CN115505581B - 一种芳胺-n乙酰转移酶的突变体及其应用 - Google Patents
一种芳胺-n乙酰转移酶的突变体及其应用 Download PDFInfo
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- CN115505581B CN115505581B CN202110670577.3A CN202110670577A CN115505581B CN 115505581 B CN115505581 B CN 115505581B CN 202110670577 A CN202110670577 A CN 202110670577A CN 115505581 B CN115505581 B CN 115505581B
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- arylamine
- aromatic amine
- acetyl transferase
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Abstract
本发明公开了一种芳胺‑N乙酰转移酶的突变体及其应用,属于酶工程及基因工程技术领域。为了解决了芳胺‑N乙酰转移酶的催化效率低的问题。本申请将来自Pseudomonas aeruginosa的芳胺‑N乙酰转移酶第231位赖氨酸突变为谷氨酰胺而获得芳胺‑N乙酰转移酶的突变体,本发明还公开了芳胺‑N乙酰转移酶突变体的酶学性质及底物特性,公开了包含芳胺‑N乙酰转移酶突变体的大肠杆菌及其构建方法和应用。本发明提供的芳胺‑N乙酰转移酶的突变体高效催化芳胺化合物发生乙酰化反应,整个过程效率高、安全环保、操作简便,可用于芳胺乙酰化的绿色生物制备。
Description
技术领域
本发明属于酶工程及基因工程技术领域,具体涉及一种芳胺-N乙酰转移酶的突变体及其应用。
背景技术
芳胺-N乙酰转移酶用于催化乙酰化反应,乙酰化反应是有机合成中常见的反应之一,其中的芳胺类化合物氨基乙酰化反应主要采用乙酰氯、乙酸酐等在酸或碱催化下进行。虽然此类化学合成的技术较为成熟,但其中仍存在很多的问题例如:乙酰氯稳定性差,价格昂贵;乙酸酐活性较低,且有副反应发生;反应过程中氨基易被氧化;反应条件苛刻且催化剂成本高,生产过程会产生大量的污水等。因此需要寻找芳胺乙酰化的绿色生物合成途径,其中包括利用高活性酶或含有酶的细胞进行催化的方法。文献Westwood I M , Holton SJ , Rodrigues-Lima F , et al. Expression, purification, characterization andstructure of Pseudomonas aeruginosa arylamine N-acetyltransferase.Biochemical Journal, 2005, 385(Pt 2):605-612.中报导的来源于铜绿假单胞菌(Pseudomonas aeruginosa)的芳胺-N乙酰转移酶(Arylamine N-acetyltransferase)是一种以Ac-CoA为辅酶的乙酰转移酶,可用于催化芳胺的氨基乙酰化反应,但是目前该酶存在的缺陷是活性较低,对底物的选择范围有限。
发明内容
本发明的目的是为了提高芳胺-N乙酰转移酶的活性和增加其对底物的选择性,解决了芳胺-N乙酰转移酶的催化效率低的问题。
本发明提供了一种芳胺-N乙酰转移酶的突变体,所述所述突变体是以来自铜绿假单胞菌(Pseudomonas aeruginosa)的芳胺-N乙酰转移酶为出发序列,对第231位的氨基酸突变获得的。
在一种实施方式中,所述突变体为将SEQ ID NO :2所示的氨基酸序列中的第231位的赖氨酸突变为谷酰胺氨酸。
本发明提供一种酶制剂,所述酶制剂包括权利要求1或2所述的突变体。
本发明提供了一种编码上述芳胺-N乙酰转移酶突变体的基因。
本发明还提供了一种重组载体,所述重组载体携带上述编码芳胺-N乙酰转移酶突变体的基因。
在一种实施方式中,所述重组载体的出发载体为pETDuet-1。
本发明还提供了一种重组微生物细胞,所述重组微生物细胞是携带上述编码芳胺-N乙酰转移酶突变体的基因或者表达芳胺-N乙酰转移酶突变体。
在一种实施方式中,所述微生物细胞为原核生物或者真核生物。
在一种实施方式中,所述原核生物为大肠杆菌。
本发明还提供了一种促进芳胺-N乙酰转移催化反应的方法,所述方法是将芳胺-N乙酰转移酶突变体或上述重组微生物细胞加入到苯胺类化合物的水溶液中,在pH=4-10,温度为30-60℃的条件下进行催化反应。
在一种实施方式中,所述方法是将芳胺-N乙酰转移酶突变体或上述重组微生物细胞加入到苯胺类化合物的水溶液中,在pH=7.5,温度为30℃的条件下进行催化反应。
芳胺-N乙酰转移酶突变体、酶制剂、编码芳胺-N乙酰转移酶突变体的基因、上述重组载体或上述重组微生物细胞在催化苯胺类化合物乙酰化中的应用。
在一种实施方式中,所述苯胺类化合物不限于对氨基苯酚、2-羟基-4氨基苯酚、2-氨基-3-羟基苯酚、2-氨基-4-氯苯酚、4-氨基-3-氯苯酚、4-氨基-3-氟苯酚、4-氨基-2-氟苯酚、5-氟邻氨基苯酚和4-氟邻氨基苯酚。
有益效果:本发明通过点突变得到一种芳胺-N乙酰转移酶突变体,表达纯化了该蛋白并构建了包含此酶的具有应用价值的重组大肠杆菌。本申请的芳胺-N乙酰转移酶的突变体对底物对氨基酚的催化活性是野生型酶的4倍。该突变体还可以高效催化芳胺类化合物生成相应的乙酰化产物,例如可以催化2-羟基-4氨基苯酚、2-氨基-3-羟基苯酚、2-氨基-4-氯苯酚、4-氨基-3-氯苯酚、4-氨基-3-氟苯酚、4-氨基-2-氟苯酚、5-氟邻氨基苯酚或4-氟邻氨基苯酚生成相应的乙酰化产物,并且整个反应安全、污染低、催化效率高、操作简便,可用于芳胺类化合物乙酰化的绿色生物制备,具有重要的工业应用潜力。
附图说明
图1为芳胺-N 酰基转移酶突变体表达蛋白的SDS-PAGE图;
图2为芳胺-N 酰基转移酶催化氨基苯酚反应,其中,i:芳胺-N 酰基转移酶突变体PANATK231Q与对氨基苯酚反应HPLC检测图;ii:野生型芳胺-N 酰基转移酶PANAT与对氨基苯酚反应HPLC检测图;iii:对乙酰氨基苯酚标准品HPLC检测图;
图3为芳胺-N乙酰转移酶催化反应示意图。
具体实施例
下面结合具体实施例对本发明做进一步说明,但本发明不受实施例的限制。
以下实施例中所用材料、试剂、仪器和方法,未经特殊说明,均为本领域中的常规材料、试剂、仪器和方法,均可通过商业渠道获得。所用酶试剂购自 NEW ENGLAND BioLabs公司,提取质粒所用的试剂盒和回收DNA片段所用的试剂盒购自美国OMEGA公司,相应的操作步骤按照产品说明书进行;所有培养基如无特别说明均用去离子水配制。
培养基配方:LB 培养基:5g/L 酵母粉,10g/L NaCl,10g/L 蛋白胨,其余为水,121℃,20min 灭菌。 在实际培养过程中,可向上述培养基中添加一定浓度的抗生素以维持质粒的稳定性,如 100 mg/L 的氨苄青霉素。
酶活测定方法为:50mM HEPES缓冲液(pH7.0),2mM底物,400μM Ac-CoA,酶20μg/mL,水浴控温30℃反应30min,加入等体积的甲醇终止反应。
用高效液相色谱检测产物反应生成量及底物减少量,产物高效液相色谱检测条件为:0-5min 15-30%乙腈(0.1% TFA)、85-70%水(0.1% TFA);5-10min 30-60%乙腈(0.1%TFA)、70-40%水(0.1% TFA);10-11min 60-80%乙腈(0.1% TFA)、40-20%水(0.1% TFA); 11-15min 80%乙腈(0.1% TFA)、20%水(0.1% TFA);15-16min 80-15%乙腈(0.1% TFA)、20%-85%水(0.1% TFA);16-20min 15%乙腈(0.1% TFA)、85%水(0.1% TFA),检测器为SPD-20A紫外-可见光检测器,检测波长250nm、270nm。
野生型芳胺-N乙酰转移酶PANAT记载在Westwood I M , Holton S J ,Rodrigues-Lima F , et al. Expression, purification, characterization andstructure of Pseudomonas aeruginosa arylamine N-acetyltransferase.Biochemical Journal, 2005, 385(Pt 2):605-612.
实施例1.制备芳胺-N乙酰转移酶突变体
1.芳胺-N乙酰转移酶的氨基酸序列(NCBI PDB:1W4T_A如SEQ ID NO:2)进行密码子优化,合成优化后的基因序列,并将序列克隆到载体pET-Duet上,获得含目的基因的重组质粒。优化后的基因序列如SEQ ID NO:1所示。
2.芳胺-N乙酰转移酶定点突变
(1)设计引物:引物1:CAGGGTCTGCAGGCGGCGAT(SEQ ID NO:3),引物2:GGTAATCGCCGCCTGCAGAC(SEQ ID NO:4)。
(2)以合成的重组质粒pETDuet-PANAT 为模板,利用引物1和引物2进行PCR扩增获得定点突变后的芳胺-N乙酰转移酶基因。
扩增体系为:
PrimeSTAR MAX Premix (2X)25μL
模板DNA1μL
引物1(25μM)1μL
引物2(25μM)1μL
ddH2O补足50μL
扩增程序为:
98℃,40s
98℃,10s;60℃,15s;72℃,60s反应30个循环
72℃,5min
将PCR产物回收获得定点突变后的芳胺-N乙酰转移酶基因。
3.构建重组载体:根据将定点突变后的芳胺-N乙酰转移酶基因(SEQ ID NO:5所示),设计引物:正向引物为TAAGGATCCGATGGGCAGCAGCCATCAT(SEQ ID NO:6所示);反向引物为TAACTCGAGCGCGCTAATTAAGCCCGCT(SEQ ID NO:7所示),利用上述引物扩增基因序列,利用BamHI和XhoI限制性内切酶酶切表达载体pETDuet-1,然后将上述的基因序列与线性化的表达载体pETDuet-1 (Novagen)连接,得到重组质粒pETDuet-PANATK231Q。
扩增基因序列的PCR体系:
PrimeSTAR MAX Premix (2X)25μL
模板DNA1μL
正向引物(25μM)1μL
反向引物(25μM)1μL
ddH2O补足50μL
扩增基因序列的PCR反应条件:
98℃,40s
98℃,10s;60℃,15s;72℃,60s反应30个循环
72℃,5min
酶切后的基因序列片段,采用 Omega DNA Extraction Kit 试剂盒回收,利用NEB T4 DNA 连接酶,将酶切回收后的目的片段和 pETDuet-1 载体连接,反应体系:Buffer(2X), 5μL;pETDuet-1线性化载体,1μL;目的片段,3μL;连接酶,1μL;总体系,10μL。连接反应条件:25 ℃,5 min。
4.将重组质粒pETDuet-PANATK231Q转化大肠杆菌E.coli BL21(DE3),步骤如下:
1)取3μL回收的PCR产物加入到于制备的大肠杆菌DH5α感受态中,轻混匀,冰浴30min。
2)在预热的42℃水浴中,45s热击处理后立即冰浴2min。
3)加入700μL不含抗生素的LB培养液,37℃培养1h使菌体复苏。
4)取200μL菌液均匀涂布在含氨苄抗生素的LB平板上。
5)37℃过夜培养,挑取单菌落测序验证,将正确突变的质粒导入到BL21(DE3)感受态中,获得含突变蛋白PANATK231Q的重组大肠杆菌BL21(DE3) -pETDuet-PANATK231Q,保存备用,利用抗生素筛选得到阳性转化子,即得到重组大肠杆菌E.coli BL21(DE3) -pETDuet-PANATK231Q为芳胺-N乙酰转移酶突变体。
实施例2.芳胺-N乙酰转移酶突变体的诱导表达及分离纯化
1)加500μL实施例1得到的重组大肠杆菌的菌液到200mL LB培养液中。37℃培养至OD600 0.8时,冰上预冷20min。再加入20μL 1M的IPTG, 16℃诱导16h。将发酵液进行离心(6000rpm/min,10min)得到菌体,用HEPES裂解液(25mM HEPES,pH7.5,0.5M NaCl,5mM咪唑)复溶菌体,高压破碎仪破碎,离心(13000rpm/min,30min)收集上清得到粗酶液。
2)将步骤1)得到的粗酶液采用镍柱纯化,洗脱方法为:将粗酶液与镍珠4℃充分孵化1h后转入到重力柱中。用洗涤液(25mM HEPES,pH7.5,0.5M NaCl,20mM咪唑)洗除杂蛋白,用洗脱液(25mM HEPES,pH7.5,0.3M NaCl,150mM咪唑)洗脱目的蛋白。纯化的蛋白如图1所示,得到的蛋白是目标蛋白,将纯化的酶保存在蛋白储存液中(25mM HEPES,pH7.5,0.3MNaCl),-80℃冻存。芳胺-N乙酰转移酶突变体的氨基酸序列如SEQ ID NO.8所示。
实施例3.检测芳胺-N乙酰转移酶突变体的酶活
以对氨基酚(底物终浓度2mM,酶浓度20μg/mL,辅因子Ac-CoA浓度400μM)为底物,加入实施例2得到的芳胺-N乙酰转移酶突变体的纯化后的蛋白PANATK231Q 或加入野生型的芳胺-N乙酰转移酶PANAT,将底物与pH4-10的50mM HEPES缓冲液在30-60℃不同的温度条件下水浴30min,测定芳胺-N乙酰转移酶突变体的酶活。
结果如图2所示,根据对乙酰氨基酚标样iii所示,i峰面积为370948,ii峰面积为92261,纯化后的蛋白PANATK231Q(图2中i所示)和PANAT(图2中ii所示)均可催化对氨基酚生成对乙酰氨基酚,且PANATK231Q催化效率是野生型PANAT的4倍。
在温度为30度,pH4-10的反应条件下,检测芳胺-N乙酰转移酶突变体的酶活,结果如表1所示,在pH7条件下芳胺-N乙酰转移酶突变体的酶活最高。
在pH为7,温度为30-60℃反应条件下,检测芳胺-N乙酰转移酶突变体的酶活,结果如表2所示,酶的最适反应温度为30℃。
表1芳胺-N乙酰转移酶突变体的相对酶活
表2芳胺-N乙酰转移酶突变体的相对酶活
实施例4.含有芳胺-N乙酰转移酶突变体的全细胞的应用
1)加500μL实施例1得到的重组大肠杆菌的菌液到200mL LB培养液中。37℃培养至OD600 0.8时,冰上预冷20min。再加入20μL 1M的IPTG, 16℃诱导16h。将发酵液进行离心(6000rpm/min,10min)得到菌体,用HEPES裂解液(25mM HEPES,pH7.5,0.5M NaCl,5mM咪唑)复溶菌体,6000rpm/min,10min离心得到去除液体得到菌体。
2)以对氨基酚为底物(底物终浓度2mM),以pH7.5 50mM HEPES为反应溶液,加入步骤1)得到的菌体BL21(DE3)-pETDuet-PANATK231Q,或者加入野生型的芳胺-N乙酰转移酶BL21(DE3)-pETDuet-PANAT,反应条件为温度30℃,pH为7,转速200rpm,反应底物浓度为2mM,细胞干重均为5g/L,进行全细胞催化产对乙酰氨基酚(反应时间1h)。
结果:在上述反应条件下,菌体BL21(DE3)-pETDuet-PANAT和BL21(DE3)-pETDuet-PANATK231Q均可催化对氨基酚生成对乙酰氨基苯酚,且芳胺-N乙酰转移酶突变体转化对氨基酚生成对乙酰氨基酚的效率提高。
3)以下列化合物为底物,2-羟基-4氨基苯酚、2-氨基-3-羟基苯酚、2-氨基-4-氯苯酚、4-氨基-3-氯苯酚、4-氨基-3-氟苯酚、4-氨基-2-氟苯酚、5-氟邻氨基苯酚、4-氟邻氨基苯酚,加入加入步骤1)得到的菌体BL21(DE3)-pETDuet-PANATK231Q,或者加入野生型的芳胺-N乙酰转移酶BL21(DE3)-pETDuet-PANAT,反应条件为温度30℃,pH为7,转速200rpm,反应底物浓度为2mM,细胞干重均为5g/L,进行全细胞催化反应(反应时间1h),催化反应如图3所示。
结果:在上述反应条件下,菌体BL21(DE3)-pETDuet-PANAT和BL21(DE3)-pETDuet-PANATK231Q均可催化步骤3)中所示的底物生成相应的乙酰氨基酚,芳胺-N乙酰转移酶突变体相对野生型芳胺-N乙酰转移酶活高。
实施例5.芳胺-N乙酰转移酶突变体表达蛋白的应用
利用实施例2获得的芳胺-N乙酰转移酶突变体表达的纯蛋白,以对氨基酚为底物,以pH7.5 50mM HEPES为反应溶液,加入芳胺-N乙酰转移酶突变体表达的纯蛋白PANATK231Q,或者加入野生型的芳胺-N乙酰转移酶PANAT,反应底物终浓度为2mM,辅因子Ac-CoA浓度400μM,进行酶催化产对乙酰氨基酚(反应时间1h)。结果:在上述条件下,纯蛋白PANATK231Q和PANAT均可催化对氨基酚生成对乙酰氨基酚,且芳胺-N乙酰转移酶突变体转化对氨基酚生成对乙酰氨基酚的效率提高。
以下列化合物为底物,2-羟基-4氨基苯酚、2-氨基-3-羟基苯酚、2-氨基-4-氯苯酚、4-氨基-3-氯苯酚、4-氨基-3-氟苯酚、4-氨基-2-氟苯酚、5-氟邻氨基苯酚、4-氟邻氨基苯酚,加入芳胺-N乙酰转移酶突变体表达的纯蛋白PANATK231Q,或者加入野生型的芳胺-N乙酰转移酶PANAT,反应条件为温度30℃转速200rpm,反应条件为温度30℃,pH为7,转速200rpm,反应底物终浓度均为2mM,辅因子Ac-CoA浓度400μM,进行酶催化反应(反应时间1h)。结果:在上述条件下,纯蛋白PANATK231Q和PANAT均可催化2中所示的底物生成相应的乙酰化氨基酚,且芳胺-N乙酰转移酶突变体相对野生型芳胺-N乙酰转移酶活高。
以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,且本发明不限于上述的实施例,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种芳胺-N乙酰转移酶的突变体及其应用
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<170> PatentIn version 3.5
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atgggcagca gccatcatca tcatcatcat agcagcggct tagttcctcg tggcagccac 60
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gcgcgtctgt tactggcgtt aggctatgaa ctggaactgc tggtggcacg tgttcgttgg 360
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gcggaaggcg aatttctggt ggatgtgggc tttggttcag cgaacccgcc tcgtgcatta 480
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catgcgggct tatatgaaag cgcggtgcgt ggtcgtagcg gttggttacc tctgtatcgc 600
tttgatctgc gcccgcagct gtggattgat tatattccgc gcaactggta taccagcacc 660
catccgcata gcgtgtttcg ccagggtctg aaagcggcga ttaccgaagg tgatctgcgt 720
ctgaccttag cggatggtct gtttggtcag cgtgcgggta acggtgaaac cctgcagcgt 780
cagttacgcg atgtggaaga actgctggat attctgcaga cccgctttcg tttacgtctg 840
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<213> Pseudomonas aeruginosa
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Claims (10)
1.一种芳胺-N乙酰转移酶的突变体,其特征在于,所述突变体是以SEQ ID NO :2所示的铜绿假单胞菌(Pseudomonas aeruginosa)的芳胺-N乙酰转移酶为出发序列,将第231位的赖氨酸突变为谷氨酰胺获得的。
2.一种酶制剂,其特征在于,所述酶制剂包括权利要求1所述的突变体。
3.编码权利要求1所述的突变体的基因。
4.一种重组载体,其特征在于,所述重组载体携带权利要求3所述的基因。
5.一种重组微生物细胞,其特征在于,所述重组微生物细胞携带权利要求3所述的基因。
6.根据权利要求5所述的重组微生物细胞,其特征在于,所述重组微生物细胞表达权利要求1所述的突变体。
7.根据权利要求5或6所述的重组微生物细胞,其特征在于,所述微生物细胞为原核生物或者真核生物。
8.一种促进芳胺-N乙酰转移酶催化反应的方法,其特征在于,所述方法是将权利要求1所述的突变体或权利要求5或6所述的重组微生物细胞加入到苯胺类化合物的水溶液中,在pH=4-10,温度为30-60℃的条件下进行催化反应。
9.根据权利要求8所述的方法,其特征在于,所述苯胺类化合物为对氨基苯酚、2-羟基-4氨基苯酚、2-氨基-3-羟基苯酚、2-氨基-4-氯苯酚、4-氨基-3-氯苯酚、4-氨基-3-氟苯酚、4-氨基-2-氟苯酚、5-氟邻氨基苯酚和4-氟邻氨基苯酚中的任意一种。
10.权利要求1所述的突变体、权利要求2所述的酶制剂、权利要求3所述的基因、权利要求4所述的重组载体或权利要求5或6所述的重组微生物细胞在催化苯胺类化合物乙酰化中的应用。
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