CN114634944A - 一种共表达载体应用于制备阿托伐他汀中间体的方法 - Google Patents
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Abstract
本发明涉及酶工程领域,涉及一株共表达羰基还原酶和葡糖脱氢酶菌株的构建,以及在(3R,5R)‑6‑氰基‑3,5‑二羟基己酸叔丁酯上的应用。羰基还原酶和辅酶循环酶共表达,节约了发酵成本,降低了发酵废液的产生量,对环境友好。
Description
技术领域
本发明属于生物制药技术领域,涉及一种KRED和辅酶循环酶共表达。
背景技术
目前合成阿托伐他汀关键中间体有化学法和酶法两种路线,与纯化学合成路线相比,酶催化反应不需要高温高压等极端催化环境,也可以减少使用对人及环境有危害的催化剂,降低了废物的产生,环境友好性好;更重要地是,酶具有优异的立体选择性,可以有效提高产率和产品的光学纯度,具有极好的产业化潜力。
传统的酶法催化需要先分别对产羰基还原酶的菌株和产辅酶循环酶的菌株进行发酵进行产酶,浪费资源,且产生较多的发酵废水,增加了污水处理的工作量。通过一株菌株共表达两种所需的酶的策略大幅度降低了能耗,减少了污水处理的工作量,节约了成本。
发明内容
本发明的目的在于提供一种阿托伐他汀中间体的制备的催化剂——生物酶合成方法。该方法利用重组羰基还原酶与辅酶循环酶在同一载体中进行共表达,实现本发明的目的的技术方案如下:
一种共表达载体制备,具体步骤如下:
将实验室保藏的突变菌株BL21(DE3)KRED06 活化后,收集质粒;并以SEQ ID NO.2和SEQ ID NO.3所示的引物序列为模板,进行PCR扩增,并经过跑胶验证后,送检测序。测序结果无误后,通过重叠PCR,分别扩增出羰基还原酶和辅酶循环酶(异丙醇脱氢酶/葡糖脱氢酶/甲酸脱氢酶)基因,并通过退火重叠连接后,在pfu作用下,延伸至完整双链,羰基还原酶基因与辅酶循环酶基因通过P2A(序列如SEQ ID NO.1所示)连接,选择pET-28a/30a等质粒中EcoRⅠ和XhoⅠ两个酶切位点,接入该段长序列,构建BL21(DE3)工程菌,获得可同时过表达羰基还原酶和辅酶循环酶的菌株。
与现有技术相比,具有以下优点:
1)羰基还原酶和辅酶循环酶共表达,节约了发酵成本,降低了发酵废液的产生量,对环境友好;
2)共表达不影响羰基还原酶本身的立体选择性,不影响反应的ee值、纯度等;
3)大幅度降低了人工成本,节约了生产成本。
具体实施方式
为了更好的理解本发明,下面结合具体的实施例对本发明进行进一步的阐述
实施例1
步骤1:酮羰基还原酶DNA序列获得
将实验室保藏的突变菌株BL21(DE3)KRED06 活化,接种于含有卡那的LB培养基中,37℃,220rpm振荡培养过夜,收集菌株于EP管中,于12000r/min,离心2-5min,收集细胞,通过天根质粒小提试剂盒,按照标准方法收集质粒;并以SEQ ID NO.2和SEQ ID NO.3所示的引物序列为模板,进行PCR扩增,并经过跑胶验证后,通过天根切胶回收试剂盒,按照标准方法回收PCR产物,送检测序。
步骤2:共表达载体制备
辅酶循环酶以GDH为例,分别扩增出KRED06 DNA序列与GDH序列,采用重叠延伸PCR方法,分别扩增出前后半段序列(引物如SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8和SEQ IDNO.9所示),PCR仪降温至58—62℃,在DNA聚合酶作用下,两端序列重叠获得 KRED06-P2A-GDH序列,并载入T载体,转入E.coli DH5α克隆细胞中。
步骤3:表达菌株构建
平板上挑选阳性克隆菌株,收集质粒,通过引物F1、R1(SEQ ID NO.10和SEQ IDNO.11所示)PCR扩增;表达质粒以pET-30a为例,按照ddH2O 31μL、缓冲液5μL、质粒DNA10μL、Fast digest (EcoRⅠ和XhoⅠ各2μL)顺序,加入到axygen PCR小管中,进行酶切反应10-20min 后终止酶切反应,跑胶验证结果,并切胶回收;
将质粒与DNA连接,反应体系:目的基因4.4μL,pET-30a3.6μL,T4连接酶1μL,10×buffer 1μL,16℃ 10-18h,获得重组质粒,并将重组质粒导入E.coliBL21(DE3)中,获得重组表达细胞。
步骤4:重组羰基还原酶和辅酶循环酶获得
将KRED06-P2A-GDH接种到含有卡那霉素抗性的LB液体培养基中,于37℃培养16h,得到种子培养液。将种子培养液接种到含卡那霉素抗性的TB液体培养基中,接种量为含卡那霉素抗性的TB液体培养基体积的1%。然后置于37℃下培养至 OD600值为0.8,加入终浓度为0.01mmol/L 的IPTG,置于30℃继续培养16h后,4000rmp,4℃下离心收集菌体,采用pH 值为7.0的100mmol/L 的PB缓冲液将收集后菌株进行洗涤并重悬,通过超声波破碎仪进行破碎,超声破碎功率为150W,运行5S,间隔5S,共运行3min,获得酮羰基还原酶与辅酶循环酶粗酶液,冻干后获得冻干粉。
步骤5:(3R,5R)-6-氰基-3,5-二羟基己酸叔丁酯的制备
在100mL锥形瓶中,加入5R-6-氰基-5羟基-3-氧代己酸叔丁酯0.2mol,NADP+0.2g,步骤(3)获得的冻干粉1.3g,葡糖1g,异丙醇4ml,pH7.0 0.1mM PB buffer 10ml,220rpm,30℃反应12h后终止反应。反应终止后经脱色、离心、萃取等操作,得到产物(3R,5R)-6-氰基-3,5-二羟基己酸叔丁酯,经气相分析,底物转化率97.98%,产物ee值99.7%。
序列表
<110> 江苏阿尔法药业有限公司
<120> 一种共表达载体应用于制备阿托伐他汀中间体的方法
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gaagctgttg tcgtccaagg agatgtcacg aaagaggaag atgtaaaaaa tatcgtgcaa 240
acggcaatta aggagttcgg cacactcgat attatgatta ataatgccgg tcttgaaaat 300
cctgtgccat ctcacgaaat gccgctcaag gattgggata aagtcatcgg cacgaactta 360
acgggtgcct ttttaggaag ccgtgaagcg attaaatatt tcgtagaaaa cgatatcaag 420
ggaaatgtca ttaacatgtc cagtgtgcac gaagtgattc cttggccgtt atttgtccac 480
tatgcggcaa gtaaaggcgg gataaagaaa atgacagaaa cattagcgtt ggaatacgcg 540
ccgaagggca ttcgcgtcaa taatattggg ccaggtgcga tcaacacgcc aatcaatgct 600
gaaaaattcg ctgaccctaa acagaaagct gatgtagaaa gcatgattcc aatgggatat 660
atcggcgaac cggaggagat cgccgcagta gcagcctggc ttgagtcgaa ggaagccagc 720
tacgtcacag gcatcacgtt attcgcggac ggcttaatga cacaatatcc ttcattccag 780
gcaggccgct aa 792
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgcctgcta cgttaaagaa 20
<210> 7
<211> 91
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aggtccaggg ttctcctcca cgtctccagc ctgcttcagc aggctgaagt tagtagctcc 60
gcttccttgg aaaattggga aggatcccca c 91
<210> 8
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccgctcgagt tagcggcctg cctggaa 27
<210> 10
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gggaattcat gcctgctacg ttaaagaa 28
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccgctcgagt tagcggcctg cctggaa 27
Claims (5)
1.一种共表达载体的制备方法,其特征在于:所述的共表达载体载入了包括一种羰基还原酶和一种辅酶NADP+循环所需的脱氢酶的基因。
2.根据权利要求1所述的共表达载体,其表达的羰基还原酶可用于不对称还原5R-6-氰基-5羟基-3-氧代己酸叔丁酯,制得光学手性醇。
3.根据权利要求1所述的共表达载体,羰基还原酶和辅酶循环所需脱氢酶的DNA连接序列为P2A,T2A、E2A,优选P2A,序列如SEQ ID NO.1所示。
4.根据权利要求1所述的共表达载体,其包含的辅酶循环酶基因为葡糖脱氢酶。
5.根据权利要求1所述的共表达载体的应用,其特征在于以5R-6-氰基-5羟基-3-氧代己酸叔丁酯为底物,在pH 7.0 的PB缓冲溶液中,在NADP+存在下,葡萄糖存在下,在权利要求2所述的羰基还原酶,权利要求4所述的脱氢酶存在下,不对称还原5R-6-氰基-5羟基-3-氧代己酸叔丁酯,获得所需的阿托伐他汀钙所需的中间体(3R,5R)-6-氰基-3,5-二羟基己酸叔丁酯。
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