CN104031873B - 用于生产异丁香酚单加氧酶的大肠埃希氏菌及构建和应用 - Google Patents
用于生产异丁香酚单加氧酶的大肠埃希氏菌及构建和应用 Download PDFInfo
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- CN104031873B CN104031873B CN201410204528.0A CN201410204528A CN104031873B CN 104031873 B CN104031873 B CN 104031873B CN 201410204528 A CN201410204528 A CN 201410204528A CN 104031873 B CN104031873 B CN 104031873B
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Abstract
本发明公开了一种用于生产异丁香酚单加氧酶的大肠埃希氏菌及构建方法和应用,该大肠埃希氏菌命名为BL21(DE3)IEM‑PP,保藏编号为CGMCC No.8918。构建方法是利用PCR技术,以土壤的全基因组DNA为模板,在PCR引物作用下扩增得到长度为1438bp的片段,经切胶回收并纯化处理后同T载体pET21(a)连接得到克隆载体重组质粒PET21(a)‑IEM的重组菌BL21(DE3)IEM‑PP,可以应用该重组菌生物转化异丁香酚生产香草醛。
Description
技术领域
本发明涉及异丁香酚单加氧酶的基因工程菌的构建及其生物催化技术领域,特别是涉及一种用于生产异丁香酚单加氧酶的大肠埃希氏菌及构建方法和应用。
背景技术
香草醛是工业中应用最广泛的香料之一,大量用于食品工业,可作为香气修饰和定香的主要原料用于食品、牙膏、香皂、烟草中;在医药化工中作为重要的原料或中间体,可用于制造治疗高血压、心脏病、皮肤病及消除口臭、利尿的常用药物;在化学工业中可作为化学助剂,用于塑料制品的抗硬化剂以及Ni,Cr,Cd等金属的电镀光亮剂;在农业生产上,香草醛可作为作物增产剂和催熟剂,并用之制备除草剂和昆虫引诱剂等,需求量很大。香草醛目前主要由化学方法制备,但用化学合成法制得的香草醛不是天然香料。天然香草醛可以从香子兰的花荚中提取,但用植物组织提取的方法生产的天然香草醛量少而价高,不能满足日益增长的需求。
随着世界各国对食品安全越来越重视,对天然香草醛的需求越来越大。天然香料是指由动植物材料经物理(包括蒸馏、溶剂萃取)方法、酶法或微生物方法得到的,可通过传统的食品加工方法(包括干燥、焙烤、发酵)加工后用于人类消费的物质。根据欧洲和美国立法,利用动植物资源通过物理方法、酶法或微生物法得到的物质才能称为天然物质(MuheimAndrea.US6,235,507.2001)。用生物法生产的香草醛,属天然产品,可以生物降解,符合消费者追求天然产品的消费心理。因此,利用生物转化技术生产生物香兰素,是一种有效的、很有前途的替代方法。
在过去的十年里报道了许多用微生物法或酶法生产香草醛的方法,一般都是通过微生物或酶将合适的前体转化为香草醛。目前,尚未有重组菌转化异丁香酚生产香草醛的任何报道。
发明内容
为了弥补上述现有技术的不足,本发明提供一种用于生产异丁香酚单加氧酶的工程菌(大肠埃希氏菌)及其构建方法和应用,该工程菌可用于生产异丁香酚单加氧酶,进而用于转化异丁香酚生产香草醛。
本发明的技术问题通过以下的技术方案予以解决:
一种用于生产异丁香酚单加氧酶的大肠埃希氏菌(Escherichia coli),该所述大肠埃希氏菌命名为BL21(DE3)IEM-PP,保藏编号为CGMCC No.8918。
本发明提供的工程菌属于大肠埃希氏菌(Escherichia coli),命名为BL21(DE3)IEM-PP,已于2014年3月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏号为CGMCC No.8918。大肠埃希氏菌(Escherichia coli)BL21(DE3)IEM-PP CGMCC No.8918简称大肠埃希氏菌BL21(DE3)IEM-PP。该大肠埃希氏菌BL21(DE3)IEM-PP可用于生产异丁香酚单加氧酶,并利用其转化异丁香酚生产香草醛。
一种上述大肠埃希氏菌的构建方法,包括如下步骤:
(1)提取土壤的全基因组DNA;
(2)根据异丁香酚单加氧酶的基因序列,设计PCR引物,所述PCR引物包括上游引物SEQ ID NO.1和下游引物SEQ ID NO.2;
(3)以所述全基因组DNA为模板,用步骤(2)中的PCR引物进行PCR扩增,得到PCR扩增产物,切胶回收得到大小为1438bp的目标基因SEQ ID NO.3;
(4)将目标基因SEQ ID NO.3插入T载体pET21(a)的Ndel和XhoI酶切位点之间,得到重组质粒PET21(a)-IEM;
(5)将重组质粒PET21(a)-IEM转化得到所述大肠埃希氏菌。
一种所述的大肠埃希氏菌在生产异丁香酚单加氧酶中的应用。
优选地,所述的应用包括如下步骤:
(1)将所述的大肠埃希氏菌接种至发酵培养基,得到菌浓度OD600nm=0.1-0.2(如0.1-0.15或0.15-0.2)的发酵初始体系;
(2)将所述发酵初始体系进行如下发酵:先在35-37℃下振荡培养3-5小时,然后加入IPTG(异丙基硫代半乳糖苷)至IPTG在所述述发酵初始体系中的终浓度为50-100μM,然后在25-30℃下继续振荡培养12-48小时,得到氨基酸序列为SEQ ID No.4异丁香酚单加氧酶。
优选地,所述发酵培养基pH为7.0-7.2(如7.0-7.1或7.1-7.2),包括10-15g/L(如10-12g/L或12-15g/L)胰蛋白胨、15-23g/L(如15-19g/L或19-23g/L)酵母提取物、5-10g/L(如5-7g/L或7-10g/L)氯化钠、10-15g/L(如10-12g/L或12-15g/L)甘油、10-17mM(如10-14mM或14-17mM)磷酸二氢钾和65-75mM(如65-70mM或70-75mM)磷酸氢二钾,余量为水。
优选地,所述大肠埃希氏菌以种子液的形式用移液器接种至所述发酵培养基,所述种子液占所述发酵培养基的体积分数为0.5-2%,即接种量为0.5-2%,进一步优选接种量为1%。
优选地,所述种子液是将所述大肠埃希氏菌接种至种子培养基,35-37℃振荡培养至菌浓度OD600nm=3-6形成。
优选地,所述种子培养基pH为7.0-7.2(如7.0-7.1或7.1-7.2),包括10-15g/L(如10-13g/L或13-15g/L)胰蛋白胨、5-8g/L(如5-7g/L或7-8g/L)酵母提取物、5-10g/L(如5-7g/L或7-10g/L)氯化钠,余量为水。
以上任一所述的振荡培养是在100-220r/min(如100-150r/min或150-220r/min)下进行。
一种香草醛的制备方法,包括如下步骤:按异丁香酚:异丁香酚单加氧酶:缓冲液=0.1~0.4g:0.2~1.2g:9~18mL加入反应容器中,在20~28℃下振摇转化12~48h,得到香草醛;所述异丁香酚单加氧酶由上述任意一项所述的应用制备得到,所述缓冲液为pH8~10.4的甘氨酸/NaOH水溶液。
优选地,甘氨酸/NaOH水溶液中甘氨酸的浓度可以为50~150mM。
优选地,在所述反应容器中,还添加DMSO、离子液体和香草醛吸附剂中的至少一种;每0.1~0.4g异丁香酚中:所述DMSO的加入量≤1mL,所述离子液体的加入量≤100μL,香草醛吸附剂的加入量≤0.6g。
加入的DMSO或离子液体可以与水互溶,可以促进异丁香酚在整个体系中的溶解度,而又不会使酶失活;加入的香草醛吸附剂可以吸附香草醛从而加快反应进程,增加产率。
优选地,所述香草醛吸附剂为树脂和壳聚糖膜中的至少一种。
本发明提供的工程菌和方法可用于生产异丁香酚单加氧酶,并用于转化异丁香酚生产香草醛,具备潜在工业化价值(生物法制香精香料),为后续的工业化研究奠定了基础。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为是从常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例一:工程菌的获得和保藏
一、工程菌的构建
1、采取深圳大学文山湖周围土样,以 Spin Kit for Soil(土壤基因组DNA提取试剂盒)提取土壤的全基因组DNA。
2、根据公知的异丁香酚单加氧酶的相关基因序列,设计PCR引物,包括上游引物SEQ ID NO.1和下游引物SEQ ID NO.2。
SEQ ID NO.1的序列如下:5’-atgctacatatggcaacgtttgaccgcaat-3’
SEQ ID NO.2的序列如下:5’-cttatctctcgaggttcttagactgccaac-3’
3、以全基因组DNA为模板,用步骤2设计的PCR引物进行PCR扩增(PCR扩增采用常规的方法:5*FastPfu缓冲液10μL、模板DNA0.5μL、引物SEQ ID NO.1为1μL、SEQ ID NO.2为1μL、dNTPS1.25μL、Transtart FastPfu DNA聚合酶1μL、ddH2O35.3μL,共50μL。PCR程序:①95℃2min;②95℃20s;③55℃20s;④72℃40s;⑤72℃5min;⑥4℃10min;⑦Cycle*30从②到④),得到PCR扩增产物,切胶回收并纯化处理得到大小为1438bp的目标基因SEQ ID NO.3。
SEQ ID NO.3的序列如下:5’-atggcaacgt ttgaccgcaa tgatccacagttgagcaggcacgatgttcc ccacccgaat agaggcgaat gtctttgacc ttgaaattga gggcgagatcccacgtgcaatcaacgggag cttcttccgc aacacccccg aacctcaggt caccacgcaa cctttccacaccttcatcgatggggatggt ttggcgtctg cttttcattt cgaagatggc caggtcgact ttgtcagccgttgggtatgt actcctcgctttgaagctga gcggtcggct cgtaaatcac tcttcggtat gtaccgcaatccgttcactg atgatccatcggtagaaggt attgatcgta cagtcgccaa caccagtatc atcactcatcacgggaaagt actggccgcaaaggaagatg gactacctta tgagcttgac ccccaaaccc tggaaacccgaggtcgttat gattacaaggggcaggtaac cagccataca catacagcgc accctaagtt cgacccccagacaggtgaaa tgttactcttcggctccgct gctaaaggcg aacgaacgct tgatatggcg tactatattgttgatcgcta cggcaaggtgacacatgaga cctggtttaa gcagccttac ggtgcattca tgcacgactttgctgtcacg cgcaactggt caatctttccgatcatgcct gcgacaaata gccttgagcg tcttaaagcaaagcagccca tttacatgtg ggagcctgagcgaggaagct atataggagt acttcctcgt cgtggtcagggcaaggacat tcgttggttc cgtgccccggcgttgtgggt tttccatgtc gtgaatgctt gggaggaagggaatagaatt ctgattgact tgatggaaag tgagattttgccgttcccat tcccgaactc gcagaaccttccatttgatc cctccaaggc tgttccgcgt ctaacccgttgggaaattga tctcaatagt ggtaacgatgagatgaaacg tacgcagcta cacgaatatt ttgcagaaatgcctatcatg gatttccgtt ttgcgctccaggatcatcgc tacgcctaca tgggggttga cgatcctcgt cgccccttagctcatcagca agctgaaaaaatctttgcct acaattcgtt aggggtttgg gacaaccatc gtaaagatta tgaactttggtttacgggaaaaatgtctgc agcgcaggaa ccggcgtttg ttcctagaag cccagatgcg cctgagggcgatggctacctactcagtgta gtagggcggc tcgatgaaga tcgtagcgat ctagttatcc ttgatacgcaatgtttggcagctgggcctg tggccactgt caagcttccc ttccgtctcc gagcagcgtt gcacggttgttggcagtctaagaactga-3’
4、将步骤3中的目标基因插入T载体pET21(a)的Ndel和XhoI酶切位点之间,得到重组质粒PET21(a)-IEM。
5、将重组质粒转化大肠杆菌BL21(DE3),得到一株重组菌(工程菌),该重组菌的生理生化性质与大肠杆菌一样,命名为BL21(DE3)IEM-PP。
二、工程菌的保藏
BL21(DE3)IEM-PP属于大肠埃希氏菌(Escherichia coli)。BL21(DE3)IEM-PP于2014年3月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏号为CGMCC No.8918。大肠埃希氏菌(Escherichia coli)BL21(DE3)IEM-PP CGMCC No.8918简称大肠埃希氏菌BL21(DE3)IEM-PP。
实施例二:应用工程菌生产异丁香酚单加氧酶
一、培养基的制备
种子培养基(pH7.0):取10g胰蛋白胨、5g酵母提取物和5g氯化钠,用水溶解并定容至1L;121℃灭菌30min。
发酵培养基(pH7.0):取10g胰蛋白胨、15g酵母提取物、5g氯化钠、10g甘油、10mM磷酸二氢钾和65mM磷酸氢二钾,用水溶解并定容至1L;121℃灭菌30min。
二、应用工程菌生产异丁香酚单加氧酶
1、将大肠埃希氏菌BL21(DE3)IEM-PP接种至种子培养基,35℃振荡培养(100r/min)至OD600nm=3,即为种子液。
2、将步骤1的种子液用移液器按1%(v/v)的接种量接种至40mL发酵培养基(可以采用250mL摇瓶),得到OD600nm=0.1的发酵初始体系;发酵过程如下(共43小时):先35℃振荡培养(100r/min)3小时,然后加入IPTG至IPTG在所述述发酵初始体系中的终浓度为50μM继续25℃振荡培养(100r/min)40小时。
3、将完成步骤2的发酵体系离心(12000r/min,5min)收集菌体,该菌体为异丁香酚单加氧酶粗酶,以SDS凝胶电泳确定50kDa处的目的蛋白条带,异丁香酚单加氧酶的氨基酸序列为SEQ ID No.4,包含478个氨基酸,其序列如下:Met Ala Thr Phe Asp Arg Asn AspPro Gln Leu Ala Gly Thr Met Phe Pro Thr Arg IleGlu Ala Asn Val Phe Asp LeuGlu Ile Glu Gly Glu Ile Pro Arg Ala Ile Asn Gly SerPhe Phe Arg Asn Thr ProGlu Pro Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile AspGly Asp Gly Leu AlaSer Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser ArgTrp Val Cys ThrPro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser Leu Phe Gly MetTyr Arg AsnPro Phe Thr Asp Asp Pro Ser Val Glu Gly Ile Asp Arg Thr Val Ala AsnThr SerIle Ile Thr His His Gly Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro TyrGluLeu Asp Pro Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln ValThr SerHis Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu Met LeuLeu Phe GlySer Ala Ala Lys Gly Glu Arg Thr Leu Asp Met Ala Tyr Tyr Ile ValAsp Arg Tyr GlyLys Val Thr His Glu Thr Trp Phe Lys Gln Pro Tyr Gly Ala PheMet His Asp Phe AlaVal Thr Arg Asn Trp Ser Ile Phe Pro Ile Met Pro Ala ThrAsn Ser Leu Glu Arg LeuLys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu ArgGly Ser Tyr Ile Gly Val Leu ProArg Arg Gly Gln Gly Lys Asp Ile Arg Trp PheArg Ala Pro Ala Leu Trp Val Phe HisVal Val Asn Ala Trp Glu Glu Gly Asn ArgIle Leu Ile Asp Leu Met Glu Ser Glu IleLeu Pro Phe Pro Phe Pro Asn Ser GlnAsn Leu Pro Phe Asp Pro Ser Lys Ala Val ProArg Leu Thr Arg Trp Glu Ile AspLeu Asn Ser Gly Asn Asp Glu Met Lys Arg Thr GlnLeu His Glu Tyr Phe Ala GluMet Pro Ile Met Asp Phe Arg Phe Ala Leu Gln Asp HisArg Tyr Ala Tyr Met GlyVal Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala GluLys Ile Phe Ala TyrAsn Ser Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr Glu LeuTrp Phe Thr GlyLys Met Ser Ala Ala Gln Glu Pro Ala Phe Val Pro Arg Ser Pro AspAla Pro GluGly Asp Gly Tyr Leu Leu Ser Val Val Gly Arg Leu Asp Glu Asp Arg SerAsp LeuVal Ile Leu Asp Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys LeuProPhe Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn
4、将步骤3获得的菌体用含有6M尿素的PDG缓冲液(包含1mM二硫苏糖醇和10%(体积分数)甘油,pH7.0,50mM磷酸盐缓冲液)震荡悬浮,超声破碎40min,离心(4℃,15,000r/min离心40min)收集上清液,以针头过滤器(孔径0.45μm)过滤后,经HiTrap Chelating HP柱纯化,收集UV280各峰组分,经SDS凝胶电泳确定50kDa处的目的蛋白,透析(甘氨酸-NaOH缓冲液)获得异丁香酚单加氧酶纯酶。
应用异丁香酚单加氧酶(可以是实施例二中制备得到的异丁香酚单加氧酶粗酶,也可以是异丁香酚单加氧酶纯酶)转化异丁香酚生产香草醛的实施例如下:
实施例三
在50mL的锥形瓶中,加入0.4g异丁香酚,异丁香酚单加氧酶1g,加入9mL的pH10.4的甘氨酸浓度为100mM的甘氨酸/NaOH缓冲液,DMSO1mL,以两层纱布覆口,在28℃,200rpm下摇床振摇转化48h,测定最终反应液中香草醛的浓度为2.4g/L。
香草醛的测定方法:
以高效液相色谱法(HPLC)测定香草醛和异丁香酚。
样品处理:反应液加入与反应液同体积的乙醇,沉淀蛋白(异丁香酚单加氧酶),并溶解底物(异丁香酚)和产物(香草醛),10,000r/min离心5min,再以乙醇稀释10-20倍(本例中稀释10倍),滤膜过滤,待测。
色谱柱:Lichrospher100RP-18柱(250mm×4mm×5μm);
流动相:体积比为13:7的甲醇和0.01%(体积分数)冰醋酸水溶液;
流速1mL/min,波长280nm处紫外检测,进样量10μL。其中香草醛和异丁香酚的出峰时间分别为3min和9min左右。
实施例四
在50mL的锥形瓶中,加入0.4g异丁香酚,异丁香酚单加氧酶1g,加入9mL的pH10.4的100mM甘氨酸/NaoH缓冲液,1-乙基-3-甲基咪唑硫酸甲酯盐60μL,以两层纱布覆口,28℃,200rpm摇床振摇转化48h,测定最终反应液中香草醛的浓度为3.0g/L。
实施例五
在50mL的锥形瓶中,加入0.4g异丁香酚,异丁香酚单加氧酶1g,加入9mL的pH10.4的100mM甘氨酸/NaoH缓冲液,壳聚糖膜0.3g,以两层纱布覆口,28℃,200rpm摇床振摇转化48h,反应结束将样品瓶中的膜取出,以10mL的去离子水冲洗,再加入30mL的17.6%(体积分数)的盐酸洗脱10h。测定洗脱液中香草醛的浓度为5g/L。
该实施例中的壳聚糖膜的制备如下:
1.称取3g的壳聚糖粉末溶于150mL2%(体积分数)的醋酸溶液中,室温下磁力搅拌5-6h充分溶解,得到明黄色的粘稠状胶体。
2.用注射器将步骤1获得的胶体挤入并平铺于圆形的塑料培养皿中(每盘约12mL胶体),-80℃冰箱中预冻12h,于冻干机中冻干24h。
3.将冻干的壳聚糖膜剪成小块(每块0.1g);在3%(质量分数)的NaOH溶液中浸泡8h后以去离子水洗至中性,平摊于两层纱布上,室温风干,样品袋中密封干燥保存,备用。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的技术人员来说,在不脱离本发明构思的前提下,还可以做出若干等同替代或明显变型,而且性能或用途相同,都应当视为属于本发明的保护范围。
Claims (6)
1.一种香草醛的制备方法,其特征在于,包括如下步骤:按异丁香酚:异丁香酚单加氧酶:缓冲液=0.1~0.4g:0.2~1.2g:9~18mL加入反应容器中,在20~28℃下振摇转化12~48h,得到香草醛;所述缓冲液为pH8~10.4的甘氨酸/NaOH水溶液;
在所述反应容器中,添加香草醛吸附剂;每0.1~0.4g异丁香酚中:香草醛吸附剂的加入量≤0.6g,所述香草醛吸附剂是壳聚糖膜。
2.如权利要求1所述的制备方法,其特征在于:所述异丁香酚单加氧酶是由大肠埃希氏菌发酵得到,所述大肠埃希氏菌命名为BL21(DE3)IEM-PP,保藏编号为CGMCC No.8918,包括如下步骤:
(1)将大肠埃希氏菌接种至发酵培养基,得到菌浓度OD600nm=0.1-0.2的发酵初始体系;
(2)将所述发酵初始体系进行如下发酵:先在35-37℃下振荡培养3-5小时,然后加入IPTG至IPTG在所述述发酵初始体系中的终浓度为50-100μM,然后在25-30℃下继续振荡培养12-48小时,得到异丁香酚单加氧酶。
3.如权利要求2所述的制备方法,其特征在于:所述发酵培养基pH为7.0-7.2,包括10-15g/L胰蛋白胨、15-23g/L酵母提取物、5-10g/L氯化钠、10-15g/L甘油、10-17mM磷酸二氢钾和65-75mM磷酸氢二钾,余量为水。
4.如权利要求2或3所述的制备方法,其特征在于:所述大肠埃希氏菌以种子液的形式用移液器接种至所述发酵培养基,所述种子液占所述发酵培养基的体积分数为0.5-2%,所述种子液是将所述大肠埃希氏菌接种至种子培养基,35-37℃振荡培养至菌浓度OD600nm=3-6形成。
5.如权利要求4所述的制备方法,其特征在于:所述种子培养基pH为7.0-7.2,包括10-15g/L胰蛋白胨、5-8g/L酵母提取物、5-10g/L氯化钠、余量为水。
6.如权利要求2所述的制备方法,其特征在于:所述的大肠埃希氏菌的构建方法包括如下步骤:
(1)提取土壤的全基因组DNA;
(2)根据异丁香酚单加氧酶的基因序列,设计PCR引物,所述PCR引物包括上游引物SEQID NO.1和下游引物SEQ ID NO.2;
(3)以所述全基因组DNA为模板,用步骤(2)中的PCR引物进行PCR扩增,得到PCR扩增产物,切胶回收得到大小为1438bp的目标基因SEQ ID NO.3;
(4)将目标基因SEQ ID NO.3插入T载体pET21(a)的Ndel和XhoI酶切位点之间,得到重组质粒PET21(a)-IEM;
(5)将重组质粒PET21(a)-IEM转化得到所述大肠埃希氏菌。
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