CN117305256A - 一种黄素依赖型单加氧酶突变体及其在制备n-羟基哌啶酸中的应用 - Google Patents
一种黄素依赖型单加氧酶突变体及其在制备n-羟基哌啶酸中的应用 Download PDFInfo
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Abstract
本发明公开了一种黄素依赖型单加氧酶突变体及其在制备N‑羟基哌啶酸中的应用,属于生物工程技术领域。本发明的黄素依赖型单加氧酶突变体是将SEQ ID NO.1所示的氨基酸序列第123位、第181位、第379位进行单突变或多突变获得的。本发明经过半理性设计,对蛋白质进行分子改造,获得反应活性提高的突变体,有利于生成目的产物。本发明构建的突变体V123G和V123G/E181K/Y379S的酶活分别为野生型FMO1的116.8%和125.4%。对黄素依赖型单加氧酶催化活性的提高使其能够应用于系统获得性抗性信号分子N‑羟基哌啶酸的工业化生产,具有重要的意义。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种黄素依赖型单加氧酶的突变体及其在催化L-哌啶酸合成N-羟基哌啶酸中的应用。
背景技术
植物代谢物在植物防御中发挥着重要作用,因为它们可以直接阻止病原体进入植物组织。系统获得性抗性是植物防御形式之一,它对除初始感染部位以外的偏远部位产生持久和广谱的免疫力。许多化合物被鉴定为系统获得性抗性信号分子,包括水杨酸、水杨酸甲酯、壬二酸、甘油-3-磷酸、烟酰胺腺嘌呤二核苷酸、哌啶酸、N-羟基哌啶酸和脱氢枞酸。
黄素依赖型单加氧酶是一种能催化氧气的一个氧原子与电子供体提供的氢原子结合生成水,而另一个氧原子插入有机分子,生成新的化合物。N-羟基-哌啶酸是哌啶酸的羟基化产物,是一种重要的系统获得性抗性信号分子,可作为天然的抗菌剂,具有抗氧化、抗菌、抗肿瘤的药理作用。因此,可以利用黄素依赖型单加氧酶催化L-哌啶酸制备具有多种生物活性功能的N-羟基-哌啶酸。然而,N-羟基-哌啶酸的生物合成在微生物中没有天然的代谢途径。
黄素依赖型单加氧酶是一种重要的能在C、S和N原子上加氧的工业酶。如2019年03月01日公布的公布号为CN109402074A的“单加氧酶突变体及其应用”专利,公开的方法是:将单加氧酶氨基酸序列突变为至少包括如下突变位点之一:第45位、第95位、第106位、第108位、第114位、第186位、第190位、第191位、第249位、第257位、第393位、第436位、第499位、第500位、第501位、第503位、第504位、第559位和第560位。该方法的主要缺点是:(1)催化对象主要是硫醚类化合物,对L-哌啶酸的N-羟基化效率较低,导致N-羟基-哌啶酸的产量较低。又如2022年04月12日公布的公布号为CN114317469A的“一种黄素单加氧酶突变体及其制备和应用”专利,公开的方法是:利用易错PCR技术对来源大蒜的黄素单加氧酶进行随机突变,并在大肠杆菌和毕赤酵母中高效表达,进一步通过发酵和提取技术获得活性提高的黄素单加氧酶。该方法的主要缺点是:(1)获得的黄素单加氧酶作用底物主要是脱氧蒜氨酸,生成的产物为蒜氨酸。
发明内容
基于现有技术存在的问题,本发明的目的在于提供一种酶活提高的黄素依赖型单加氧酶突变体,包括该突变体的编码基因,含有该基因的重组载体,以及重组载体转化得到的重组基因工程菌,及其在催化合成N-羟基-哌啶酸中的应用。
本发明采用的技术方案是:
本发明提供一种黄素依赖型单加氧酶突变体,所述突变体是将SEQ ID NO.1所示氨基酸序列第123位、第181位、第379位进行单突变或多突变获得的。所述突变体采用定点突变获得。所述SEQ ID NO.1所示氨基酸序列编码基因的核苷酸序列为SEQ ID NO.2所示。
进一步,所述突变体为下列之一:(1)将SEQ ID NO.1所示氨基酸序列第123位缬氨酸突变为甘氨酸(V123G,SEQ ID NO.3);(2)将SEQ ID NO.1所示氨基酸序列第181位谷氨酸突变为赖氨酸(E181K,SEQ ID NO.4);(3)将SEQ ID NO.1所示氨基酸序列第379位酪氨酸突变为丝氨酸(Y379S,SEQ ID NO.5);(4)将SEQ ID NO.1所示氨基酸序列第123位缬氨酸突变为甘氨酸,第181位谷氨酸突变为赖氨酸,第379位酪氨酸突变为丝氨酸(V123G/E181K/Y379S,SEQ ID NO.6)。
本发明还提供一种黄素依赖型单加氧酶突变体编码基因,由编码基因构建的重组载体,及重组基因工程菌,所述重组载体按如下方法构建:将黄素依赖型单加氧酶突变体基因插入pET-21a载体的BamHI与SacI位点,构建含黄素依赖型单加氧酶突变体基因的重组质粒。所述工程菌按如下方法制备:将构建的重组质粒转入宿主菌Escherichia coli BL21(DE3)得到重组工程菌。
本发明提供所述黄素依赖型单加氧酶突变体在催化L-哌啶酸合成N-羟基哌啶酸中的应用,所述的应用为:以含黄素依赖型单加氧酶突变体编码基因的工程菌经发酵培养后获得的湿菌体或湿菌体破碎后提取的酶作为生物催化剂,以L-哌啶酸为底物,加入辅因子NADPH0.1~2.5mM,以pH为7.5~8.5的缓冲液为反应介质构成转化体系,在25~45℃、100~300rpm/min条件下进行转化反应,反应结束后,在3500~8000rpm/min条件下进行离心,收集上清。
进一步,转化体系中底物的加入终浓度以缓冲液体积计为15~80mM,催化剂的用量以湿菌体重量计,所述湿菌体加入量以缓冲液体积计为10~25g/L,其中湿菌体含水质量为86~92%。
进一步,湿菌体加入量优先为20g/L。
进一步,NADPH的浓度优选为2.0mM。
进一步,缓冲液为pH 8.0的Tris-HCl缓冲液。
进一步,反应条件优选为30℃、200rpm/min条件下反应4h。
进一步,反应结束后,离心条件优选为8000rpm/min。
进一步,所述湿菌体在制备方法为:含黄素依赖型单加氧酶突变体编码基因的工程菌接种至含终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm/min条件下震荡培养8~10h,获得种子液。将种子液以1%的接种量接种至新鲜的含有终浓度100mg/L氨苄青霉素的LB液体培养基中,30℃、200rpm下培养至菌体OD600达到0.6~0.8,加入终浓度1.0mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG),30℃、200rpm下诱导培养10~12h,于4℃、8000rpm离心10min,收集菌体,用pH为7.5~8.5、50mM的Tris-HCl缓冲液重悬、洗涤3次,得到湿菌体。
本发明的有益之处在于:本发明通过非理性和理性设计对黄素依赖型单加氧酶FMO1进行分子改造,获得反应活性提高的突变体,有利于生成目的产物。本发明构建的突变体V123G/E181K/Y379S催化L-哌啶酸时,反应的主要产物为N-羟基哌啶酸,将野生型FMO1活力记为100%,黄素依赖型单加氧酶突变体活力为野生型FMO1的125.4%。对黄素依赖型单加氧酶催化活性的提高使其能够应用于系统获得性抗性信号分子N-羟基哌啶酸的工业化生产,具有重要的意义。
具体实施方式
下面结合具体实施方式,进一步说明本发明。
实施例1
构建黄素依赖型单加氧酶FMO1基因第123位氨基酸密码子突变的突变体。
为了对黄素依赖型单加氧酶FMO1基因的第123位氨基酸密码子进行野生型以外的19种突变,设计了引物V123A、V123R、V123N、V123D、V123C、V123Q、V123E、V123G、V123H、V123I、V123L、V123K、V123M、V123F、V123P、V123S、V123T、V123W、V123Y、V123-R。引物序列如下所示:
SEQ ID NO.7,V123A:5’-GCTgacctcggtgcttatggcaac-3’
SEQ ID NO.8,V123R:5’-CGAgacctcggtgcttatggcaac-3’
SEQ ID NO.9,V123N:5’-AACgacctcggtgcttatggcaac-3’
SEQ ID NO.10,V123D:5’-GACgacctcggtgcttatggcaac-3’
SEQ ID NO.11,V123C:5’-TGCgacctcggtgcttatggcaac-3’
SEQ ID NO.12,V123Q:5’-CAGgacctcggtgcttatggcaac-3’
SEQ ID NO.13,V123E:5’-GAGgacctcggtgcttatggcaac-3’
SEQ ID NO.14,V123G:5’-GGTgacctcggtgcttatggcaac-3’
SEQ ID NO.15,V123H:5’-CATgacctcggtgcttatggcaac-3’
SEQ ID NO.16,V123I:5’-ATCgacctcggtgcttatggcaac-3’
SEQ ID NO.17,V123L:5’-CTGgacctcggtgcttatggcaac-3’
SEQ ID NO.18,V123K:5’-AAGgacctcggtgcttatggcaac-3’
SEQ ID NO.19,V123M:5’-ATGgacctcggtgcttatggcaac-3’
SEQ ID NO.20,V123F:5’-TTCgacctcggtgcttatggcaac-3’
SEQ ID NO.21,V123P:5’-CCTgacctcggtgcttatggcaac-3’
SEQ ID NO.22,V123S:5’-TCGgacctcggtgcttatggcaac-3’
SEQ ID NO.23,V123T:5’-ACTgacctcggtgcttatggcaac-3’
SEQ ID NO.24,V123W:5’-TGGgacctcggtgcttatggcaac-3’
SEQ ID NO.25,V123Y:5’-TACgacctcggtgcttatggcaac-3’
SEQ ID NO.26,V123-R:5’-CATCAGTGGAGTTTCGCCATC-3’
将黄素依赖型单加氧酶(FMO1,GenBank登录号:XP_009149401.1)基因(氨基酸序列为SEQ ID NO.1,核苷酸序列为SEQ ID NO.2)插入pET-21a载体的BamHI与SacI位点,构建重组质粒pET21a-FMO1。以重组质粒pET21a-FMO1为模板,利用上述引物进行全质粒PCR扩增,在该基因第123位进行单位点定点饱和突变。
实施例2
构建黄素依赖型单加氧酶FMO1基因第181位氨基酸密码子突变的突变体。
为了对黄素依赖型单加氧酶FMO1基因的第181位氨基酸密码子进行野生型以外的19种突变,设计了引物E181A、E181R、E181N、E181D、E181C、E181Q、E181G、E181H、E181I、E181L、E181K、E181M、E181F、E181P、E181S、E181T、E181W、E181Y、E181V、E181-R。引物序列如下所示:
SEQ ID NO.27,E181A:5’-GCTctattcaaaggaaaagtgatgc-3’
SEQ ID NO.28,E181R:5’-CGActattcaaaggaaaagtgatgc-3’
SEQ ID NO.29,E181N:5’-AACctattcaaaggaaaagtgatgc-3’
SEQ ID NO.30,E181D:5’-GACctattcaaaggaaaagtgatgc-3’
SEQ ID NO.31,E181C:5’-TGCctattcaaaggaaaagtgatgc-3’
SEQ ID NO.32,E181Q:5’-CAGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.33,E181G:5’-GGTctattcaaaggaaaagtgatgc-3’
SEQ ID NO.34,E181H:5’-CATctattcaaaggaaaagtgatgc-3’
SEQ ID NO.35,E181I:5’-ATCctattcaaaggaaaagtgatgc-3’
SEQ ID NO.36,E181L:5’-CTGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.37,E181K:5’-AAGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.38,E181M:5’-ATGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.39,E181F:5’-TTCctattcaaaggaaaagtgatgc-3’
SEQ ID NO.40,E181P:5’-CCTctattcaaaggaaaagtgatgc-3’
SEQ ID NO.41,E181S:5’-TCGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.42,E181T:5’-ACTctattcaaaggaaaagtgatgc-3’
SEQ ID NO.43,E181W:5’-TGGctattcaaaggaaaagtgatgc-3’
SEQ ID NO.44,E181Y:5’-TACctattcaaaggaaaagtgatgc-3’
SEQ ID NO.45,E181V:5’-GTCctattcaaaggaaaagtgatgc-3’
SEQ ID NO.46,E181-R:5’-CGGCCCTTTATTCGCTGGAAAC-3’
以重组质粒pET21a-FMO1为模板,利用上述引物进行全质粒PCR扩增,在该基因第181位进行单位点定点饱和突变。
实施例3
构建黄素依赖型单加氧酶FMO1基因第379位氨基酸密码子突变的突变体。
为了对黄素依赖型单加氧酶FMO1基因的第379位氨基酸密码子进行野生型以外的19种突变,设计了引物Y379A、Y379R、Y379N、Y379D、Y379C、Y379Q、Y379E、Y379G、Y379H、Y379I、Y379L、Y379K、Y379M、Y379F、Y379P、Y379S、Y379T、Y379W、Y379V、Y379-R。引物序列如下所示:
SEQ ID NO.47,Y379A:5’-GCTgatggcaagaagaagctcaaag-3’
SEQ ID NO.48,Y379R:5’-CGAgatggcaagaagaagctcaaag-3’
SEQ ID NO.49,Y379N:5’-AACgatggcaagaagaagctcaaag-3’
SEQ ID NO.50,Y379D:5’-GACgatggcaagaagaagctcaaag-3’
SEQ ID NO.51,Y379C:5’-TGCgatggcaagaagaagctcaaag-3’
SEQ ID NO.52,Y379Q:5’-CAGgatggcaagaagaagctcaaag-3’
SEQ ID NO.53,Y379E:5’-GAGgatggcaagaagaagctcaaag-3’
SEQ ID NO.54,Y379G:5’-GGTgatggcaagaagaagctcaaag-3’
SEQ ID NO.55,Y379H:5’-CATgatggcaagaagaagctcaaag-3’
SEQ ID NO.56,Y379I:5’-ATCgatggcaagaagaagctcaaag-3’
SEQ ID NO.57,Y379L:5’-CTGgatggcaagaagaagctcaaag-3’
SEQ ID NO.58,Y379K:5’-AAGgatggcaagaagaagctcaaag-3’
SEQ ID NO.59,Y379M:5’-ATGgatggcaagaagaagctcaaag-3’
SEQ ID NO.60,Y379F:5’-TTCgatggcaagaagaagctcaaag-3’
SEQ ID NO.61,Y379P:5’-CCTgatggcaagaagaagctcaaag-3’
SEQ ID NO.62,Y379S:5’-TCGgatggcaagaagaagctcaaag-3’
SEQ ID NO.63,Y379T:5’-ACTgatggcaagaagaagctcaaag-3’
SEQ ID NO.64,Y379W:5’-TGGgatggcaagaagaagctcaaag-3’
SEQ ID NO.65,Y379V:5’-GTCgatggcaagaagaagctcaaag-3’
SEQ ID NO.66,Y379-R:5’-ACCAGTCGCCAGTATCACAAC-3’
以重组质粒pET21a-FMO1为模板,利用上述引物进行全质粒PCR扩增,在该基因第379位进行单位点定点饱和突变。
实施例4
黄素依赖型单加氧酶突变体V123G活力测定。
将测序正确的含突变体基因的工程菌接种到含有终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm下培养8h,以1%(v/v)接种量接种至新鲜的含有终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm培养至菌体OD600达到0.8,加入终浓度0.5mM的异丙基-β-D-硫代吡喃半乳糖苷,30℃、200rpm下诱导培养12h,于4℃、8000rpm离心10min,收集湿菌体。
反应体系组成为:20mL 50mM pH 8.0Tris-HCl缓冲液,湿菌体0.2g,2.0mM NADPH,50mM L-哌啶酸。于30℃、200rpm/min条件下反应2h。取样1mL,加入20μL 2M HCl终止反应,8000rpm离心2min,取上清。经高效液相色谱HPLC分析样品中产物N-羟基哌啶酸和未反应底物L-哌啶酸的浓度,并计算酶活。
黄素依赖型单加氧酶活力定义为标准反应条件(30℃、200rpm)下,每分钟生成1μmol N-羟基哌啶酸所需要的细胞量定义为一个酶活力单位(1U)。
黄素依赖型单加氧酶突变体V123G的酶活为野生型FMO1的116.8%。
实施例5
黄素依赖型单加氧酶突变体V123G/E181K/Y379S活力测定。
将测序正确的含突变体基因的工程菌接种到含有终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm下培养8h,以1%(v/v)接种量接种至新鲜的含有终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm培养至菌体OD600达到0.6,加入终浓度1.0mM的异丙基-β-D-硫代吡喃半乳糖苷,30℃、200rpm下诱导培养12h,于4℃、8000rpm离心10min,收集湿菌体。
反应体系组成为:20mL 50mM pH 8.0Tris-HCl缓冲液,湿菌体0.2g,1.0mM NADPH,60mM L-哌啶酸。于30℃、200rpm/min条件下反应4h。取样1mL,加入20μL 2M HCl终止反应,8000rpm离心2min,取上清。经高效液相色谱HPLC分析样品中产物N-羟基哌啶酸和未反应底物L-哌啶酸的浓度,并计算酶活。
黄素依赖型单加氧酶活力定义为标准反应条件(30℃、200rpm)下,每分钟生成1μmol N-羟基哌啶酸所需要的细胞量定义为一个酶活力单位(1U)。
黄素依赖型单加氧酶突变体V123G/E181K/Y379S的酶活为野生型FMO1的125.4%。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以权利要求为准。
Claims (8)
1.一种黄素依赖型单加氧酶突变体,其特征在于所述突变体是将SEQ ID NO.1所示氨基酸序列第123位、第181位、第379位氨基酸中的一位或多位进行突变获得的。
2.如权利要求1所述的黄素依赖型单加氧酶突变体,其特征在于所述突变体为下列之一:(1)将SEQ ID NO.1所示氨基酸序列的第123位缬氨酸突变为甘氨酸;(2)将SEQ ID NO.1所示氨基酸序列的第181位谷氨酸突变为赖氨酸;(3)将SEQ ID NO.1所示氨基酸序列的第379位酪氨酸突变为丝氨酸;(4)将SEQ ID NO.1所示氨基酸序列的第123位缬氨酸突变为甘氨酸,同时将第181位谷氨酸突变为赖氨酸,同时将第379位酪氨酸突变为丝氨酸。
3.一种权利要求1所述黄素依赖型单加氧酶突变体的编码基因。
4.一种权利要求3所述黄素依赖型单加氧酶突变体的编码基因构建的重组基因工程菌。
5.一种权利要求1所述黄素依赖型单加氧酶突变体在催化L-哌啶酸制备N-羟基哌啶酸中的应用。
6.如权利要求5所述的应用,其特征在于所述的应用以含黄素依赖型单加氧酶突变体的基因工程菌经发酵培养获得的湿菌体,湿菌体细胞或湿菌体破碎后提取的纯酶为催化剂,以L-哌啶酸为底物,以pH为7.5~8.5、50mM的Tris-HCl缓冲液为反应介质构成反应体系,在25~45℃、100~300rpm/min条件下进行转化反应,反应结束后,在3500~8000rpm/min条件下进行离心,收集上清,获得N-羟基哌啶酸。
7.如权利要求5所述的应用,其特征在于所述底物加入终浓度以缓冲液体积计为15~80mM;催化剂的用量以湿菌体重量计,所述湿菌体加入量以缓冲液体积计为10~25g/L。
8.如权利要求5所述的应用,其特征在于所述湿菌体按如下方法制备:将含黄素依赖型单加氧酶突变体编码基因的工程菌接种至含终浓度100mg/L氨苄青霉素的LB液体培养基中,37℃、200rpm/min条件下震荡培养8~10h,获得种子液;将种子液以1%的接种量接种至新鲜的含有终浓度100mg/L氨苄青霉素的LB液体培养基中,30℃、200rpm下培养至菌体OD600达到0.6~0.8,加入终浓度1.0mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG),30℃、200rpm下诱导培养10~12h,于4℃、8000rpm离心10min,收集菌体,用pH为7.5~8.5、50mM的Tris-HCl缓冲液重悬、洗涤3次,得到湿菌体。
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