CN113430219B - 水稻色氨酸脱羧酶及其生产方法 - Google Patents
水稻色氨酸脱羧酶及其生产方法 Download PDFInfo
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- CN113430219B CN113430219B CN202110710832.2A CN202110710832A CN113430219B CN 113430219 B CN113430219 B CN 113430219B CN 202110710832 A CN202110710832 A CN 202110710832A CN 113430219 B CN113430219 B CN 113430219B
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- tryptophan decarboxylase
- ala
- rice
- fermentation culture
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Abstract
本发明公开了一种水稻色氨酸脱羧酶及其生产方法,属于生物工程技术领域。编码水稻色氨酸脱羧酶的基因TDC的核苷酸序列如SEQ ID NO.1所示。将SEQ ID NO.1所示的基因TDC连入质粒pEGX‑4t‑J,获得重组表达载体;再将获得的重组表达载体导入大肠杆菌,获得重组菌;将重组菌接种至培养基中进行发酵培养,获得发酵培养液;向发酵培养液中加入诱导剂进行诱导培养,获得诱导培养物;对诱导培养物进行分离纯化,即可制备得到水稻色氨酸脱羧酶。本发明生产得到的水稻色氨酸脱羧酶表达水平高、催化活性好。
Description
技术领域
本发明涉及生物工程技术领域,具体涉及一种水稻色氨酸脱羧酶及其生产方法。
背景技术
色氨酸脱羧酶(Tryptophan decarboxylase,TDC)是一种5'-磷酸吡哆醛(Pyridoxal-5’-phosphate,PLP)依赖型的脱羧酶,可以催化色氨酸脱羧后产生色胺(王鹏等,2014;López-Meyer et al.,1997)。色氨酸脱羧酶可以来源于植物、动物以及微生物。在植物体内,色氨酸脱羧酶属于植物大类中芳香族氨基酸脱羧酶(Aromatic amino aciddecarboxylases,AAADs)家族蛋白中的一类(江晶洁等,2019),依赖于辅因子5'-磷酸吡哆醛的作用下实现催化色氨酸脱羧形成色胺的功能,目前已见报道于喜树、长春花、短小蛇根草、番茄、印度人参、美丽帽柱木等多种植物中(Jadaun et al.,2017;Pang et al.,2018;Tossaton et al.,2013;Yamazaki et al.,2003)。色氨酸脱羧酶基因在陆生植物中往往以多基因家族形式存在,不同植物来源的色氨酸脱羧酶的氨基酸序列同源性存在差异,催化色氨酸脱羧产生色胺的活性也存在不同。
目前我国的色胺类产品还是主要依赖于进口(陈宁等,2017),价格相当昂贵,亟需开发出一种绿色的、低成本的、高效的生物合成色胺的新技术路线。因此通过对色氨酸脱羧酶的分离纯化进行生物催化合成色胺将具有重要的应用前景。
发明内容
针对上述现有技术,本发明的目的是提供一种水稻色氨酸脱羧酶及其生产方法。
为实现上述目的,本发明采用如下技术方案:
本发明的第一方面,提供一种编码水稻色氨酸脱羧酶的基因TDC,所述基因TDC的核苷酸序列如SEQ ID NO.1所示。
本发明的第二方面,提供上述基因TDC编码的水稻色氨酸脱羧酶,其氨基酸序列如SEQ ID NO.2所示。
本发明的第三方面,提供一种生产水稻色氨酸脱羧酶的方法,包括以下步骤:
(1)将SEQ ID NO.1所示的编码水稻色氨酸脱羧酶的基因TDC连入质粒pEGX-4t-J,获得重组表达载体;再将获得的重组表达载体导入大肠杆菌,获得重组菌;
(2)将重组菌接种至培养基中进行发酵培养,发酵培养至发酵培养液稀释100倍后的OD600值为0.18-0.20,获得发酵培养液;向发酵培养液中加入诱导剂进行诱导培养,获得诱导培养物;对诱导培养物进行高压均质处理,制备得到水稻色氨酸脱羧酶。
优选的,步骤(1)中,所述质粒pEGX-4t-J是由如下方法构建而成:
以pEGX-4T-1为出发质粒,利用pflmⅠ和btgⅠ分别将质粒pEGX-4T-1的第3250和4869位点进行酶切,酶切之后再连入长度为100bp的人造链;所述质粒pEGX-4t-J的长度为3449bp,仅在第3249位点处存在SpeⅠ酶切位点,仅在第3349位点处存在Bsc91Ⅰ酶切位点。
优选的,步骤(2)中,所述培养基的组成为:甘油12g/L,酵母膏5g/L,蛋白胨5g/L,KH2PO4 2g/L,K2HPO4 15g/L,(NH4)2SO4 1.5g/L,MgSO4 0.5g/L,CaCl2 0.015g/L,ZnCl20.06g/L。
优选的,步骤(2)中,发酵培养的条件为:pH控制在7.0,罐压0.05MPa,温度33℃,通风比1:1。
优选的,步骤(2)中,诱导培养的条件为:加入诱导剂IPTG,使IPTG的终浓度100mg/L,诱导温度22℃,诱导培养时间2-6h。
优选的,步骤(2)中,高压均质处理的条件为:均质压力15,000PSI,均质流量400L/Hr,控制温度为30℃。
本发明的有益效果:
(1)本发明从营养匮乏的水稻叶片中克隆得到色氨酸脱羧酶的编码基因。为了使编码基因能够更适用于原核表达系统,本发明对编码基因进行了密码子优化,采用优化后的色氨酸脱羧酶编码基因进行原核表达,目的蛋白色氨酸脱羧酶的表达量显著提高。
(2)本发明以pGEX-4T-1为出发质粒,pGEX-4T-1为诱导型质粒,需要加入诱导物诱导后才能表达目的基因,因此可以控制目的基因的表达时间。本发明进一步对pGEX-4T-1进行了改造,将质粒pEGX-4T-1双酶切之后又连接一段100bp的碱基,这样改造处理的好处是减小质粒大小,原质粒4969bp,经过改造后只有3449bp。而且在3249和3349分别有SpeⅠ和Bsc91Ⅰ两个酶切位点,方便目的基因的插入。
(3)现有色氨酸脱羧酶的发酵生产中普遍采用的是LB培养基,本发明还对发酵培养基的组成进行了优化,本发明优化后的培养基更适宜于重组菌的生长繁殖,采用本发明的培养基进行发酵培养,可有效提高色氨酸脱羧酶的表达量。
(4)现有技术中通过原核表达的色氨酸脱羧酶,一般还需要采用柱层析进行纯化,其纯化过程复杂,难以实现工业化生产。而本发明将诱导培养物进行高压均质处理之后即可作为色氨酸脱羧酶进行使用,无需柱层析纯化的过程。
附图说明
图1:优化前的密码子相对适应度图。
图2:优化后的密码子相对适应度图。
图3:本发明的表达载体pGEX-4t-J的结构示意图。
图4:本发明构建的色氨酸脱羧酶基因原核表达载体的结构示意图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
术语说明:
通风比:一分钟内通过单位体积培养液的无菌空气体积;例如,装有18m3培养液的发酵罐,若每分钟通入无菌空气18L,则称通风比为1:1。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例详细说明本申请的技术方案。
实施例1:水稻色氨酸脱羧酶(TDC)基因的克隆及密码子优化
选取营养匮乏水稻的叶片,通过CTAB法提取材料的总RNA,根据从GenBank EST数据库(https://www.ncbi.nlm.nih.gov/genbank/GenBank)中所获得的完整cDNA ORF序列信息设计引物(TDCsense和TDCantisense),以反转录合成的cDNA为模板,进行PCR扩增全长开放读码框。
TDCsense:5’-GTAAGGTATAGTATCATC-3’;(SEQ ID NO.4)
TDCantisense:5’-GTGAGTTAGCTAGGTTGGTGTG-3’。(SEQ ID NO.5)
水稻色氨酸脱羧酶(TDC)基因的cDNA全长包含完整开放读码框为1533bp,核苷酸序列如SEQ ID NO.3所示;编码507个氨基酸的多肽,理论分子质量为56.21ku。
为了使水稻色氨酸脱羧酶的编码基因能更好的适应于原核表达系统,本发明进一步对水稻色氨酸脱羧酶的编码基因进行了密码子优化。经密码子优化后的水稻色氨酸脱羧酶的编码基因的核苷酸序列如SEQ ID NO.1所示;实际编码507个氨基酸的多肽,其氨基酸序列如SEQ ID NO.2所示。
优化前的密码子相对适应度图如图1所示;优化后的密码子相对适应度图如图2所示。
实施例2:表达载体pGEX-4t-J的构建:
以质粒pGEX-4T-1为出发质粒,先采用pflmⅠ在3250位点处进行单酶切,3’末端再切2个,5’末端切6个,3’末端加A,5’末端加CTAGT;再保护;再用btgⅠ在4869位点处进行单酶切,5’末端切4个再加AAGA,去掉所有的保护;再加上5’-CTAGT……G-3’的全长100bp的人造链,人造链中间省略的序列,其只要不包含SpeⅠ和Bsc91Ⅰ两个酶切位点,中间省略的序列可任意选择。
用SpeⅠ和Bsc91Ⅰ对质粒pGEX-4t-J进行双酶切并鉴定,双酶切后pEGX-4t-J出现3349和100两条,则证明表达载体pGEX-4t-J构建成功。
实施例3:水稻色氨酸脱羧酶的发酵生产
(1)使用SpeⅠ和Bsc91Ⅰ对质粒pGEX-4t-J进行双酶切,再通过DNA连接酶将优化后的SEQ ID NO.1所示的TDC基因整合到双酶切后的表达载体pGEX-4t-J上,得到重组表达载体pEGX-TDC;再将获得的重组表达载体导入大肠杆菌B21(DE3),获得重组菌。
(2)将重组菌接种至培养基中进行发酵培养,发酵培养的条件为:pH控制在7.0,罐压0.05MPa,温度33℃,通风比1:1,发酵培养至发酵培养液稀释100倍后的OD600值为0.18-0.20,获得发酵培养液;
将发酵培养液降温至22℃,向发酵培养液中加入诱导剂IPTG,使IPTG的终浓度100mg/L,进行诱导培养2h,获得诱导培养物;将诱导培养物进行高压均质处理,分离取上清液,即制备得到水稻色氨酸脱羧酶(水稻色氨酸脱羧酶为水溶性的,其主要存在于上清液中);
所述培养基的组成为:甘油12g/L,酵母膏5g/L,蛋白胨5g/L,KH2PO4 2g/L,K2HPO415g/L,(NH4)2SO4 1.5g/L,MgSO4 0.5g/L,CaCl2 0.015g/L,ZnCl2 0.06g/L。
发酵培养采用30吨发酵罐,诱导培养后的诱导培养物的体积约21L,高压均质处理的条件如下:
(1)均质压力:压力15,000PSI;
(2)均质流量:400L/Hr;
(3)卫生级别:接触物料部件的材质都是经FDA&GMP认可的316L和17-4PH不锈钢、碳化钨、超高分子聚乙烯和PEEK等,支持CIP;
(4)温度控制:30℃;
(5)液压式动力传输;
(6)进料方式:系统直接连接管路。
对比例1:
将实施例3中整合到双酶切后的表达载体pGEX-4t-J上的TDC基因替换为SEQ IDNO.3所示的核苷酸序列;基于成本考虑,发酵培养采用的是1L发酵罐;其余操作同实施例3。
对比例2:
将实施例3中的培养基替换为LB液体培养基;基于成本考虑,发酵培养采用的是1L发酵罐;其余操作同实施例3。
对比例3:
将实施例3中的培养基的组成调整为:酵母膏5g/L,蛋白胨5g/L,KH2PO4 2g/L,K2HPO4 15g/L,(NH4)2SO4 1.5g/L,MgSO4 0.5g/L,CaCl2 0.015g/L,ZnCl2 0.06g/L;基于成本考虑,发酵培养采用的是1L发酵罐;其余操作同实施例3。
对比例4:
将实施例3中的培养基的组成调整为:甘油12g/L,酵母膏5g/L,蛋白胨5g/L,(NH4)2SO4 1.5g/L,MgSO4 0.5g/L,CaCl2 0.015g/L,ZnCl2 0.06g/L;基于成本考虑,发酵培养采用的是1L发酵罐;其余操作同实施例3。
对实施例3、对比例1-对比例4生产得到的色氨酸脱羧酶的含量及酶活进行考察;其中,色氨酸脱羧酶的含量是利用植物色氨酸脱羧酶(TDC)检测试剂盒(购自于上海臻科生物科技有限公司)测定色氨酸脱羧酶(TDC)的浓度进行表征,其检测原理为:用纯化的色氨酸脱羧酶(TDC)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入色氨酸脱羧酶(TDC),再与HRP标记的色氨酸脱羧酶(TDC)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的色氨酸脱羧酶(TDC)呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中色氨酸脱羧酶(TDC)浓度。检测方法按检测试剂盒说明书操作。
色氨酸脱羧酶的酶活的计算方法如下:
取1g L-色氨酸+0.01g辅酶PLP,加0.900g色氨酸脱羧酶(实施例3、对比例1-对比例4生产得到),溶解,用稀硫酸和NaOH溶液调pH为6.0,定容100mL,25℃水浴。通过液相色谱测定反应开始后5-30min的色胺并绘制曲线,最终计算出酶活力。
结果见表1。
表1:
组别 | 色氨酸脱羧酶(TDC)浓度 | 色氨酸脱羧酶(TDC)酶活 |
实施例3 | 0.02μmol/L | 1.10-1.45U |
对比例1 | 0.012μmol/L | 0.96-1.01U |
对比例2 | 3nmol/L | 0.95-1.03U |
对比例3 | 5nmol/L | 0.96-1.01U |
对比例4 | 5nmol/L | 1.00-1.12U |
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 新泰市佳禾生物科技有限公司,汕头市佳禾生物科技有限公司
<120> 水稻色氨酸脱羧酶及其生产方法
<130> 2021
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1533
<212> DNA
<213> 人工序列
<400> 1
ctagttgttg atatgggttc tctggacacc aacccgaccg ctttctctgc tttcccggct 60
ggtgaaggtg aaaccttcca gccgctgaac gctgacgacg ttcgttctta cctgcacaaa 120
gctgttgact tcatctctga ctactacaaa tctgttgaat ctatgccggt tctgccgaac 180
gttaaaccgg gttacctgca ggacgaactg cgtgcttctc cgccgaccta ctctgctccg 240
ttcgacgtta ccatgaaaga actgcgttct tctgttgttc cgggtatgac ccactgggct 300
tctccgaact tcttcgcttt cttcccgtct accaactctg ctgctgctat cgctggtgac 360
ctgatcgctt ctgctatgaa caccgttggt ttcacctggc aggcttctcc ggctgctacc 420
gaaatggaag ttctggctct ggactggctg gctcagatgc tgaacctgcc gacctctttc 480
atgaaccgta ccggtgaagg tcgtggtacc ggtggtggtg ttatcctggg taccacctct 540
gaagctatgc tggttaccct ggttgctgct cgtgacgctg ctctgcgtcg ttctggttct 600
gacggtgttg ctggtctgca ccgtctggct gtttacgctg ctgaccagac ccactctacc 660
ttcttcaaag cttgccgtct ggctggtttc gacccggcta acatccgttc tatcccgacc 720
ggtgctgaaa ccgactacgg tctggacccg gctcgtctgc tggaagctat gcaggctgac 780
gctgacgctg gtctggttcc gacctacgtt tgcgctaccg ttggtaccac ctcttctaac 840
gctgttgacc cggttggtgc tgttgctgac gttgctgctc gtttcgctgc tggttgcacc 900
tctacccgtc gtaccccggc tgctcgtgct tctgctcgtt cttctggtac cacctctacc 960
gcttggtctg cttggacccc gtctgcttaa gctccgacca acggttaata accggcttct 1020
accgctccgg cttctacctg cgctaccccg accgcttctc cggctccgtc tcgtccgacc 1080
cgttctacct ctcgtaccac cccggctacc ccggctcgtt ctccgacctc tcgtacctgc 1140
cgttctgctt ctgctgctgc ttctggtggt tcttcttctg gttggtcttg cgctccgacc 1200
gcttctccgt cttgccgttc tacctctggt gctacctctc cgtggccgcg ttgctctcgt 1260
acctcttctg ctgctaccac cggttctcgt tcttcttgcc gtggtacctc tctgtcttct 1320
gcttctggtt ctggtccggg tcgtcgtcgt cgtcgtgacg gtggtggtcg tcgtcgtggt 1380
gaaccgcgtg ctgacggtgc tgctgaacag gaccgtcagg gtgttcgtgg tgctcacggt 1440
ggtcgtcgtc aggttcgtgc tgctctgcgt ggtggtctgg ttgctgctgg tcgtgcttct 1500
cgtgctgaac gtgttggtgc tcaccaggaa gac 1533
<210> 2
<211> 507
<212> PRT
<213> 人工序列
<400> 2
Met Gly Ser Leu Asp Thr Asn Pro Thr Ala Phe Ser Ala Phe Pro Ala
1 5 10 15
Gly Glu Gly Glu Thr Phe Gln Pro Leu Asn Ala Asp Asp Val Arg Ser
20 25 30
Tyr Leu His Lys Ala Val Asp Phe Ile Ser Asp Tyr Tyr Lys Ser Val
35 40 45
Glu Ser Met Pro Val Leu Pro Asn Val Lys Pro Gly Tyr Leu Gln Asp
50 55 60
Glu Leu Arg Ala Ser Pro Pro Thr Tyr Ser Ala Pro Phe Asp Val Thr
65 70 75 80
Met Lys Glu Leu Arg Ser Ser Val Val Pro Gly Met Thr His Trp Ala
85 90 95
Ser Pro Asn Phe Phe Ala Phe Phe Pro Ser Thr Asn Ser Ala Ala Ala
100 105 110
Ile Ala Gly Asp Leu Ile Ala Ser Ala Met Asn Thr Val Gly Phe Thr
115 120 125
Trp Gln Ala Ser Pro Ala Ala Thr Glu Met Glu Val Leu Ala Leu Asp
130 135 140
Trp Leu Ala Gln Met Leu Asn Leu Pro Thr Ser Phe Met Asn Arg Thr
145 150 155 160
Gly Glu Gly Arg Gly Thr Gly Gly Gly Val Ile Leu Gly Thr Thr Ser
165 170 175
Glu Ala Met Leu Val Thr Leu Val Ala Ala Arg Asp Ala Ala Leu Arg
180 185 190
Arg Ser Gly Ser Asp Gly Val Ala Gly Leu His Arg Leu Ala Val Tyr
195 200 205
Ala Ala Asp Gln Thr His Ser Thr Phe Phe Lys Ala Cys Arg Leu Ala
210 215 220
Gly Phe Asp Pro Ala Asn Ile Arg Ser Ile Pro Thr Gly Ala Glu Thr
225 230 235 240
Asp Tyr Gly Leu Asp Pro Ala Arg Leu Leu Glu Ala Met Gln Ala Asp
245 250 255
Ala Asp Ala Gly Leu Val Pro Thr Tyr Val Cys Ala Thr Val Gly Thr
260 265 270
Thr Ser Ser Asn Ala Val Asp Pro Val Gly Ala Val Ala Asp Val Ala
275 280 285
Ala Arg Phe Ala Ala Trp Val His Val Asp Ala Ala Tyr Ala Gly Ser
290 295 300
Ala Cys Ile Cys Pro Glu Phe Arg His His Leu Asp Gly Val Glu Arg
305 310 315 320
Val Asp Ser Ile Ser Met Ser Pro His Lys Trp Leu Met Thr Cys Leu
325 330 335
Asp Cys Thr Cys Leu Tyr Val Arg Asp Thr His Arg Leu Thr Gly Ser
340 345 350
Leu Glu Thr Asn Pro Glu Tyr Leu Lys Asn His Ala Ser Asp Ser Gly
355 360 365
Glu Val Thr Asp Leu Lys Asp Met Gln Val Gly Val Gly Arg Arg Phe
370 375 380
Arg Gly Leu Lys Leu Trp Met Val Met Arg Thr Tyr Gly Val Ala Lys
385 390 395 400
Leu Gln Glu His Ile Arg Ser Asp Val Ala Met Ala Lys Val Phe Glu
405 410 415
Asp Leu Val Arg Gly Asp Asp Arg Phe Glu Val Val Val Pro Arg Asn
420 425 430
Phe Ala Leu Val Cys Phe Arg Ile Arg Ala Gly Ala Gly Ala Ala Ala
435 440 445
Ala Thr Glu Glu Asp Ala Asp Glu Ala Asn Arg Glu Leu Met Glu Arg
450 455 460
Leu Asn Lys Thr Gly Lys Ala Tyr Val Ala His Thr Val Val Gly Gly
465 470 475 480
Arg Phe Val Leu Arg Phe Ala Val Gly Ser Ser Leu Gln Glu Glu His
485 490 495
His Val Arg Ser Ala Trp Glu Leu Ile Lys Lys
500 505
<210> 3
<211> 1533
<212> DNA
<213> 水稻
<400> 3
ctagttgttg atatgggcag cttggacacc aaccccacgg ccttctccgc cttccccgcc 60
ggcgagggtg aaaccttcca gccgctcaac gccgatgatg tccggtccta cctccacaag 120
gcggtggact tcatctcgga ctactacaag tccgtggagt ccatgccggt gctgcccaat 180
gtcaagccgg ggtacctgca ggacgagctc agggcctcgc cgccgacgta ctcggcgccg 240
ttcgacgtca ccatgaagga gctccggagc tccgtcgtcc ccgggatgac gcactgggcg 300
agccccaact tcttcgcgtt tttcccctcc acgaatagtg cggccgccat tgccggcgac 360
ctcatcgcgt cggcgatgaa cacggtcggg ttcacgtggc aggcgtcgcc ggcggccacc 420
gagatggagg tgctcgcgct ggactggctc gcgcagatgc tcaacctgcc gacgagcttc 480
atgaaccgca ccggcgaggg gcgtggcacc ggcggtgggg ttattctggg gacgaccagc 540
gaggcgatgc tcgtcacgct cgttgccgcg cgcgacgccg cgctgcggcg gagcggcagc 600
gacggcgtgg cgggactcca ccggctcgcc gtgtacgccg ccgaccagac gcactccacg 660
ttcttcaagg cgtgccgcct cgccgggttt gatccggcga acatccggtc gatccccacc 720
ggggccgaga ccgactacgg cctcgacccg gcgaggctgc tggaggcgat gcaggccgac 780
gccgacgccg ggctggtgcc cacctacgtg tgcgccacgg tgggcaccac gtcgtccaac 840
gccgtcgacc cggtgggcgc cgtggccgac gtcgcggcga ggttcgccgc tgggtgcacg 900
tcgacgcggc gtacgccggc agcgcgtgca tctgcccgga gttcaggcac cacctcgacg 960
gcgtggagcg cgtggactcc atcagcatga gcccccacaa atggctgatg acctgcctcg 1020
actgcacctg cctctacgtg cgcgacaccc accgcctcac cggctccctc gagaccaacc 1080
cggagtacct caagaaccac gccagcgact ccggcgaggt caccgacctc aaggacatgc 1140
aggtcggcgt cggccgccgc ttccgggggc tcaagctctg gatggtcatg cgcacctacg 1200
gcgtcgccaa gctgcaggag cacatccgga gcgacgtcgc catggccaag gtgttcgagg 1260
acctcgtccg cggcgacgac aggttcgagg tcgtcgtgcc gaggaacttc gctctcgtct 1320
gcttcaggat cagggccggg ccggcgccgc cgccgcgacg gaggaggacg ccgacgaggc 1380
gaaccgcgag ctgatggagc ggctgaacaa gaccggcaag gcgtacgtgg cgcacacggt 1440
ggtcggcggc aggttcgtgc tgcgcttcgc ggtgggctcg tcgctgcagg aagagcatca 1500
cgtgcggagc gcgtgggagc tcatcaagaa gac 1533
<210> 4
<211> 18
<212> DNA
<213> 人工序列
<400> 4
gtaaggtata gtatcatc 18
<210> 5
<211> 22
<212> DNA
<213> 人工序列
<400> 5
gtgagttagc taggttggtg tg 22
Claims (5)
1.一种生产水稻色氨酸脱羧酶的方法,其特征在于,包括以下步骤:
(1)将SEQ ID NO.1所示的编码水稻色氨酸脱羧酶的基因TDC连入质粒pEGX-4t-J,获得重组表达载体;再将获得的重组表达载体导入大肠杆菌,获得重组菌;
(2)将重组菌接种至培养基中进行发酵培养,发酵培养至发酵培养液稀释100倍后的OD600值为0.18-0.20,获得发酵培养液;向发酵培养液中加入诱导剂进行诱导培养,获得诱导培养物;对诱导培养物进行高压均质处理,制备得到水稻色氨酸脱羧酶;
步骤(1)中,所述质粒pEGX-4t-J是由如下方法构建而成:
以pEGX-4T-1为出发质粒,利用pflmⅠ和btgⅠ分别将质粒pEGX-4T-1的第3250和4869位点进行酶切,酶切之后再连入长度为100bp的人造链;所述质粒pEGX-4t-J的长度为3449bp,仅在第3249位点处存在SpeⅠ酶切位点,仅在第3349位点处存在Bsc91Ⅰ酶切位点。
2.根据权利要求1所述的方法,其特征在于,步骤(2)中,所述培养基的组成为:甘油12g/L,酵母膏 5g/L,蛋白胨 5g/L,KH2PO4 2g/L,K2HPO4 15g/L,(NH4)2SO4 1.5 g/L,MgSO4 0.5 g/L,CaCl2 0.015 g/L,ZnCl2 0.06 g/L。
3.根据权利要求1所述的方法,其特征在于,步骤(2)中,发酵培养的条件为:pH控制在7.0,罐压0.05MPa,温度33℃,通风比1:1。
4.根据权利要求1所述的方法,其特征在于,步骤(2)中,诱导培养的条件为:加入诱导剂IPTG,使IPTG的终浓度100mg/L,诱导温度22℃,诱导培养时间2-6h。
5.根据权利要求1所述的方法,其特征在于,步骤(2)中,高压均质处理的条件为:均质压力15,000PSI,均质流量400L/Hr,控制温度为30℃。
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