CN116606824A - 一种异丁香酚单加氧酶突变体iem-f305w-l470e、工程菌及应用 - Google Patents
一种异丁香酚单加氧酶突变体iem-f305w-l470e、工程菌及应用 Download PDFInfo
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Abstract
本发明公开了一种异丁香酚单加氧酶突变体IEM‑F305W‑L470E、工程菌及应用,属于生物技术领域。异丁香酚单加氧酶基因来源于恶臭假单胞菌(Pseudomonasputida),其核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。所述突变体是在SEQ ID NO:2所示氨基酸序列第305位、第470位发生单突变或多位点突变获得的。本发明构建的异丁香酚单加氧酶并构建工程菌株,筛选到的突变体酶活较原始最高提高634.6%,并用于对羟基苯甲醛的酶法合成,产物浓度最高可达2.48g/L,转化率最大可达81.3%。
Description
技术领域
本发明涉及异丁香酚单加氧酶突变体、工程菌及应用,属于生物技术领域。
背景技术
对羟基苯甲醛(p-Hydroxybenzaldehyde,简称p-HBA),可以用于合成心脑血管药物艾司洛尔、口服抗菌素羟氨苄基青霉素(阿莫西林)和抗菌磺胺增效剂三甲氧基苄胺嘧啶等医药原料,还可用于合成乙基香兰素、洋茉莉醛、茴香醛和覆盆子酮等许多珍贵香料。广泛用于医药、农药、香料、化工、电镀、液晶材料等领域,前景广阔。目前,p-HBA主要通过化学法合成,以苯酚、对甲基苯酚、对羟基苯甲醇及对羟基苯乙酸为原料,存在着成本高、环境污染严重、合成选择性差、产率低及工艺不成熟等诸多问题。
微生物法合成p-HBA具有极大的开发应用前景,符合绿色化学的理念要求。2006年,Sachan等使用拟青霉属菌类降解香豆酸合成对羟基苯甲醛、对羟基苯甲酸、原儿茶酸,然而产率低,且组分复杂。酶在生物合成和代谢过程中表现出的高活性、高选择性、高专一性和高效性,极具应用价值。异丁香酚单加氧酶(isoeugenol monooxygenase,Iem)是一种单加氧酶,特异性氧化异丁香酚的侧链碳碳双键转化生成香草醛和乙醛。定向进化是改善蛋白质活性与功能的有效工具,已成功改造各种酶分子定向进化是一种能改善酶的性能,如底物的特异性、热稳定性及酶活力等。
发明内容
本发明针对现有对羟基苯甲醛化学合成工艺存在的缺陷,提供一种产异丁香酚单加氧酶突变体、重组菌及其全细胞作为催化剂,用于对羟基苯甲醛的合成。
本发明是通过如下技术方案实现的:
本发明提供了一种异丁香酚单加氧酶突变体IEM-F305W-L470E,异丁香酚单加氧酶基因来源于恶臭假单胞菌(Pseudomonas putida)IE27,其密码子优化后的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示;所述突变体是在SEQ ID NO:2所示氨基酸序列第305位苯丙氨酸突变为色氨酸以及第470位亮氨酸突变为谷氨酸(IEM-F305W-L470E)的异丁香酚单加氧酶突变体。
本发明还涉及一种所述异丁香酚单加氧酶突变体的编码基因。
本发明提供含有所述编码基因的工程菌。
本发明还提供一种所述异丁香酚单加氧酶突变体在催化对羟基苯乙烯合成对羟基苯甲醛中的应用。
进一步,所述的应用方法为以含异丁香酚单加氧酶突变体基因的工程菌经发酵离心收获的菌体细胞作为全细胞催化剂,催化体系为:对羟基苯乙烯1~5g/L,二甲基亚砜(DMSO)100g/L,硫酸亚铁1~5mM,pH为9.5,反应体积为20mL,装入500mL三角瓶置于空气摇床敞口反应,摇床转速200r/min,温度为30~40℃。
进一步,所述转化体系中,催化剂的用量以湿菌体重量计为50~100g/L。
进一步,含异丁香酚单加氧酶突变体基因的工程菌湿菌体按如下方法制备:将含异丁香酚单加氧酶突变体基因的重组大肠杆菌接入LB斜面培养基,37℃培养12~16h;接斜面菌种于LB液体种子培养基,37℃、200r/min振荡培养4~8h;按体积比1~5%接种量将种子液接入含50mL发酵培养基的500mL三角瓶中,37℃、220r/min振荡培养4h,降温至20~30℃并加入1mmol/L异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)继续诱导表达16h,发酵结束后10000r/min、4℃离心10min收集菌体,无菌生理盐水洗涤菌体两次,得异丁香酚单加氧酶突变体全细胞催化剂。
进一步,所述LB斜面培养基的制备方法:含有终浓度为酵母浸粉5g/L、蛋白胨10g/L、NaCl 5g/L、氨苄西林100mg/L、琼脂20g/L的混合溶液,调节pH6.8~7.0,于121℃高压蒸汽灭菌20min制得;
进一步,所述LB液体种子培养基的制备方法:含有终浓度为酵母浸粉5g/L、蛋白胨10g/L、NaCl 5g/L和氨苄西林100mg/L的混合溶液,调节pH7.0~7.2,于121℃高压蒸汽灭菌20min制得;
进一步,所述发酵培养基的制备方法:含有甘油20g/L、蛋白胨10g/L、酵母浸粉5g/L、麦芽浸粉2g/L、MgSO42 g/L、KH2PO415 g/L、NH4Cl 2g/L和FeSO450 mg/L的混合溶液,调节pH6.7~7.0,于121℃高压蒸汽灭菌20min制得。
本发明与现有技术相比的有益效果是:
本发明采用易错PCR技术成功突变来源于恶臭假单孢菌(Pseudomonas putida)IE27的异丁香酚单加氧酶并构建工程菌株,筛选到的突变体酶活较原始最高提高634.6%,并用于对羟基苯甲醛的酶法合成,产物浓度最高可达2.48g/L,转化率最大可达81.3%。同时本发明所述的生产方法所使用的原料廉价易得,且生产工艺简单易行、生产成本较低。建立的全细胞转化体系,解决了化学法合成对羟基苯甲醛的成本高、环境污染严重、合成选择性差、产率低的问题,实现了高产率绿色生产对羟基苯甲醛。
具体实施方式
下面通过实施例来对本发明的技术方案做进一步解释,但本发明的保护范围不受实施例任何形式上的限制。
实施例1:表达载体和工程菌构建
通过文献报道和基因序列同源性,在NCBI数据库中查询并选择恶臭假单孢菌(Pseudomonas putida)IE27的异丁香酚单加氧酶(GenBank:BAF62888.1),全基因合成异丁香酚单加氧酶IEM(基因序列为SEQ ID No.1,氨基酸序列为SEQ ID No.2),设计引物,利用同源重组进行PCR扩增,亚克隆到载体pET32a的相应位点,获得重组质粒pET32a-IEM。通过热激转化E.coli BL21(DE3),得到表达异丁香酚单加氧酶的重组大肠杆菌E.coli BL21(DE3)/pET32a-IEM。
以重组载体pET32a-IEM作为模板,利用易错PCR技术构建随机突变文库。正向引物IEM-F为5’-acaaggccatggctgatatcggatccatggcaacgtttgaccgcaatg-3’(加粗斜体为同源臂),反向引物IEM-R为5’-tcagtggtggtggtggtggtgctcgagtcagttcttagactgccaac-3’(加粗斜体为同源臂)。易错PCR的反应体系为:PCR Grade Water 39μL,10×TITANIUM TaqBuffer 5μL,MnSO48 mM 1μL,dGTP 2mM1μL,50×Diversify dNTPMix 1μL,Primer mix1μL,模板1μL,TITANIUM TaqPolym 1μL,共50μL体系,其中反应体系中的Primer mix为实例1中所述上下游引物各0.5μL的混合溶液。易错PCR反应条件为:94℃30s,1个循环;94℃30s,68℃1min30s,25个循环;68℃1min,1个循环。将易错PCR扩增产物用1%的琼脂糖凝胶检测,并用AxyPrep DNA凝胶回收试剂盒进行纯化。纯化后的片段与双酶切的载体pET32a(BamH I和Xho I)进行同源重组反应,反应体系为:4μL 5×CE II Buffer,2μL Exase II,用ddH2O补齐20μL。混匀后37℃,融合30min,立即置于冰上冷却10min,然后转化大肠杆菌BL21(DE3)感受态。并置于37℃,200r/min条件下培养1h进行活化,将活化后重组细胞涂布于含100mg/L氨苄西林抗性的LB平板上,37℃倒置培养过夜,获得异丁香酚单加氧酶突变体表达文库。
从获得的异丁香酚单加氧酶突变体表达文库菌落中利用高通量筛选方法,从这些单菌落中筛选高活性突变体菌株。其具体筛选方法为:从平板上随机挑取1800个单菌落,接入96深孔板,培养基为LB液体培养基,37℃,180r/min培养6h后,按5%接种量接入发酵培养基,37℃,220r/min培养6h后加入IPTG至终浓度为0.5mM并降温至20℃诱导表达;离心收集菌体,测定酶活。标准酶活检测体系:湿细胞催化剂10g/L,对羟基苯乙烯2g/L,二甲基亚砜(DMSO)100g/L,硫酸亚铁1mM,反应pH为9.5,总体系为1mL。单位酶活的定义:在标准的反应条件下,每分钟生成1μmol对羟基苯甲醛所需要的酶量为一个酶活单位U。筛选得到1株高活性异丁香酚单加氧酶突变菌株,经测序,为SEQ ID NO:2所示氨基酸序列中第305位苯丙氨酸突变为色氨酸以及第470位亮氨酸突变为谷氨酸(IEM-F305W-L470E)的异丁香酚单加氧酶突变体,对应的工程菌株为E.coli BL21(DE3)/pET32a-IEM-F305W-L470E。突变株酶活如表1所示。
表1异丁香酚单加氧酶突变体酶活
实施例2:利用重组菌发酵生产异丁香酚单加氧酶突变体及酶法转化对羟基苯乙烯生产对羟基苯甲醛
(1)将IEM突变株接入LB斜面培养基37℃培养12h;接斜面菌种于LB液体种子培养基,37℃、200r/min振荡培养6h,按5%接种量将种子液接入含50mL发酵培养基的500mL三角瓶中,37℃、220r/min振荡培养4h,降温至25℃并加入1mmol/L异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)继续诱导表达16h。发酵结束后10000r/min、4℃离心10min收集菌体,无菌生理盐水洗涤菌体两次,分别得到异丁香酚单加氧酶突变体全细胞催化剂。
(2)利用异丁香酚单加氧酶突变体全细胞催化生产对羟基苯甲醛,其转化条件为:全细胞催化剂的添加量50g/L,对羟基苯乙烯2g/L,二甲基亚砜(DMSO)100g/L,硫酸亚铁1mM,pH为9.5,反应体积为20mL,装入500mL三角瓶置于空气摇床敞口反应,摇床转速200r/min,30℃下反应3h。结果如表2所示,E.coli BL21(DE3)/pET32a-IEM-F305W-L470E作为催剂,对羟基苯甲醛浓度为1.35g/L,摩尔转化率为66.5%。
表2IEM突变株转化对羟基苯乙烯生产对羟基苯甲醛
实施例3:利用重组菌发酵生产异丁香酚单加氧酶突变体(IEM-F305W-L470E)及酶法转化对羟基苯乙烯生产对羟基苯甲醛
(1)将IEM突变体IEM-F305W-L470E接入LB斜面培养基37℃培养14h;接斜面菌种于LB液体种子培养基,37℃、200r/min振荡培养6h,按体积比3%接种量将种子液接入含50mL发酵培养基的500mL三角瓶中,37℃、220r/min振荡培养4h,降温至20℃并加入0.5mmol/L异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)继续诱导表达16h。发酵结束后10000r/min、4℃离心10min收集菌体,无菌生理盐水洗涤菌体两次,分别得到异丁香酚单加氧酶突变体全细胞催化剂。
(2)利用异丁香酚单加氧酶突变体IEM-F305W-L470E全细胞催化生产对羟基苯甲醛,其转化条件为:全细胞催化剂的添加量50g/,对羟基苯乙烯3g/L,二甲基亚砜(DMSO)100g/L,硫酸亚铁1mM,pH为9.5,反应体积为20mL,装入500mL三角瓶置于空气摇床敞口反应,摇床转速200r/min,30℃下反应5h。结果对羟基苯甲醛浓度为2.48g/L,摩尔转化率为81.3%。
SEQ ID No.1:
atggcaacctttgatcgtaatgatccgcagctggcaggtaccatgtttccgacccgtattgaagcaaatgtttttgacctgga
aatcgagggtgaaatcccgcgtgcaattaatggtagcttttttcgtaataccccggaaccgcaggttaccacccagccgttt
catacctttattgatggtgatggtctggccagcgcatttcattttgaagatggtcaggttgatttcgtgagccgttgggtttgtac
cccgcgttttgaagcagaacgtagcgcacgtaaaagcctgtttggtatgtatcgtaatccgtttaccgatgacccgagcgtt
gaaggtattgatcgtaccgttgcaaataccagcattattacccatcatggcaaagtgctggcagcaaaagaagatggtctgc
cgtatgaattagatccgcagaccctggaaacccgtggtcgttatgattataaaggtcaggttaccagccatacccataccgc
acatccgaaatttgatccgcagaccggtgaaatgctgctgtttggtagcgcagcaaaaggtgaacgtaccctggatatggc
atattatattgtggaccgctatggcaaagttacccatgaaacctggtttaaacagccgtatggtgcatttatgcatgactttgca
gttacccgtaattggagcatttttccgattatgccggcaaccaatagcctggaacgtctgaaagcaaaacagccgatttatat
gtgggaaccggaacgtggtagctatattggtgttctgccgcgtcgtggtcagggtaaagatattcgttggtttcgtgcaccg
gcactgtgggtttttcatgttgttaatgcatgggaagagggtaaccgcattctgattgatctgatggaaagcgaaattctgccg
tttccgtttccgaatagccagaatctgccgtttgatccgagcaaagcagttccgcgtctgacccgttgggaaattgatctgaa
tagcggtaatgatgagatgaagcgtacccagctgcatgaatattttgccgaaatgccgattatggactttcgctttgcactgc
aggatcaccgttatgcatatatgggtgttgatgacccgcgtcgtccgctggcacatcaacaagcagaaaaaatttttgccta
caacagcctgggcgtgtgggataatcatcgtaaagattatgagctgtggttcaccggtaaaatgagcgcagcacaggaac
cggcatttgttccgcgtagcccggatgcacctgaaggtgatggttatctgctgagcgttgttggtcgtctggatgaagatcgt
agcgatctggttattctggatacccagtgtctggcagcaggtccggttgcaacagttaaactgccgtttcgtctgcgtgcagc
actgcatggttgttggcagagcaaaaattaa
SEQ ID No.2:
MATFDRNDPQLAGTMFPTRIEANVFDLEIEGEIPRAINGSFFRNTPEPQVTTQP
FHTFIDGDGLASAFHFEDGQVDFVSRWVCTPRFEAERSARKSLFGMYRNPFT
DDPSVEGIDRTVANTSIITHHGKVLAAKEDGLPYELDPQTLETRGRYDYKGQ
VTSHTHTAHPKFDPQTGEMLLFGSAAKGERTLDMAYYIVDRYGKVTHETWF
KQPYGAFMHDFAVTRNWSIFPIMPATNSLERLKAKQPIYMWEPERGSYIGVLP
RRGQGKDIRWFRAPALWVFHVVNAWEEGNRILIDLMESEILPFPFPNSQNLPF
DPSKAVPRLTRWEIDLNSGNDEMKRTQLHEYFAEMPIMDFRFALQDHRYAYM
GVDDPRRPLAHQQAEKIFAYNSLGVWDNHRKDYELWFTGKMSAAQEPAFVP
RSPDAPEGDGYLLSVVGRLDEDRSDLVILDTQCLAAGPVATVKLPFRLRAAL
HGCWQSKN。
Claims (10)
1.一种异丁香酚单加氧酶突变体IEM-F305W-L470E,异丁香酚单加氧酶基因来源于恶臭假单胞菌IE27,其密码子优化后的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ IDNO:2所示,其特征在于,所述突变体IEM-F305W-L470E的氨基酸序列是在SEQ ID NO:2所示氨基酸序列的第305位苯丙氨酸突变为色氨酸以及第470位亮氨酸突变为谷氨酸。
2.一种基因,其特征在于,所述基因编码权利要求1所述的异丁香酚单加氧酶突变体IEM-F305W-L470E。
3.一种工程菌,其特征在于,所述工程菌含有权利要求2所述基因。
4.一种应用,其特征在于,权利要求1所述异丁香酚单加氧酶突变体IEM-F305W-L470E在催化对羟基苯乙烯合成对羟基苯甲醛中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的应用方法为以权利要求3所述的工程菌经发酵离心收获的菌体细胞作为全细胞催化剂,催化体系的成分及浓度为对羟基苯乙烯1~5g/L、二甲基亚砜100g/L和硫酸亚铁1~5mM,催化体系的pH为9.5,反应体积为20mL,将所述催化体系装入容器,置于空气摇床,敞口反应,摇床转速200r/min,温度为30~40℃。
6.根据权利要求5所述的应用,其特征在于,所述催化体系中,全细胞催化剂的用量以湿菌体重量计为50~100g/L。
7.根据权利要求6所述的应用,其特征在于,全细胞催化剂按如下方法制备:将含权利要求2所述的异丁香酚单加氧酶突变体IEM-F305W-L470E基因的重组大肠杆菌接入LB斜面培养基,37℃培养12~16h;接斜面菌种于LB液体种子培养基,37℃、200r/min振荡培养4~8h;按体积比1~5%接种量将种子液接入含50mL发酵培养基中,37℃、220r/min振荡培养4h,降温至20~30℃并加入1mmol/L异丙基硫代-β-D-半乳糖苷继续诱导表达16h,发酵结束后离心收集菌体,无菌生理盐水洗涤菌体两次,得异丁香酚单加氧酶突变体IEM-F305W-
L470E全细胞催化剂。
8.根据权利要求7所述的应用,其特征在于,所述LB斜面培养基的制备方法:将含有终浓度为5g/L酵母浸粉、10g/L蛋白胨、5g/LNaCl、100mg/L氨苄西林、20g/L琼脂的混合溶液,调节pH6.8~7.0,于121℃高压蒸汽灭菌20min制得。
9.根据权利要求7所述的应用,其特征在于,所述LB液体种子培养基的制备方法:将含有终浓度为5g/L酵母浸粉、10g/L蛋白胨、5g/LNaCl和100mg/L氨苄西林的混合溶液,调节pH7.0~7.2,于121℃高压蒸汽灭菌20min制得。
10.根据权利要求7所述的应用,其特征在于,所述发酵培养基的制备方法:将含有20g/L甘油、10g/L蛋白胨、5g/L酵母浸粉、2g/L麦芽浸粉、2g/LMgSO4、15g/LKH2PO4、2g/LNH4Cl和50mg/LFeSO4的混合溶液,调节pH6.7~7.0,于121℃高压蒸汽灭菌20min制得。
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