CN111876404B - 一种醛缩酶突变体及其编码基因和应用 - Google Patents
一种醛缩酶突变体及其编码基因和应用 Download PDFInfo
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- CN111876404B CN111876404B CN202010752289.8A CN202010752289A CN111876404B CN 111876404 B CN111876404 B CN 111876404B CN 202010752289 A CN202010752289 A CN 202010752289A CN 111876404 B CN111876404 B CN 111876404B
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- aldolase
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Abstract
本发明公开了一种醛缩酶突变体及其编码基因和在生产他汀类药物中间体中的应用,属于分子生物学技术领域。所述醛缩酶突变体的氨基酸序列如SEQ ID NO.3所示。本发明在嗜热菌Thermotoga maritima野生型醛缩酶的基础上,对其编码基因引入定点突变,将184位编码苯丙氨酸的密码子(TTT)突变为编码异亮氨酸的密码子(ATT),将233位的丝氨酸(Ser)突变为丙氨酸(Ala),获得的醛缩酶突变体在以乙醛和氯乙醛为底物的醛缩反应活性较有显著改善,催化效率提高了0.86倍,该突变体具有良好的工业应用前景,解决了野生型嗜热菌的醛缩酶催化转化率不高,活性不足,底物亲和性较差等问题。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及一种醛缩酶突变体及其编码基因和在生产他汀类药物中间体中的应用。
背景技术
他汀类药物是最经典和有效的降脂类药物,其结构中双手性侧链的合成是整个合成工艺的关键步骤,双手性中心大大增加了他汀侧链合成的难度,其化学合成方法存在工艺复杂、立体选择性较差、环境污染大等缺陷,而生物催化方法具有反应条件温和、立体选择性高、环境友好等显著优势,是近年来合成他汀侧链中间体的重要途径。
目前,已经发现有生物酶对羟醛缩合反应有催化效果,其中研究较为广泛的是大肠杆菌的醛缩酶DERA,能催化生成具有两个手性中心的产物,例如能催化乙醛和氯乙醛发生羟醛缩合反应,生成6-氯-(3R,5S)-二羟基醛,此物质是他汀类药物侧链合成的一个关键前体物质。但由于大肠杆菌的醛缩酶存在醛缩反应活性不高,抗底物变性能力差等问题,难以适应大规模的工业化生产。
根据现有报道,引入位点突变是改造野生型DERA酶以提高其催化活性的有效手段,如专利文献CN103409402A公开了一种高性能催化羟醛缩合反应的DERA酶,该酶针对野生型大肠杆菌DERA酶引入一个或多个位点突变:V29A、T142P、M185V、K196E、F199I,相比野生型大肠杆菌DERA酶,突变体具有提高的底物耐受能力以及更高效的催化能力。以该酶作为催化剂可以很好地解决高浓度底物导致的DERA酶失活问题。
专利文献CN10450832A公开了一种产DERA酶的短乳杆菌,保藏编号CGMCCNO.10135,通过对野生型短乳杆菌DERA酶氨基酸序列的第29位、第78位或第163位引入单位点突变,获得酶活性显著提高的突变体。
嗜热菌Thermotoga maritima所表达的醛缩酶具有良好的热稳定性,但其催化活性仍然偏低,底物亲和性差,难以适应大规模的工业化生产。
发明内容
本发明的目的在于提供一种具有高催化活性,底物亲和性好的生物催化合成他汀类药物中间体的醛缩酶。
为实现上述目的,本发明采用如下技术方案:
本发明提供一种醛缩酶突变体,其氨基酸序列如SEQ ID NO.3所示。
本发明利用定点突变技术,对嗜热菌Thermotoga maritima野生型醛缩酶DERAThm的编码基因进行突变,将233位编码丝氨酸的密码子(AGT)突变为编码丙氨酸的密码子(GCA),在此基础上,将184位编码苯丙氨酸的密码子(TTT)突变为编码异亮氨酸的密码子(ATT),得到氨基酸序列如SEQ ID NO.3所示的双位点突变体。上述突变位点序号基于包括起始密码子编码的氨基酸序列。
本发明研究表明,相较于嗜热菌Thermotoga maritima野生型醛缩酶,上述醛缩酶突变体在以乙醛和氯乙醛为底物的醛缩反应的活性显著改善,DERAThm(F184I,S233A)催化效率提高了86%。
本发明提供了一种编码所述的醛缩酶突变体的基因,其核苷酸序列如SEQ IDNO.4所示。
所述醛缩酶突变体的编码基因通过如下方式获得:
以嗜热菌Thermotoga maritima野生型醛缩酶的编码基因为模板,利用定点突变引物进行PCR扩增,筛选目的突变基因。
所述184位点定点突变的引物为:
上游引物:5’-CTTCCACGGGAATTGGAACAGGAG-3’;
下游引物:5’-CTCCTGTTCCAATTCCCGTGGAAG-3’。
所述233位点定点突变引物为:
上游引物:5’-GAATAGGAACGGCATCGGGAGTTAAGATC-3’;
下游引物:5’-CTTAACTCCCGATGCCGTTCCTATTCTATCAG-3’。
本发明还提供了包含所述编码基因的表达单元、重组载体和转化子。
作为优选,所述表达单元的启动子可以为T7启动子、lac启动子或araBAD启动子。所述重组载体的原始载体可采用PET28a。所述转化子的宿主细胞为大肠杆菌(E.coli)BL21。
本发明还提供了所述的醛缩酶突变体在生产他汀类药物中间体6-氯-(3R,5S)-二羟基醛中的应用。
所述应用包括:以含醛缩酶突变体编码基因的工程菌经发酵培养获得的湿菌体经细胞破碎后得到的酶为催化剂,以乙醛和氯乙醛为底物,反应介质为0.2M磷酸-咪唑缓冲液,在25℃和搅拌条件下进行催化反应,反应结束后,获得含他汀类药物中间体6-氯-(3R,5S)-二羟基醛的混合液,经分离纯化获得6-氯-(3R,5S)-二羟基醛。
所述工程菌为含醛缩酶突变体质粒pET28a(+)-DERAThm(F184I,S233A)的大肠杆菌BL21,发酵培养的方法包括:将所述工程菌接种于含卡那霉素的LB培养基中,37℃下培养至OD600=0.6~1时,加入终浓度为0.8mM的异丙基-β-D-硫代吡喃半乳糖苷,28℃诱导培养10-12h,离心,收集菌体得到所述湿菌体。
反应体系中,底物乙醛和氯乙醛的浓度分别为200mM和100mM,搅拌条件为700rpm,酶的添加量以湿菌体计约为20mg/ml。
本发明具备的有益效果:
本发明在嗜热菌Thermotoga maritima野生型醛缩酶DERAThm的基础上,对其编码基因引入定点突变,将184位编码苯丙氨酸的密码子(TTT)突变为编码异亮氨酸的密码子(ATT),将233位的丝氨酸(Ser)突变为丙氨酸(Ala),获得的醛缩酶突变体DERAThm(F184I,S233A)在以乙醛和氯乙醛为底物的醛缩反应活性较有显著改善,催化效率提高了0.86倍,该突变体具有良好的工业应用前景,解决了野生型嗜热菌的醛缩酶催化转化率不高,活性不足,底物亲和性较差等问题。
附图说明
图1为DERAThm(F184I,S233A)蛋白表达的SDS-PAGE凝胶电泳结果,其中M为蛋白marker,泳道1为蛋白表达产物。
图2为不同浓度氯乙醛和2,4-二硝基苯肼溶液反应示意图,从左往右第一孔为:氯乙醛0mg/L,第二孔为:1mg/L氯乙醛,第三孔为:5mg/L氯乙醛。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
一、合成嗜热菌Thermotoga maritima野生型醛缩酶编码基因
本发明所使用的嗜热菌Thermotoga maritima野生型醛缩酶基因DERAThm是由人工合成来的,其基因序列来源于NCBI。利用该人工合成基因的目的是为了得到突变醛缩酶基因,对载体无特异性要求。
二、单突变体的制备
根据突变醛缩酶基因的碱基序列,设计在第233位点进行突变的引物:
233位点上游引物:5’-GAATAGGAACGGCATCGGGAGTTAAGATC-3’;
233位点下游引物:5’-CTTAACTCCCGATGCCGTTCCTATTCTATCAG-3’。
PCR反应体系为:纯净水29.5μL;KD plus buffer 10μL;DMSO 2.5μL;上游引物1μL;下游引物1μL;dNTPs 4μL;模板1μL;KD plus 1μL;总体积为50μL。
所用PCR条件为:94℃变性2min后进入扩增循环,即95℃变性45s,52℃退火60s,68℃延伸7min,共循环25次,最后68℃延伸7min。
将上述获得的定点突变PCR产物,经过DpnI消化模板DNA,将消化后的PCR产物转化至大肠杆菌(E.coli)BL21化学感受态细胞中,涂布到含有40μg/ml卡那霉素的LB琼脂平板上,37℃培养15个小时。从平板上挑取单菌落到5ml LB液体培养基(含有40μg/ml卡那霉素)的试管中,于温度为37℃,转速为250rpm条件下培养过夜。菌液经DNA测序,获得233位点的突变菌株。
三、双突变体的制备
以上述得到S233A突变体为模板,进行F184I位点的叠加突变,所使用的引物序列为:
184位点上游引物:5’-CTTCCACGGGAATTGGAACAGGAG-3’;
184位点下游引物:5’-CTCCTGTTCCAATTCCCGTGGAAG-3’。
PCR反应体系为:纯净水29.5μL;KD plus buffer 10μL;DMSO 2.5μL;上游引物1μL;下游引物1μL;dNTPs 4μL;模板1μL;KD plus 1μL;总体积为50μL。
所用PCR条件为:94℃变性2min后进入扩增循环,即95℃变性45s,50℃退火60s,68℃延伸7min,共循环25次,最后68℃延伸7min。
将上述获得的定点突变PCR产物,经过DpnI消化模板DNA,将消化后的PCR产物转化至大肠杆菌(E.coli)BL21化学感受态细胞中,涂布到含有40μg/ml卡那霉素的LB琼脂平板上,37℃培养15个小时。从平板上挑取单菌落到5ml LB液体培养基(含有40μg/ml卡那霉素)的试管中,于温度为37℃,转速为250rpm条件下培养过夜。菌液经DNA测序,获得双突变菌株DERAThm(F184I,S233A)。
四、醛缩酶双突变体的诱导表达
取构建好的醛缩酶双突变体质粒pET28a(+)-DERAThm(F184I,S233A)、野生型pET28a(+)-DERAThm分别导入大肠杆菌BL21(DE3)感受态细胞,得到表达醛缩酶的重组工程菌。挑取菌体于5m1含有终浓度为40μg/ml卡那霉素的试管中培养过夜。将上述过夜培养液转接到装有50ml TB培养液(含有40μg/ml卡那霉素)摇瓶中,于温度为37℃,转速为250rpm条件下培养至OD600值为0.6-1左右,加入终浓度0.8mM的IPTG诱导,于温度为28℃,转速为250rpm条件下诱导过夜。
五、醛缩酶双突变体粗酶液的制备
8000rpm离心10min收集发酵完毕的菌液,弃上清,菌体用冰预冷的PBS洗涤1次,40ml 0.2M磷酸-咪唑缓冲液(pH7.0)重悬,高压破胞,10000rpm离心10min,取上清,即为粗酶液。其中含有突变酶DERAThm(F184I,S233A)的粗酶液的电泳检测结果见图1。
六、醛缩酶双突变体的蛋白纯化
收集发酵完毕的菌液,8000rpm离心10min收集菌体,弃上清,菌体用冰预冷的buffer A(50mM NaH2PO4-Na2HPO4,300mM NaCl,pH7.0)重悬,超声破碎,然后4℃15,000rpm离心30分钟。然后将上清加入到预先洗好Ni-NTA beads中,4℃滚轴混合一个小时。将含有beads的菌液转移到柱子中,用buffer B(50mM NaH2PO4-Na2HPO4,300mM NaCl,5mMImidazole,pH7.0)清洗3次,然后用Elution Buffer(50mM NaH2PO4-Na2HPO4,300mM NaCl,150mM Imidazole,pH7.0)洗脱蛋白,脱盐,获得醛缩酶突变体蛋白。
七、醛缩酶双突变体的活性检测
氯乙醛可与2,4-二硝基苯肼发生显色反应(图2),因此可用该方法检测醛缩酶突变体的活性。在总体积5mL反应体系中,加入适量醛缩酶双突变体蛋白,50mM乙醛和25mM氯乙醛,25℃,700rpm条件下催化反应30min。反应液稀释1000倍后取1.6mL稀释液,加入300μL2,4-二硝基苯肼溶液(0.1%),避光静置20min,加入100μL KOH溶液(100g/L),避光静置10min。测定OD528吸光度,并计算酶活。
结果显示,醛缩酶突变体质粒pET28a(+)-DERAThm(F184I,S233A)的酶活较野生型提高了0.86倍,S233A单突变体较野生型提高了0.62倍。
实施例2
他汀类药物侧链前体的合成
在100ml反应体系中,加入20ml实施例1中制备的粗酶液,流加或分批加入乙醛和氯乙醛至终浓度为200mM和100mM,25℃,700rpm条件下催化反应12h。以在相同条件下制备大肠杆菌DERA、嗜热菌Pyrobaculum arophilum DERA、野生型DERAThm粗酶液作为对照。
在反应液中加入两倍体积的丙酮,10000rpm离心10min,取上清,旋转蒸发掉丙酮,乙酸乙酯萃取3次,合并乙酸乙酯,旋蒸,得到黄色的油状液体,即为他汀类药物侧链前体的粗产物。产物可用气质联用检测,条件参数为:进样280℃;检测器280℃;柱温:100℃2min,升温10℃/min至250℃,250℃2min,HP-5柱。
结果显示,经大肠杆菌DERA、嗜热菌Pyrobaculum arophilum DERA、野生型DERAThm以及双突变体DERAThm(F184I,S233A)催化反应,反应结束后进行分离纯化,分别得到黄色油状物质0.24g,0.49g,0.75g和1.29g,表明双突变体DERAThm(F184I,S233A)的催化效率较野生型得到了显著提升,并显著优于大肠杆菌、嗜热菌Pyrobaculum arophilum等来源的DERA。
序列表
<110> 浙大宁波理工学院
<120> 一种醛缩酶突变体及其编码基因和应用
<160> 9
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atgatagagt acaggattga ggaggcagta gcgaagtaca gagagttcta cgaattcaag 60
cccgtcagag aaagcgcagg tattgaagat gtgaaaagtg ctatagagca cacgaatctg 120
aaaccgtttg ccacaccaga cgatataaaa aaactctgtc ttgaagcaag ggaaaatcgt 180
ttccatggag tctgtgtgaa tccgtgttat gtgaaactgg ctcgtgaaga actcgaagga 240
accgatgtga aagtcgtcac cgttgttggt tttccactgg gagcgaacga aactcggacg 300
aaagcccatg aggcgatttt cgctgttgag agtggagccg atgagatcga tatggtcatc 360
aacgttggca tgctcaaggc aaaggagtgg gagtacgttt acgaggatat aagaagtgtt 420
gtcgaatcgg tgaaaggaaa agttgtgaag gtgatcatcg aaacgtgcta tctggatacg 480
gaagagaaga tagcggcgtg tgtcatttcc aaacttgctg gagctcattt cgtgaagact 540
tccacgggat ttggaacagg aggggcgacc gcagaagacg ttcatctcat gaaatggatc 600
gtgggagatg agatgggtgt aaaagcttcc ggagggatca gaaccttcga ggacgctgtt 660
aaaatgatca tgtacggtgc tgatagaata ggaacggcat cgggagttaa gatcgttcag 720
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tccacgggaa ttggaacagg aggggcgacc gcagaagacg ttcatctcat gaaatggatc 600
gtgggagatg agatgggtgt aaaagcttcc ggagggatca gaaccttcga ggacgctgtt 660
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atgatagagt acaggattga ggaggcagta gcgaagtaca gagagttcta cgaattcaag 60
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aaaccgtttg ccacaccaga cgatataaaa aaactctgtc ttgaagcaag ggaaaatcgt 180
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tccacgggat ttggaacagg aggggcgacc gcagaagacg ttcatctcat gaaatggatc 600
gtgggagatg agatgggtgt aaaagcttcc ggagggatca gaaccttcga ggacgctgtt 660
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<213> 人工序列(Artificial Sequence)
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<210> 9
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ctcctgttcc aattcccgtg gaag 24
Claims (8)
1.一种醛缩酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.3所示。
2.一种编码如权利要求1所述的醛缩酶突变体的基因,其特征在于,其核苷酸序列如SEQ ID NO.4所示。
3.一种包含如权利要求2所述基因的表达单元。
4.如权利要求3所述的表达单元,其特征在于,启动子为T7启动子、lac启动子或araBAD启动子。
5.一种包含如权利要求3所述表达单元的重组载体。
6.一种包含如权利要求5所述的重组载体的转化子。
7.如权利要求6所述的转化子,其特征在于,宿主细胞为大肠杆菌。
8.如权利要求1所述的醛缩酶突变体在生产他汀类药物中间体6-氯-(3R,5S)-二羟基醛中的应用。
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