术领域
本发明涉及一种利用包涵体蛋白CipA与目标蛋白融合实现酶的固定化的方法，本发明还涉及实现目标蛋白的循环利用以及连续的催化反应，属于生物工程技术领域。
背景技术
随着分子生物学、细胞工程和发酵工程的发展，重组蛋白的表达效率显著提高。尽管取得了这样的成功，开发一种大规模、简便且高效的蛋白纯化方法以及能更加高效的使用酶生产出所需要的产物仍然是一项巨大的挑战。固定化可以增加酶的稳定性，且可以使其余反应体系中的其他物质分离，最重要的是可以大大提高酶的重复使用率，并且适合大规模生产。
常见的固定化酶的方法有包埋法、吸附法、共价结合法及交联法等。传统的固定化方法将可溶性的酶固定，都需要选择固定载体。在选择载体的过程中，既要考虑载体的形状大小、化学稳定性、亲疏水性、机械迁都及官能团种类等要求，又要根据其应用途径选择不同的材料。现在研究中主要用到的载体材料有无机、有机及复合载体材料三种。这些方法均需要用物理或化学的方法使酶与载体结合，如此看来，传统的酶固定化方法由于载体的需要，需要考虑的问题众多，而且有些载体的成本较高，甚至有可能影响酶的结构与活性，因此在酶固定化的过程中存在很多限制。
利用CipA包涵体蛋白，并将其与一个反应体系中的几种酶分别融合,且使这几种融合蛋白在同一质粒中表达。并利用CipA的自组装功能得到三种融合蛋白的聚集体，其存在形式为沉淀包涵体，通过破碎、离心弃去上清，从而获得纯度较高的沉淀包涵体。
将一个反应链上的多个蛋白与CipA融合，将这个含有多种目标蛋白的聚集体灌注到层析柱中，并将其填充至底部装有垫片的层析柱中，将配置好的反应液通过蠕动泵注入层析柱以此使反应可以不断进行。这一方法在很多蛋白中都具有普遍适用性，且省去了固定化酶所需要的载体的选择。
发明内容
本发明的目的在于提供一种简单高效的酶固定化的方法，该方法中使用到质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP，其表达产物为CipA-KivD-CipA-LeuDH-CipA-AdhP PCIs，通过离心可获得单一的包涵体蛋白复合体，从而达到纯化的效果。将该包涵体蛋白填充至底部装有垫片的层析柱中，将配置好的反应液通过蠕动泵注入层析柱以此使反应可以不断进行。从而实现酶的固定化及持续的催化反应。
本发明的另一个目的是提供上述质粒的构建方法和固定化酶的反应方法，其包括下述步骤：
(1)构建质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP，采用OE-PCR方法合成了 cipA(CAE13869.1)、leuDH(E.C.4.2.1.33)、kivD(E.C.4.1.1.1)及adhP(E.C.1.11.1.2)基因。然后分别用OE-PCR 将cipA的3'端和leuDH的5'端连接，cipA的3'端和kivD的5'端连接以及将cipA的3'端和adhP的5' 端连接。然后，用Gibson组装法分别将cipA-leuDH、cipA-kivD和cipA-adhP克隆到pET-28a中。
(2)表达蛋白CipA-KivD-CipA-LeuDH-CipA-AdhP，将质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP 导入大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液。次日，将种子液接种于200mL新鲜TB培养基中，37°С培养2h左右，使OD600达到0.5-0.8，在培养液中加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。
(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-KivD-CipA-LeuDH-CipA-AdhP PCIs。
(4)将所获得CipA-KivD-CipA-LeuDH-CipA-AdhP PCIs填充至底部装有垫片的层析柱中，将层析柱上下两端橡胶管连接至蠕动泵。配制反应体系(50mM Tris-HCl，pH＝6.5,0.5M/L Valine,0.2g/L TPP,1mM MgCl2,0.25mM NAD+,0.5mM NADPH)，将反应体系通过蠕动泵自下而上注入填充了固定化酶的层析柱。
(5)利用Agilent GC测定产物异丁醇的产量，并根据需要调整流速或将反应体系循环注入层析柱以提高底物转化率。
附图说明
图1为原理流程图；
图2为装备结构示意图；
图3为异丁醇产量检测结果，其中x轴表示时间，单位为h，y轴表示产物的转化率，单位为％。
序列表：
序列1，包涵体蛋白CipA的氨基酸序列；
序列2，异丙基苹果酸脱氢酶LeuDH的氨基酸序列；
序列3，丙酮酸脱羧酶KivD的氨基酸序列；
序列4，乙醇脱氢酶AdhP的氨基酸序列；
具体实施方式
以下实例是对本发明的进一步说明，并不构成对本发明实质内容的限制。
下述实例中所使用的试验方法如无特殊说明，均为常规方法。
下述实例中所用的材料、试剂等，如无特殊说明，均可从商业途径获取。
实施例1
构建质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP
(1)为构建质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP，采用OE-PCR方法合成了cipA(加入号:CAE13869.1)、leuDH(E.C.4.2.1.33)和kivD基因。然后分别用OE-PCR将cipA的3'端和leuDH的5'端连接，cipA的3'端和kivD的5'端连接以及将cipA的3'端和adhP的5'端连接。然后，用Gibson组装法分别将cipA-leuDH、cipA-kivD和cipA-adhP克隆到pET-28a中。
(2)表达蛋白CipA-KivD-CipA-LeuDH-CipA-AdhP，将质粒pET-28a-cipA-kivD-cipA-leuDH-cipA-adhP 导入大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液。次日，将种子液接种于200mL新鲜TB培养基中，37℃培养2h左右，使OD600达到0.5-0.8，在培养液中加入0.5mM异丙基-β-D-硫半乳糖苷(IPTG)，30℃培养10h。
(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris-HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得CipA-KivD-CipA-LeuDH-CipA-AdhP PCIs。
(4)通过SDS-PAGE验证CipA-KivD-CipA-LeuDH-CipA-AdhP蛋白表达的情况。SDS-PAGE浓缩胶和分离胶配方为：5％浓缩胶(3.4mL ddH2O，830μL 30％丙烯酰胺，630μL 1MTris-HCl(pH 6.8)，50 μL 10％SDS，50μL 10％过硫酸铵，5μL TEMED)和10％分离胶(4mLddH2O，3.3mL 30％丙烯酰胺， 2.5mL 1.5M Tris-HCl)，100μL 10％SDS，100μL 10％过硫酸铵，5μL TEMED。
蛋白表达的培养基组成为：酵母提取物24g/L，蛋白胨12g/L，KH2PO4 12.54g/L，K2HPO4 2.31g/L，甘油4mL/L，高温高压灭菌。
实施例2
酶的固定化及异丁醇的生产和检测
将所获得CipA-KivD-CipA-LeuDH-CipA-AdhP PCIs填充至底部装有垫片的层析柱中，将层析柱上下两端橡胶管连接至蠕动泵。配制反应体系(50mM Tris-HCl，pH＝6.5,0.5M/L Valine,0.2g/L TPP,1mM MgCl2, 0.25mM NAD+,0.5mM NADPH)，将反应体系通过蠕动泵自下而上注入填充了固定化酶的层析柱。
异丁醇检测，使用配备火焰离子化检测器的Agilent 6890气相色谱仪。毛细管柱为DB-FFAP(30 m×0.32mm×0.25μm，安捷伦科技)，色谱条件为80℃，3min，利用增加梯度(115℃/min)增加到230℃，并在230℃保持1min，氮气作为载气。进样器和检测器分别维持在250和280℃。取反应后上清液1μL 进样，其中进样分流比为1：30，正戊醇为内标。
在1.9min时获得异丁醇的气相色谱峰，产率可达到90％以上。
序列表
序列1：CipA
MINDMHPSLIKDKDIVDDVMLRSCKIIAMKVMPDKVMQVMVTVLMHDGVCEEMLLKWNLLDNRGMA IYKVLMEALCAKKDVKISTVGKVGPLGCDYINCVEISM
序列2：LeuDH
MAEKFIKHTGLVVPLDAANVDTDAIIPKQFLQKVTRTGFGAHLFNDWRFLDEKGQQPNPDFVLNFPQYQ GASILLARENFGCGSSREHAPWALTDYGFKVVIAPSFADIFYGNSFNNQLLPVKLSDAEVDELFALVKANP GIHFDVDLEAQEVKAGEKTYRFTIDAFRRHCMMNGLDSIGLTLQHDDAIAAYEAKQPAFMN*
序列3：Kivd
MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISRKDMKWVGNANELNASYMADGYARTKKAAAF LTTFGVGELSAVNGLAGSYAENLPVVEIVGSPTSKVQNEGKFVHHTLADGDFKHFMKMHEPVTAARTLLT AENATVEIDRVLSALLKERKPVYINLPVDVAAAKAEKPSLPLKKENSTSNTSDQEILNKIQESLKNAKKPIV ITGHEIISFGLEKTVSQFISKTKLPITTLNFGKSSVDEALPSFLGIYNGKLSEPNLKEFVESADFILMLGVKLT DSSTGAFTHHLNENKMISLNIDEGKIFNESIQNFDFESLISSLLDLSEIEYKGKYIDKKQEDFVPSNALLSQD RLWQAVENLTQSNETIVAEQGTSFFGASSIFLKPKSHFIGQPLWGSIGYTFPAALGSQIADKESRHLLFIGDG SLQLTVQELGLAIREKINPICFIINNDGYTVEREIHGPNQSYNDIPMWNYSKLPESFGATEERVVSKIVRTEN EFVSVMKEAQADPNRMYWIELILAKEDAPKVLKKMGKLFAEQNKS
序列4：AdhP
MKAAVVTKDHHVDVTYKTLRSLKHGEALLKMECCGVCHTDLHVKNGDFGDKTGVILGHEGIGVVAEV GPGVTSLKPGDRASVAWFYEGCGHCEYCNSGNETLCRSVKNAGYSVDGGMAEECIVVADYAVKVPDGL DSAAASSITCAGVTTYKAVKLSKIRPGQWIAIYGLGGLGNLALQYAKNVFNAKVIAIDVNDEQLKLATEM GADLAINSHTEDAAKIVQEKTGGAHAAVVTAVAKAAFNSAVDAVRAGGRVVAVGLPPESMSLDIPRLVLD GIEVVGSLVGTRQDLTEAFQFAAEGKVVPKVALRPLADINTIFTEMEEGKIRGRMVID。
序列表
<110> 北京理工大学
<120> 一种高效简便的酶固定化的方法
<141> 2019-09-20
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Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu
20 25 30
Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn
35 40 45
Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys
50 55 60
Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val
65 70 75 80
Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile
85 90 95
Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His
100 105 110
His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu
115 120 125
Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val
130 135 140
Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val
145 150 155 160
Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro
165 170 175
Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln
180 185 190
Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro
195 200 205
Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr
210 215 220
Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn
225 230 235 240
Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile
245 250 255
Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser
260 265 270
Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr
275 280 285
Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn
290 295 300
Ile Asp Glu Gly Lys Ile Phe Asn Glu Ser Ile Gln Asn Phe Asp Phe
305 310 315 320
Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys
325 330 335
Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala
340 345 350
Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln
355 360 365
Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380
Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu
385 390 395 400
Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile
405 410 415
Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430
Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn
435 440 445
Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu
450 455 460
Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr
465 470 475 480
Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Glu Arg Val Val Ser
485 490 495
Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala
500 505 510
Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys
515 520 525
Glu Asp Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu
530 535 540
Gln Asn Lys Ser
545
<210> 5
<211> 333
<212> PRT
<213> Escherichia coli
<400> 5
Met Lys Ala Ala Val Val Thr Lys Asp His His Val Asp Val Thr Tyr
1 5 10 15
Lys Thr Leu Arg Ser Leu Lys His Gly Glu Ala Leu Leu Lys Met Glu
20 25 30
Cys Cys Gly Val Cys His Thr Asp Leu His Val Lys Asn Gly Asp Phe
35 40 45
Gly Asp Lys Thr Gly Val Ile Leu Gly His Glu Gly Ile Gly Val Val
50 55 60
Ala Glu Val Gly Pro Gly Val Thr Ser Leu Lys Pro Gly Asp Arg Ala
65 70 75 80
Ser Val Ala Trp Phe Tyr Glu Gly Cys Gly His Cys Glu Tyr Cys Asn
85 90 95
Ser Gly Asn Glu Thr Leu Cys Arg Ser Val Lys Asn Ala Gly Tyr Ser
100 105 110
Val Asp Gly Gly Met Ala Glu Glu Cys Ile Val Val Ala Asp Tyr Ala
115 120 125
Val Lys Val Pro Asp Gly Leu Asp Ser Ala Ala Ala Ser Ser Ile Thr
130 135 140
Cys Ala Gly Val Thr Thr Tyr Lys Ala Val Lys Leu Ser Lys Ile Arg
145 150 155 160
Pro Gly Gln Trp Ile Ala Ile Tyr Gly Leu Gly Gly Leu Gly Asn Leu
165 170 175
Ala Leu Gln Tyr Ala Lys Asn Val Phe Asn Ala Lys Val Ile Ala Ile
180 185 190
Asp Val Asn Asp Glu Gln Leu Lys Leu Ala Thr Glu Met Gly Ala Asp
195 200 205
Leu Ala Ile Asn Ser His Thr Glu Asp Ala Ala Lys Ile Val Gln Glu
210 215 220
Lys Thr Gly Gly Ala His Ala Ala Val Val Thr Ala Val Ala Lys Ala
225 230 235 240
Ala Phe Asn Ser Ala Val Asp Ala Val Arg Ala Gly Gly Arg Val Val
245 250 255
Ala Val Gly Leu Pro Pro Glu Ser Met Ser Leu Asp Ile Pro Arg Leu
260 265 270
Val Leu Asp Gly Ile Glu Val Val Gly Ser Leu Val Gly Thr Arg Gln
275 280 285
Asp Leu Thr Glu Ala Phe Gln Phe Ala Ala Glu Gly Lys Val Val Pro
290 295 300
Lys Val Ala Leu Arg Pro Leu Ala Asp Ile Asn Thr Ile Phe Thr Glu
305 310 315 320
Met Glu Glu Gly Lys Ile Arg Gly Arg Met Val Ile Asp
325 330

Claims (3)

1.一种高效简便的酶固定化方法及应用该方法来循环利用酶，并实现酶连续催化的方法，其特征是利用包涵体蛋白CipA与靶蛋白融合，从而使体外反应所需靶蛋白聚集成沉淀，将沉淀复合体装柱，并在柱子两端连接蠕动泵，制备成可重复利用酶并实现连续反应的反应器。
2.根据权利要求所述一种高效简便的酶固定化方法，其特征采用如下步骤：
(1)构建表达包涵体融合蛋白CipA-靶蛋白的质粒pET-28a-cipA-基因A-cipA-基因B-cipA-基因C…，采用OE-PCR方法合成了cipA(CAE13869.1)、基因A、基因B及基因C等基因。然后分别用OE-PCR将cipA的3'端和目标基因的5'端连接以及将cipA的3'端和kivD的5'端连接，然后，用Gibson组装法分别将cipA-leuDH和cipA-kivD克隆到pET-28a中。
(2)表达重组蛋白，将质粒转化到大肠杆菌BL21(DE3)中，挑取单菌落接种于5mL LB培养基中，加入50μg/mL卡那霉素，37℃过夜培养获得种子液，次日，将种子液接种于200mL新鲜TB培养基中，37℃培养2h左右，使OD600达到0.5-0.8，在培养液中加入0.5mM异丙基-B-D-硫半乳糖苷(IPTG)，30℃培养10h；
(3)获得重组蛋白，将(2)中表达完蛋白的发酵液，离心(4000×g，4℃，20min)，弃上清，获得菌体，加入50mL缓冲液(50mM Tris HCl，pH 7.5)重悬，超声破碎后离心(10000×g，4℃，20min)，弃上清，最终获得包涵体融合蛋白；
(4)将沉淀复合体装柱，并在柱子两端连接蠕动泵，制备成可重复利用酶并实现连续反应的反应器。
3.权利要求1所述的方法，其特征在于：将包涵体蛋白CipA与目的蛋白融合，以实现蛋白的固定化，并通过两端可连接蠕动泵的层析柱，通过注入反应液，以实现连续反应及酶的重复利用。
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